188 32 46MB
English Pages 862 [864] Year 1986
Cysteine Proteinases and Their Inhibitors
Cysteine Proteinases and Their Inhibitors Proceedings of the International Symposium Portoroz, Yugoslavia, September 15 -18,1985 Editor Vito Turk
W G DE
Walter de Gruyter • Berlin • New York 1986
Editor Vito Turk, Professor, Dr. Institute Jozef Stefan Department of Biochemistry Jamova 39 61111 Ljubljana Yugoslavia
Cysteine proteinases and their inhibitors. Based on the First International Symposium on Cysteins Proteinases and Their Inhibitors. Includes bibliographies and indexes. 1. Cysteine proteinases-Congresses. 2. Cysteine proteinases-InhibitorsCongresses. I. Turk, Vito. II. International Symposium on Cysteine Proteinases and Their Inhibitors (1st: 1985 : Portoroz, Slovenia) [DNLM: 1. Cysteine-anatagonists & inhibitors-congresses. 2. Peptide Peptidohydrolases-congresses. 3. Protease Inhibitors-congresses. QU 60C9971985] QP609.C94C 1986 574.19'256 86-29132 ISBN 0-89925-263-X (U.S.)
CIP-Kurztitelaufnahme der Deutschen
Bibliothek
Cysteine proteinases and their inhibitors : proceedings of the internai, symposium Portoroz, Yugoslavia, September 15-18, 1985. / ed. Vito Turk. - Berlin ; New York : de Gruyter, 1986. ISBN 3-11-010724-4 N E : Turk, Vito [Hrsg.]
Copyright © 1986 by Walter de Gruyter & Co., Berlin 30. All rights reserved, including those of translation into foreign languages. N o part of this book may be reproduced in any form - by photoprint, microfilm or any other means nor transmitted nor translated into a machine language without written permission from the publisher. Printing: Gerike G m b H , Berlin. - Binding: D. Mikolai, Berlin. - Printed in Germany.
PREFACE
Proteinases and their inhibitors play an important role in protein degradation processes. Recently, there has been a tremendous growth of interest in cysteine
(thiol) proteinases and their newly dis-
covered protein inhibitors. The results of these studies have been published in many journals of biochemical, biological and clinical orientation, but have never been collected. The First International Symposium on Cysteine Proteinases and their Inhibitors, which was held in Portoroz, Yugoslavia, September 15-18, 1985, represented a unique opportunity for bringing together the most active groups working in this field. The list of participants clearly indicates the success of this meeting. Their results presented in this book reflect the current level of knowledge in this very active field. A variety of new ideas were expressed in the lively and stimulating discussions. The organizers of the Symposium would like to express their thanks and appreciation to all the participants. I sincerely hope that this book will be very useful in clarifying many problems, including the classification and nomenclature of the protein inhibitors of cysteine proteinases. What is more, I hope that the presented papers will stimulate further research in the areas covered in this book and in the medical sciences in general. Lubljana, August 1986
Vito Turk
ACKNOWLEDGEMENT The Organizers would like to express thanks to all of them who encouraged and supported this Symposium. Special thanks go to Dr. Hans Fritz for his activities before the conference. The meeting could not have benn possible without the financial support of the J. Stefan Institute (Ljubljana), Research Council of Slovenia, Krka, Pharmaceutical and Chemical Works (Novo mesto), Lek, Chemical and Pharmaceutical Work (Ljubljana),, TOK, Factory for Production of Organic Acids (Ilirska Bistrica), Jugolaboratorija (Beograd), E. I. Du Pont de Nemours and Co. (Wilmington, USA), Applied Biosystems (Foster City, USA) and Gruenenthal GmbH (Aachen, FRG). Gratitude is expressed also to Dr. Igor Kregar for his help in the preparation of this book. It is a pleasure to acknowledge the excellent secretarial assistance of Maja Lozar-Stamcar and Natasa Prislan.
O R G A N I Z I N G
C O M M I T T E E
V. Turk J. Brzin J. Kos M. Kotnik I. Kregar B. Lenarcic M. Lozar-Stamcar, Secretary
CONTENTS Preface
III
Acknowledgements Organizing Committee
V VII
NOMENCLATURE AND CLASSIFICATION OF THE PROTEINS HOMOLOGOUS WITH THE CYSTEINE PROTEINASE INHIBITOR CHICKEN CYSTATIN A.J. Barrett, H. Fritz, W. Müller-Esterl, A. Grubb, S. Isemura, M. Järvinen, N. Katunuma, W. Machleidt, M. Sasaki, V. Turk
1
GENERAL CYSTEINE PROTEINASES Isolation, properties , primary structure and cloning Human cathepsins B, H and L: characterization by amino acid sequences and some kinetics of inhibition by the kininogens W. Machleidt, A. Ritonja, T. Popovic, M. Kotnik, J. Brzin, V. Turk, I. Machleidt, W. Muller-Esterl
3
Tentative amino acid sequence of bovine spleen cathepsin B B. Meloun, J. Pohl, V. Kostka
19
Inhibition of human liver cathepsins B, H and L by the human
^ cysteine proteinase inhibitor
M. Pagano, R. Engler
31
Human kidney cathepsin L M. Kdtnik, T. Popovic, V. Turk
43
Latent human cathepsin L R. W. Mason
51
Cloning of a bovine protein homologous with cysteine proteinases and identification of the gene G. Salvesen, N. Gay, J. Walker
55
X Immunological studies on the secreted forms of cathepsin B J.S. Mort, A.D. Recklies
63
A study of the peptidyldipeptidase activity of bovine spleen cathepsin B using synthetic substrates J. Pohl, S. Davinic, I. Blaha, P. Strop, V. Kostka
73
Comparison of characteristics and drug-induced modifications of rat spleen cathepsins B and H K. Yamamoto, Y. Kato
79
Cystein proteinase activities in embryonic chicken sekeletal muscle I. Sohar, E. Fekete, J.W.C. Bird, G. Yorke, F.J. Roisen
89
Histone degradation by lysosomal proteases R. Chatterjee, M. Lones, G. Kalnitsky
97
Properties of a cysteine proteinase from human thyroids A.D. Dunn
111
Primary structure and function of calcium activated neutralprotease K. Suzuki, S. Ohno, F. Emori, S. Imajoh, H. Kawasaki
121
Identification of two alkaline cysteine proteinases from rat skeletal muscle B. Dahlmann, L. Kuehn, H. Reinauer
133
Multiple forms, structure and specificity of clostripain B. Keil, A.M. Gilles
147
Enzymatic and immunological identification of a cysteine proteinase procoagulant in human melanoma B. Casali, C.G. Passerini, A. Falanga, G. Fossati, N. Semeraro, M.B. Donati, S.G. Gordon
163
Thyroid cysteine proteinases that catalyze release of thyroxine from thyroglobulin and thyroxine-containing peptide H. Nakagawa, S. Ohtaki Proteases in African
179 trypanosomes
J.D. Lonsdale-Eccles, D.J. Grab
189
XI Characterization of a cysteinyl proteinase from the human parasite, Schistosoma mansoni C.L. Chappell, M.H. Dresden
199
Studies on a proteinase inactivating enzyme from the basidiomycete Coprinus atramentarius K. Otto, C. Lipperheide
209
GENERAL CYSTEINE PROTEINASES OF DIFFERENT ORIGIN Localization and biological role Distributions and localizations of lysosomal cysteine proteinases and cystatins N. Katunuma, E. Kominami
219
Functional shares of cathepsins B, H and L in autophagy and heterophagy E. Kominami, T. Obshita, N. Katunuma
229
Post mortem localization of lysosomal peptide hydrolase, cathepsin B A. Lacourt, A. Obled, C. Deval, A. Ouali, C. Valin
239
Bifunctional derivatives of L-trans-epoxysuccinyl-L-leucylamido-(4-amino)butane (Ep-459) as potential localization agents for cysteine proteinases C.G. Knight, S. Yamamoto
2^9
Cysteine proteinases and bone resorption J.M. Délaissé, P. Ledent, Y. Eeckhout, G. Vaes
259
The role of collagen-degrading cysteine proteinases in connective tissue metabolism D.J. Etherington, R.A. Maciewicz, M.A.J. Taylor, R.J. Wardale, I.A. Silver, R.A. Murrills, D. Pugh
269
The role of cathepsins H and B, and inhibitors leupeptin and CPI in proliferative activities of non-malignant cells in culture A. Suhar, V. Turk, M. Korbelik, D. Petrovic, J. Skrk, P. Schauer
283
XII Viruses as vectors for cysteine proteases B. Korant, T. Towatari, L. Ivanoff, C. Kettner, A. Cordova, S. Petteway, Jr
293
MECHANISM OF ACTION OF CYSTEINE PROTEINASES Natural structural variation in the cysteine proteinases as an aid to the study of mechanism by reactivity probe kinetics, catalysis kinetics and spectroscopic methods K. Brocklehurst
307
Mechanism of action of cysteine proteases: 1/ differences from serine enzymes;2/ the second thiol group of chymopapain L. Polgar, B. Asbotb, I. Korodi
327
Proposal of the double regulation mechanism for the action of calpain R. Kannagi, T. Sakihama, T. Murachi
339
Control mechanism of calcium-activated neutral protease (CANP) activity G. Kawashima, M. Inomata, K. Imahori
359
GENERAL CYSTEINE PROTEINASE INHIBITORS Isolation, properties and primary structure The mammalian cysteine proteinase inhibitors. Structural diversity and evolutionary origin. W. Muller-Esterl, H. Fritz, J.Kellermann, F. Lottspeich, W. Machleidt, V. Turk
369
Kininogens as thiol proteinase inhibitors M. Sasaki, I. Ohkubo, C. Namikawa, S. Higashiyama, H. Ishiguro, M. Kunimatsu, K. Kurachi, H. Shiokawa, T. Takasawa
393
Cystatin-like domains of LMW-kininogen, and speculations on the evolution of cystatins G. Salvesen. C. Parkes, N.D. Rawlings, M.A. Brown, A.J. Barrett, M. Abrahamson, A. Grubb
413
XIII Human stefins and cystatins: their properties and structural relationships V. Turk, J. Brzin, B. Lenarcic, A. Sali, W. Machleidt
429
Isolation of kininogens using affinity chromatography J. Brzin, M. Trstenjak, A. Ritonja, W. Machleidt, V. Turk
443
Characterization of low molecular mass cysteine proteinase inhibitors from human amniotic fluid S.T. Rohrlich, Z. Seigfried, P. Mignatti, W. Machleidt, H. Levy, D.B. Rifkin
455
Properties and structure of human spleen stefin B - a low molecular weight protein inhibitor of cysteine proteinases B. Lenarcic, A. Ritonja, A. Sali, M. Kotnik, V. Turk, W. Machleidt
473
Amino acid sequence of the cysteine proteinase inhibitor cystatin B from human liver A. Ritonja, W. Machleidt, A.J. Barrett
489
Cystatin S and the related cysteine proteinase inhibitors in human saliva S. Isemura, E. Saitoh, K. Sanada, M. Isemura, S. Ito
497
Cystatin C (Post i f Globulin) in serum from patients with autoimmune diseases A. Cattaneo, J.L. Sansot, D. Prevot, C. Blanc, Y. Manuel, A. Colle
507
Differential actions of human cystatin C on different functions of granulocytes J.L. Sansot, J.J. Bourgarit, C. Blanc, Y. Manuel, A. Colle, J. Leung-Tack, J.L. Mege
517
Potentiation of excretion of canine cystatin-C (Post-gamma globulin) T. Sekine, M.D. Poulik
527
The 43 kDa papain inhibitor in human tissues V.K. Hopsu-Havu, A. Rinne, M. Jarvinen
535
XIV Small and high molecular weight proteinase inhibitors from bovine muscle A. Ouali, L. Bige
A. Obled, A. Lacourt, C. Valin
5^5
Low molecular weight protein inhibitors of cysteine proteinases from bovine parotid glands N. Cimerman, J. Brzin, V. Turk
555
The interaction of papain molecule with thiol proteinase inhibitors from newborn rat epidermis T. Samejima, H. Kaji, A. Takeda
561
Chicken egg white cystatin as a ligand for affinity chromatography J. Kos, V. Arbanas, V. Turk, A.V. Maksimenko
569
Distribution of the egg white cystatin in chicken V. Curin, J. Babnik, J. Kos, F. Gubensek, V. Turk
577
Isolation and characterization of chicken egg white low-Mr kininogen J. Kos, M. Dolinar, V. Turk
583
Papain inhibition by snake venoms F. Aragon-Ortiz, A. Ritonja, V. Turk, F. Gubensek
593
Cysteine proteinase inhibitors from fish liver J. Dentes, Lj. Vitale
603
Cysteine proteinase inhibitors from sea anemone B. Lenarcic, M. Kokalj, V. Turk
609
Characterization of low molecular weight and high molecular weight endogenous inhibitors of calcium activated neutral protease K. Imahori, S. Kawashima, M. Nakamura
617
Calpain inhibition by peptide epoxides and the effect of autolysis C. Parkes, A.A. Kembhavi, A.J. Barrett
631
EST, a new analog of E-64, can prolong the life span of dystrophic hamsters, UM-X7.1 M. Tamai, K. Matsumoto, K. Oguma, I. Koyama, S. Omura, K. Hanada, S. Ishiura, H. Sugita
633
XV GENERAL CYSTEINE PROTEINASE INHIBITORS Localization, biological function and kinetics Cystatins A and B in normal and pathologically altered human tissues M. Jarvinen, A. Rinne, M. Alavaiko, V.K. Hopsu-Havu
6^+9
Chicken and rat muscle cystatins and their localization in cultured myoblasts L. Wood, J.W.C. Bird, G. Yorke, F.J. Roisen
667
Distribution and solubilization of a high molecular weight cysteine proteinase inhibitor from rat epidermis K. Fukuyama, S. Toku, S. Nakano, W.L. Epstein
685
Possible biological functions of protein proteinase inhibitors J. G. Bieth
693
A curve-fitting approach to the determination of kinetic constants of proteinase inhibitors W. Machleidt, I. Machleidt, W. Muller-Esterl, J. Brzin, M. Kotnik, T. Popovic, V. Turk
705
Inhibition of cathepsins B, H and L by rat thiostatin, the circulating o61cysteine proteinase inhibitor, and by an active fragment F. Gauthier, T, Moreau, N. Gutman, A. El Moujahed, F. Esnard
719
CYSTEINE PROTEINASES AND THEIR INHIBITORS IN TUMORS Tumor cysteine proteinases and their inhibitors B.F. Sloane, T.T. Lah, N.A. Day, J. Rozhin, Y. Bando, K.V. Honn
729
Thiol protease inhibitor released from human malignant melanoma Y. Nishida, H. Tsushima, N. Toki, H. Sumi, H. Mihara
751
XVI
PROTEINASES AND THEIR INHIBITORS Medical aspects Cystatin C (if" -trace) amyloidosis 0. Jensson, A. Arnason, L. Thorsteinsson, I. Petursdottir, G. Gudmundsson,H. Blondal, A. Grubb, H. Lofberg, W. Luyendijk, G.T.A.M. Bots, B. Frangione
761
Some biochemical aspects of chymopapain treatment of sciatica D.J. Buttle
779
Granulocyte proteinases as mediators of unspecific proteolysis in inflammation: a review H. Fritz, M. Jochum, K.H. Duswald, H. Dittmer, H. Kortmann, S. Neumann and H. Lang
785
LIST OF PARTICIPANTS
809
AUTHOR INDEX
815
SUBJECT INDEX
821
NOMENCLATURE AND CLASSIFICATION OF THE PROTEINS HOMOLOGOUS WITH THE CYSTEINE PROTEINASE INHIBITOR CHICKEN CYSTATIN Alan J. Barrett Department of Biochemistry, Strangeways Research Laboratory, Worts Causeway, Cambridge CB1 4RN, U. K. Hans Fritz, Werner Muller-Esterl Institute of Clinical Chemistry and Clinical Biochemistry, University of Munich, Nussbaumstrasse 20, 8000 Munich 2, FRG Anders Grubb Department of Clinical Chemistry, University of Lund, Malmo General Hospital, S-214 01 Malmo, Sweden Satoko Isemura Department of Oral Biochemistry, Nippon Dental University, Niigata Faculty, Niigata 851, Japan Mikko Jarvinen University of Oulu, Department of Pathology, Kajaanintie 52D, SF-90220 Oulu 22, Finland Nobuhiko Katunuma Institute for Enzyme Research, The University of Tokushima, School of Medicine, Kuramoto, Tokushima 770, Japan Werner Machleidt Institute of Physiological Chemistry, Physical Biochemistry and Cell Biology, University of Munich, D 8000 Munich 2, Goethestrasse 33, FRG Makoto Sasaki Department of Biochemistry, Nagoya City University Medical School, Mizuho-Ku, Nagoya, Aichi ^67, Japan Vito Turk Department of Biochemistry, J. Stefan Institute, University E. Kard.elj of Ljubljana, Jamova 39, 61000 Ljubljana, Yugoslavia The subject and the nomenclature of the proteins homologous with chicken cystatin, most if not all of which are inhibitors of cysteine proteinase, was discussed at the First International Symposium on Cysteine Proteinases and Their Inhibitors (Portoroz, Yugoslavia: September 15-18, 1985). The great majotity of scientists who have published work on these proteins anywhere in the world
Cysteine Proteinases and their Inhibitors © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany
2 were present. Several points were discussed, and agreement was reached as follows: 1. It was agreed that the proteins that can be shown statistically to have an evolutionary relationship to chicken cystatin form a distinct superfamily (as defined by Dayhoff et al., 1983). This should be called the cystatin superfamily. 2. The known proteins of the cystatin superfamily can be seen to comprise three protein families ( as defined by Dayhoff et al., 1983). 3. Family 1, which may also be called the stefin family, contains the proteins that are distinguished within the superfamily by the lack of disulphide bonds, and by close sequence relationships to each other. Known proteins of this family have been named "human stefin", "human cystatin A", "rat cystatin«6", "human cystatin B" and "rat cystatin/i ". Family 2, which may also be called the cystatin family, so long as confusion with the name of the superfamily is avoided, contains proteins that have two disulphide bonds. These include proteins that have been named "chicken cystatin", "human cystatin", "human cystatin C", "human cystatin S" and"beef colostrum cystatin". 5. Family 3 comprises the plasma kininogens, and may therefore also be called the kininogen family. Known members of this family, which contain 9 disulphide bonds, have been named "human
-thiol proteinase inhibitor", "hu-
man JL, 2-thiol proteinase inhibitor", "human -raa>i)cíjffl•-tía; • ^fHfHa)tí Aifna) tí o cn dtí tuPrH h tí ^[Hotí>-Hra raa-P) 3a* CM • CD CD CO UJ UJ Iii g -p*Hoiûa)tí-Hra o raft% M M M fíhHtí a ) pi -h tí œ••> tstí¡>o-dtí-ho ajh H OOO OOOOO pqpi ® "dtí ra-p |> M > £ ai 3 œ o OOOEHtJKOtí tí ¿i o H o H * tt * oc ooo • h ra ra ,qtí oo'd a) q ti f n c u tío fH ® >^ X c o u. ai ^a)cd O u. u. £ ^oh cdo ^Ph,tíofcHa).típ!q o K) » M M- u. u. ooo ra ' H tí E H c d ^ -p ao > Ai ® • tí ai a ) • ra^cpq OOO O 0£ Û£ O a>tíeh h^tíA o>o o o p^Ho^g SO3a>cs^-pm -HAití -Hra H H H OÛ.O.Û. CO q CO CO co DZZ c\i p in • H a ) c d ra tó-Htí•>rao-HfnaP)),tí joqû CM|Z M M 111 H •a!ra ^ I*-'**! oc oc oc -Ci O h ftli-P *o-ha;^ oMAicji>-Pracd MM M 0)ÜOO o o olui CMoooAiajrao cd•Hï> P cd cö~ • tíH -P'-v'ti ^ -H ÍHcd H© > ** m «H O > ** 0 3• o O® 3 o aj 3 o • o • s o S c d P( CD OC X CD £C I CD CD Oí I CD ai fH^Htí cd C î=d- .pl>3 ra co a: i cd
26 h i g h l y probable, also i n view of the recent data on the
carbo-
hydrate m o i e t y of cathepsin B f r o m porcine spleen. In contrast, the m e t h i o n i n e residues w h i c h are also of m i n o r occurrence
in
cathepsins B^ occupy different positions v a r y i n g w i t h source of the enzyme. D i s t r i b u t i o n of Half-Cystines and Methionines in C a t h e p s i n B f r o m Bovine S p l e e n a n d Other Sources Figure 3 shows a schematical representation of the polypeptide backbone of cathepsin B w i t h the positions of h a l f - c y s t i n e s ; the m e t h i o n i n e residues a n d the five fragments resulting from cleavage at these residues are also shown. A s m e n t i o n e d above the active site cysteine occupies p o s i t i o n 29. From our data (12) Cys-14 and Cys-43 of the light chain of bovine
spleen
cathepsin B are l i n k e d by a disulfide bond. A close spatial contact of these residues seems to emerge also from computer graphics m o d e l i n g of m o l e c u l e s of various cysteine
proteinases
r e p o r t e d b y Kamphuis a n d coworkers (22). Still another
disulfide
b o n d is s u p p o s e d to h o l d together the two-chain f o r m of cathepsin B and to link Cys-26 w i t h Cys-71. A n identical link exists b e t w e e n the sequentially homologous regions i n related cysteine proteinases
(22). Information o n the remaining disulfide bonds
is lacking. Bonds b e t w e e n Cys-62 and Cys-119 and b e t w e e n Cys-63 and Cys-67 have b e e n p o s t u l a t e d to exist in cathepsins B based on their sequential h o m o l o g y w i t h p a p a i n a n d actinidine. puter graphics modeling of cysteine proteinases
Com-
(22) seems to
indicate that Cys-100, Cys-132, Cys-108 and Cys-128 are l o c a l i z e d close together i n the three-dimensional structure; direct evidence of disulfide bonds linking these residues, h o w e v e r , is lacking. M o r e o v e r , there are no disulfide bonds i n the
sequen-
tially homologous positions of p a p a i n a n d cathepsin H. I n this study a peptide containing residues 236 through 254 was isolated f r o m the tryptic digest. Positions 240 and 252 of the peptide are occupied by half-cystines and there is a methionine at p o s i t i o n 250. In other experiments we have shown that two cyanogen bromide fragments, CB4 a n d CB5 can be separated only after their r e d u c t i o n a n d carboxymethylation. This f i n d i n g leeds u s to conclude that i n this particular p r e p a r a t i o n of cathepsin
27
Figure 3. Distribution of half-cystines and. methionines in bovine spleen cathepsin B. A schematic representation of the polypeptide backbone of the enzyme molecule is shown. Half-cystines are marked by empty circles, the active site cysteine by a full circle. The positions of methionines (JSC) and of the cyanogen bromide fragments (CB1-CB5) are also shown. The residues are numbered as described in Figure 2.
28 B Cys 240 and. Cys 252 are linked by a disulfide bond. If this is the case there is still one "odd" half-cystine in position 148 (peptide 145-151) in bovine spleen cathepsin B for which we do not find a partner. It remains to be shown whether or not this is smother cysteine bearing a free SH-group in this enzyme.
Conclusions The data shown in Figure 2 indicate a high degree of homology in amino acid sequence of cathepsin B from various sources. However, half-cystine residues Cys-148 and Cys 252 were found in bovine cathepsin B only. The existence of a disulfide bond between these residues is at the present state of our knowledge excluded by the finding of the link between Cys-240 and Cys-252. All the cathepsins B studied so far show even numbers of half-cystine residues determined by sequence studies. Since at least one of these residues must bear a free SH-group we are faced with the problem of one unpaired half-cystine. The existence of such a residue seems to be suggested by the data of Snellmann (23). Before all the disulfide bonds are determined unambiguously several other explanations are possible, such as the formation of artifacts during enzyme isolation or disulfide interchange between closely localized half-cystines in the three-dimensional structure of the native enzyme.
References 1. Ritonja, A., T. Popovic, V. Turk, K. Wiedenmann, W. Machleidt. 1985. FEBS Lett. 181, 169-172. 2. Takio, K. , T. Towatari, N. Katunuma, D.C. Teller, K. Titani. 1983. Proc. Uatl. Acad. Sci. USA 80, 3666-3670. 3. Takahashi, T., A.H. Dehdarani, P.G. Schmidt, J. Tang. 1984. J. Biol. Chem. 259, 9874-9882. 4. Takahashi, T., P.G. Schmidt, J. Tang. 1984. J. Biol. Chem. 259, 6059-6062. 5. Takahashi, T., A. Yomezawa, A.H. Dehdarani, J. Tang. 1985. Federation Proc. 44, 874.
29 6. Keilovä, H. , B. Keil. 1969. FEBS Lett. 4, 295-298. 7. Keilovä, H. 1971. In: Tissue Proteinases (A.J. Barrett and. J.T. Dingle, eds.) N o r t h - H o l l a n d Publishing Co., A m s t e r d a m - L o n d o n , pp. 45-68. 8. K e i l o v a , H . , V. Tomäsek. 1973. FEBS Lett. 29, 335-338. 9. Keilova, H . , V. Tomäsek. 1974. Biochim. Biophys. A c t a 334, 179-186. 10. Keilova, H . , V. Tomäsek. 1975- Collect. Czech. Chem. Commun. 40, 218-224. 11. K e i l o v ä , H . , V. Tomäsek. 1977* Acta biol. med. germ. 36, 1873-1881. 12. Pohl, J., M . Baudys, V. Tomäsek, V. Kostka. 1982. FEBS Lett. 142, 23-26. 13- Turk, V . , J. Brzin, M . Kopitar, I. Kregar, P. Locnikar, M. L o n g e r , T. P o p o v i c , A. R i t o n j a , Lj. V i t a l e , W. M a c h l e i d t , T. Giraldi, G. Sava. 1983. In: Proteinase Inhibitors: M e d i c a l a n d Biological Aspects (N. Katunuma, H. Umezawa, H. Holzer, eds.) J a p a n Sei. Soc. Press, Tokyo, S p r i n g e r - V e r l a g , Berlin, pp. 125-134. 14. Erlanger, B.F., A . G . Cooper, W. Cohen. 1956. Biochemistry 5, 190-196. 15. Bieber, F.A., G. Braunitzer. 1984. Hoppe-Seyler's Z. Physiol. Chem. 365, 321-334. 16. Edman, P., G. Begg. 1967. Eur. J. Biochem. 1, 80-91. 17. Chang, J.Y., D. Brauer, B. Wittmaim-Liebold. 1978. FEBS Lett. 93, 205-214. 18. Kostka, V . , F.H. Carpenter. 1964. J. Biol. Chem. 239, 1 7 9 9 -1803. 19. Shechter, Y., M. Rubinstein, A. Patchornik. 1977. B i o c h e m i s t r y 16, 1424-1430. 20. S a n Segundo, B., S.J. Chan, D.F. Steiner. 1985. Proc. Natl. Acad. Sei. USA 82, 2320-2324. 21. Gay, N.J., J.E. Walker. 1985. Biochem. J. 225, 707-712. 22. Kamphuis,I.G., J. Drenth, E.N. Baker. 1985. J. Mol. Biol. 182. 317-329. 23. Snellmann, 0. 1971. In: Tissue Proteinases (A.J. Barrett a n d J.T. Dingle, eds.) N o r t h - H o l l a n d Publishing Co., A m s t e r d a m - L o n d o n , pp. 29-38.
INHIBITION
OF HUMAN L I V E R
CATHEPSINS
B , H AND L BY THE HUMAN a 2
CYSTEINE
PROTEINASE
INHIBITOR.
M. Pagano, R. Engler Laboratoire de Biochimie, Université Paris 6, 15 rue de l ' E c o l e de Médecine, 75270 PARIS Cedex 06, FRANCE.
1.
Introduction
Plasma proteinase i n h i b i t o r s are involved in the regulation of e x t r a c e l l u l a r a c t i v i t i e s of proteinases. weight glycoproteins lation.
They are, in mammalian species, high molecular
biosynthetized in the l i v e r and secreted into the c i r c u -
The biological function of t h i s group
of component
, which are often
acute phase reactants, would be to counteract the degradative a c t i v i t i e s of t i s s u e proteinases liberated by injured c e l l s .
A l o t of studies were performed
on the plasma i n h i b i t o r s of serine proteinases (cq proteinase i n h i b i t o r , the s o - c a l l e d olj a n t i t r y p s i n , and cq antichymotrypsin for example)
(1-2).
On the contrary, l i t t l e i s known about the plasma i n h i b i t o r s of cysteine proteinases.
The f i r s t report in t h i s f i e l d was S a s a k i ' s paper on the papain i n h i -
b i t o r of human serum, a 2 CPI ( 3 ) .
This was followed by some studies on the pu-
r i f i c a t i o n of t h i s protein (4-5).
But the weak i n h i b i t i o n of human l i v e r ca-
thepsin B was very puzzling.
This fact has increased the d i f f i c u l t y for the
understanding of the biological role of a 2 CPI. We have found that a 2 CPI i n h i b i t s human l i v e r cathepsins L and H in such a way that i t could be of physiological relevance.
For the cathepsin L, we have
shown that t h i s i n h i b i t o r acts as a r e v e r s i b l e i n h i b i t o r which i s able to t r a n s fer the enzyme to plasma a 2 macroglobulin
(6-7-8).
The i n h i b i t o r y properties of a 2 CPI on the lysosomal cysteine proteinases have been reported by other authors (9).
A discrepancy was found for the i n t e r a c -
tion between human l i v e r cathepsin L and a 2 CPI.
Consequently, i t has been men-
tioned that our p u r i f i e d cathepsin L was " e i t h e r a severely modified form of cathepsin L or a d i f f e r e n t enzyme" by the same group (10).
For a better under-
standing, we have reinvestigated a l l the properties of our p u r i f i e d cathepsin L. Here we describe the reexamination of human l i v e r cathepsin L and we summarize our f u l l study on the i n h i b i t o r y properties of a 2 CPI.
Cysteine Proteinases and their Inhibitors © 1 9 8 6 Walter d e Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y
32 Abbreviations : &2 CPI : a2 Cysteine Proteinase Inhibitor, ct2_i and Z-Arg-Arg-NHMEC : Benzyloxycarbonyl-L-Argi nyl-L-Argi ni ne-4-Methyl-7-coumarylamide. Z-Phe-Arg-NHMEC : Benzyloxycarbonyl-L-Phenylalanyl-L-Argi ni ne-4-Methyl7-coumaryl amide. Arg-NHMEC : L-Arginine-4-Methyl-7-coumarylamide. NH2MEC : 4-Methyl-7-coumarylamide. Z-Phe-PheCHN2 : Benzyloxycarbonyl-L-Phenylalanyl-L-Phenylalanine-Diazomethane. Z-Arg-Arg-NNAP : Benzyloxycarbonyl-L-Arginyl-L-Argi nine-2-Naphthylami de. Bz-Arg-NNAP : N-Benzoyl-D-L-Arginine-2-Naphthylamide. E64 : L-3-Carboxy-trans-2-3-epoxypropyl-Leucyl-amino (4-guanidino) butane.
2.
Materials and Methods :
2.1.
Materials :
Z-Phe-Arg-NHMEC, Arg-NHMEC, NH2MEC, E64 were supplied by the Protein Research Foundation (Osaka, JAPAN). Z-Arg-Arg-NHMEC, Z-Arg-NNAP, Z-Phe-Phe-CHN2 and Bz-Arg-NNAP were purchased from Bachem Feinchemikalien AG (Bubendorf, SWITZERLAND). CM32 cellulose and ultrogel ACA44 were obtained from Whatman (Maidstone, Kent, GREAT BRITAIN) and IBF (Gennevilliers, FRANCE). Ampholines pH 3.5-10.0 were from LKB (Bromma, SWEDEN). All other reagents were of analytical grade. Fluorescence measurements were carried out with a Kontron SFM 25 spectrofluorometer at Xex. 340nm and Xem. 433nm.
The Atx. and the Xem. values were de-
termined with NH2MEC also used for the calibration.
For initial rate assay, a
Tarkan 600 recorder was used. 2.2.
Purification of lysosomal cysteine proteinases :
All the operations were performed at 4°C.
A piece of frozen human liver (lOOg)
was homogeneised in 250 ml of 3% NaCl , ImM EDTA pH=3.80 with 0, 2% X 100 in a Waring Blendor Apparatus.
triton
Then the pH of the homogenate was adjus-
ted to 3.80 by adding 1M HC1 and enzymes were extracted by stirring overnight at 4°C. 3000g.
The insoluble material was removed by centrifugation 15 min. at A 30-70% saturation (NH4) 2SO4 fraction was taken.
The pellet was
33 resuspended in 50 ml 0.1M acetate buffer pH 5.30 and extensively dialysed against the same buffer.
After c e n t r i f u g a t i o n , the supernatant was applied to
a column (3.0 x 25 cm) of CM-32 c e l l u l o s e equilibrated in the acetate buffer 0,1M pH 5.30.
Cathepsins B and H emerged in the breakthrough peak.
was used for the
This peak
separation of both enzymes according to Schwartz and Barrett
(11) by means of a DEAE Sephadex chromatography.
Cathepsin L which binds
t i g h t l y to the CM-32 column was eluted with a phosphate buffer, 0,15M pH 7.50 and concentrated q u i c k l y .
The three separated enzymes were subsequently p u r i -
f i e d by gel f i l t r a t i o n on a ACA-44 column (2.5 x 75 cm) equilibrated with a 0.15M acetate buffer pH 4.60, 0.5M NaCl, ImM EDTA, 0,5mM DTT.
Two successive
gel f i l t r a t i o n steps were required to complete the p u r i f i c a t i o n . 2.3.
Enzyme assays :
For the active s i t e t i t r a t i o n of human l i v e r cathepsin L, 5 M1 of enzyme s o l u tion (1,5 yM , protein concentration) were preincubated 30 min. at 25°C with increasing volume of 1,46 yM
E64 (1 to 20 y l ) in 100 yl of a c t i v a t i o n buffer
(0.1M phosphate pH 6.0, ImM DTT, 2mM EDTA).
20 yl of t h i s mixture was added
to 980 yl of a c t i v a t i o n buffer containing Z-Phe-Arg-NHMEC (9 yM, final tration). kcat
concen-
Appearance of product was recorded continuously during 10 min.
The
and the Km values against Z-Phe-Arg-NHMEC were determined by the same
method as described above.
For comparison, these experiments were also c a r r i e d
out with 9 yM of Z-Arg-Arg-NHMEC instead of Z-Phe-Arg-NHM EC. ments were made up
These measure-
with 0,2nM of active enzyme.
The s p e c i f i c a c t i v i t i e s of the enzyme was measured by stopped assays in the following way : Cathepsin L (0,1 yM active f i n a l concentration) in 400 yl of a c t i v a t i o n buffer was incubated with 0,1 mM of substrate during 15 min. at 40°C.
The three following fluorogenic substrates were used : Z-Phe-Arg-NHMEC,
Z-Arg-Arg-NHMEC, Arg-NHMEC. (12). rial"
The assay was stopped and diluted as described in
The fluorescence measurement was carried out as described in the "Matesection.
For the Naphthylamide substrates (Z-Arg-Arg-NNAP and Bz-Arg-NNAP), the concent r a t i o n s used were 0,16 mM and 0,4 mM respectively in the same incubation conditions.
Then the reaction was stopped and samples were processed as in (6-7).
The i n h i b i t i o n of t h i s enzyme by Z-Phe-Phe-CHNg
was assayed with 0,5 yM of
i n h i b i t o r and 10 nM of enzyme by using Z-Phe-Arg-NHMEC as substrate.
34 2.4.
Gel electrofocusing of human l i v e r cathepsin L.
The p u r i f i e d enzyme (25 yg) was subjected to i s o e l e c t r o f o c u s i n g in acrylamide gels as previously described (6). blue.
A gel was stained with Coomassie b r i l l i a n t
Another one was cut in 5 mm s t r i p s which were immersed in 500 yl
tion buffer containing 0.1 mM Z-Phe-Arg-NHMEC.
activa-
After one hour of incubation
time at 40 c C, the fluorescence was measured as already described.
The pH gra-
dient was obtained after incubation of 5 mm s t r i p s during two days i n 1 ml of d i s t i l l e d water at 4°C. 2.5.
SDS gel electrophoresis of human l i v e r cathepsin L.
This was carried out in 15% acrylamide gels as previously mentioned
(6).
Samples (40 ug)were incubated overnight with 1% SDS and 1% DTT before b o i l i n g for 5 min. 3.
Results and d i s c u s s i o n .
3.1.
P u r i f i c a t i o n and properties of human l i v e r cathepsin L.
By gel e l e c t r o f o c u s i n g , t h i s enzyme exhibited several c l o s e l y spaced bands in the pH range 5.2 - 6.2 when the gel was stained by Coomassie b r i l l i a n t blue. The major disk was at pH 5.8 which corresponded to the maximal a c t i v i t y Z-Phe-Arg-NHMEC, as shown on F i g . 1 .
A lower a c t i v i t y against t h i s
against
substrate
was also present between pH 5.2 - 6.2 associated with the protein s t a i n i n g . A s i m i l a r a c t i v i t y pattern was shown for the rabbit l i v e r enzyme (13) and the human l i v e r enzyme has been described to c o n s i s t in multiple forms in the pH range 5.7 - 6.3 (10).
For the rat l i v e r enzyme, a range 5.5 - 6.1 was repor-
ted (14). SDS-polyacrylamide-gel-electrophoresis
under reducing conditions showed one
band of protein at Mr 30 000, according to our previous r e s u l t s (6) (Fig. 2). The enzyme was obtained as a s i n g l e chain form.
The human l i v e r enzyme has
been described by Mason et A1. (10) as a protein of Mr 29 000 which exhibited two chains of Mr 25 000 and 5000 after reduction, but in that case an a u t o l y s i s step (37°C-4H) was used at the beginning of the p u r i f i c a t i o n procedure. Active s i t e t i t r a t i o n of human l i v e r cathepsin L (Fig. 3a and 3b) shows that the enzyme was 40% active : 2 yl of 1.46 yM E64 i n h i b i t e d all the active enzyme
present in 5 yl of 1.5 yM p u r i f i e d protein.
This l a t t e r r e s u l t allows
us to define the k i n e t i c constants of the enzyme : I n i t i a l
rate assay with
35
Gel
Length
(Cm)
Fig. 1. :
Analytical
gel electrofocusing of purified human liver cathepsin L.
The enzyme (25 ug) was run over a pH gradient 3.5-10. A gel was stained with Coomassie brillant blue.
(Top).
The enzymatic activity was measured on gel strips (5 mm) after lh. incubation time with
O.lwMZ-Phe-Arg-NHMEC.
pH gradient
: •
Enzymatic activity :
The major isozyme was located at pH 5.8.
O
36
M r x id3 94
66
45
20.1
14.4 —DYE
b
a
Fig. 2. : SDS gel electrophoresis of human l i v e r cathepsin L under reducing conditi The enzyme (40 yg) was run on 15% (w/v) acrylamide g e l s . Samples were processed as described in the experimental
part.
Reference proteins are shown for comparison. (a) : Standards proteins,
(b) : Human l i v e r cathepsin L.
37
Fig. 3. : Active-site titration of human liver cathepsin L. 5 yl of enzyme solution were preincubated 30 min. at 25°C with increasing amount (1 to 20 yl) of 1.46 yM E64 in 100 yl of activation buffer. A 20 yl aliquot was added in a cuvette thermostated at 25°C containing Z-Phe-Arg-NHMEC (9 yM) in 980 yl of activation buffer and the fluorescence of free NH2MEC was recorded. (a) : Appearance of product versus time. (b) : Titration curve.
38 0,2 nM of enzyme
at 25°C gave a k c a t > value of 12.5 S" 1 and a K m value of 2
pM against Z-Phe-Arg-NHMEC. On the other hand, by the same assay method, the activity against Z-Arg-ArgNHMEC was found to be very low (5% of the activity against Z-Phe-Arg-NHMEC). (Fig. 4). The kinetic data are similar to those reported by other groups (10-12-13) and underline the requirement of an hydrophobic residue in P2 for cathepsin L. Stopped assays at 40°C for 15 min. gave similar results and no activity was found against Arg-NHMEC.
The peptide diazomethane, Z-Phe-Phe-CHN2, inhibited
the purified enzyme in such a way that the presence of cathepsin B in the cathepsin L preparation could be excluded (Table 1)
(14-15).
All the data presented above are in agreement with our previous report on human liver cathepsin L (6) and there is no evidence that this enzyme is a modified form of cathepsin L.
3.2.
Study of the inhibition of human cathepsins B, H and L by a2 CPI.
The ct2 CPI used in our work was isolated from pleural or ascitic fluids.
By
analytical gel electrofocusing, we have found a similar pi than that reported for the plasma inhibitor. (3-4-5-6). An antiserum was raised in the rabbit against the purified protein.
By elec-
troimmunoassay, the plasma concentration was found to be 7.5 yM ± 2.5. protein is not an acute phase reactant in the human.
This
The rat homologue, oq
CPI is an acute phase reactant identical to the a^ globulin of Darcy, the socalled a} major acute phase globulin (16). After purification, the human inhibitor gave two distinct components, ct2 CPI and a^ CPI.
In plasma, and also in exsudates, a2 CPI was found to be the major
component (3-4-5-6-17) by using a monospecific antiserum against both inhibitors.
The a-^ CPI would be a diiner which appears during the isolation (4-8).
Recently, it has been found that ag CPI is identical to the LMr kininogen (18) and it has been pointed out that a^ CPI may be identical with the HMr kininogen (19). This latter point may give a possible explanation for the poor immunological reaction of cq CPI in human plasma : HMr kininogen is present as a complex with prokallikrein, which is tightly bound (20) and LMr kininogen is present in a free form in the plasma. The poor immunological reaction in the oq region of the plasma may be provoked by the HMr kininogen-prekallikrein complex, and the appearance of a^ CPI during the course of complex.
the purification is probably linked to the disruption of this
39
F i g . 4. : Comparison of the cathepsin L a c t i v i t y a g a i n s t Z-Phe-Arg-NHMEC and Z-Arg-Arg-NHMEC. 0.2 nM of active enzyme was used in the conditions described above ( F i g . 3) with 9 uM Z-Phe-Arg-NHMEC or Z-Arg-Arg-NHMEC. kcat.
was
calculated by using a c a l i b r a t i o n curve drawn with i n c r e a s i n g con-
c e n t r a t i o n s (1 to 100 nM) of free
NH2MEC.
The amount of NH2MEC l i b e r a t e d in these c o n d i t i o n s was 2.5 n N . S " 1 when Z-Phe-Arg-NHMEC was the s u b s t r a t e .
40 Table 1 Specific activities of human liver cathepsin L against synthetic substrates and inhibition of this enzyme by Z-Phe-Phe-CHN2The assays were carried out at pH 6.0, 40°C for 15 min. in the conditions described in the experimental part.
Substrates
Specific activities 111 = 1 yM P. minting" 1 active enzyme.
Z-Phe-Arg-NHMEC
2710
Z-Arg-Arg-NHMEC
136 0
Arg-NHMEC Z-Arg-Arg-NNAP
0.38
Bz-Arg-NNAP
0,025
Inhibition test : Residual activity against Z-Phe-Arg-NHMEC
0
Table 2 Kinetic constants for the interaction of cig CPI with cathepsins H and L. Data from (7) and (8). Ki (nM)
ki ( M _ 1 S _ 1 )
(S" 1 )
Cathepsi n H
0.9
2.10
1.10"
3.0
8.10*
7.10"
Cathepsin L
41
I f t h i s assumption i s v a l i d , a j CPI i s not a dimer o f cig CPI as p r e v i o u s l y cribed.
l y s t e p s o f the p u r i f i c a t i o n by another group (9) dance
des-
Plasma K a l l i k r e i n has been shown to contaminate a CPI d u r i n g the e a r : t h i s result i s in accor-
with the above e x p l a n a t i o n .
The i n h i b i t o r y p r o p e r t i e s o f a C P I - K i n i n o g e n s a g a i n s t c y s t e i n e p r o t e i n a s e s were also recently discovered
(6-7-8-9).
These p r o t e i n s were d e s c r i b e d as papain and f i c i n i n h i b i t o r s shown that a probable p h y s i o l o g i c a l
(3-4-5).
We have
f u n c t i o n o f these i n h i b i t o r s was to r e g u -
l a t e both c a t h e p s i n s H and L a c t i v i t i e s
(6-7-8).
these i n t e r a c t i o n s are summarized i n Table 2.
All
the k i n e t i c c o n s t a n t s
found that these i n h i b i t o r s were a c t i n g as r e v e r s i b l e i n h i b i t o r s t h i s p r o t e i n a s e to plasma 012 m a c r o g l o b u l i n .
of
For c a t h e p s i n L , we have a l s o transferring
From these r e s u l t s , we p o s t u l a t e
t h a t a CPI may have a r e g u l a t o r y r o l e on the lysosomal c a t h e p s i n s H and L i n the e x t r a c e l l u l a r space d u r i n g inflammatory The weak i n h i b i t i o n o f c a t h e p s i n B was an structural
processes. intriguing result.
But the f u l l
study o f c a t h e p s i n j B , H and papain (21) may g i v e an e x p l a n a t i o n :
C a t h e p s i n H i s more c l o s e l y r e l a t e d to papain than c a t h e p s i n B. T h i s l a t t e r p r o t e i n a s e would be the most s t r u c t u r a l l y modified p r o t e i n a s e
in
the papain s u p e r - f a m i l y . The i n h i b i t i o n o f c a l p a i n s reported by S a s a k i et A1. (22) was another p o i n t f o r the b i o l o g i c a l
role of a CPI.
important
These p r o t e i n a s e s may be r e l e a s e d i n
the blood by a l o t o f t i s s u e s and they are f u l l y a c t i v e i n the
intravascular
space. In c o n c l u s i o n , as a working h y p o t h e s i s , we suppose t h a t both b i o l o g i c a l
func-
t i o n s o f a C P I - K i n i n o g e n s may be l i n k e d : For example, on one hand, i n the k i d n e y , membrane-bound k a l l i k r e i n k i n i n s from LMr k i n i n o g e n , which c o n t r o l hand, u r i n a r y d e r i v a t i v e s
renal c i r c u l a t i o n
releases
; and on the o t h e r
o f a CPI were found with i n h i b i t o r y p r o p e r t i e s
(17).
References. 1.
T r a v i s , J . and S a l v e s e n , G.S.
1983. Ann. Rev. Biochem. 52, 655-709.
2.
Arnaud,P. and Gianazza, E. 1984. In : Marker P r o t e i n s in Inflammation, V o l . 2 (P. Arnaud, J . Bienvenu and P. L a u r e n t , e d s . ) . Walter de G r u y t e r , B e r l i n , pp. 181-203.
3.
S a s a k i , M., M i n a k a t a , K . , Yamamoto,H., Niwa,M., Kato,T. and I t o , N . Biochem. B i o p h y s . Res. Commun. 76, 917-924.
1977.
42 4.
Jarvinen, M. 1979. FEBS Lett. 108, 461-464.
5.
Ryley, H.C. 1979. Biochem.Biophys.Res.Commun. 89, 871-878.
6.
Pagano,M. and Engler, R. 1982. FEBS Lett. 138, 307-310.
7.
Pagano,M. and Engler, R. 1984. FEBS Lett. 166, 62-66.
8.
Pagano,M., Esnard,F., Engler,R. and Gauthier.F. 1984. Biochem.J. 220, 147-155.
9.
Gounaris.A.D., Brown,M.A. and Barrett,A.J. 1984. Biochem.J. 221,445-452.
10.
Mason,R.W., Green,G.D.J, and Barrett,A.J. 1985. Biochem.J. 226, 233-241.
11.
Schwartz,W.N. and Barrett,A.J. 1980. Biochem.J. 191, 487-497.
12.
Barrett,A.J. and Kirschke,H. 1981. Methods Enzymol. 80, 535-561.
13.
Mason,R.W., Taylor,M.A.J. and Etherington,D.J. 1984. Biochem.J. 217, 209-217.
14.
Kirschke,H. Locnikar,P. and Turk,V. 1984. FEBS Lett. 174, 123-127.
15.
Kirschke.H. and Shaw,E. 1981. Biochem.Biophys.Res.Commun. 101, 454-458.
16.
Esnard,F. and Gauthier.F. 1983. J.Biol.Chem. 258, 12443-12447.
17.
Taniguchi ,K., Ito,J. and Sasaki,M. 1981. J.Biochem. (Tokyo). 89, 179-184.
18.
0hkubo,I., Kurachi.K., Takasawa,T., Shiokawa,H. and Sasaki,M. 1984. Biochemistry. 25, 5691-5697.
19.
Muller-Esterl ,W., Fritz,H., Machleidt.W., Ritonja.A., Brzin,J., Kotnik.M., Turk,V., Kellerman,J. and Lottspeich,F. 1985. FEBS Lett. 182, 310-314.
20.
Kato.H., Nagasawa,S. and Iwanaga.S. 1981. Methods Enzymol. 80, 172-198.
21.
Takio,K., Towatari,T., Katunuma.N., Teller,D.C. and Titani,K. 1983. Proc.Natl.Acad.Sci. USA. 80, 3666-3670.
22.
Sasaki ,M., Taniguchi ,K., Suzuki,K. and Imahori,K. 1983. Biochem.Biophys. Res.Commun. 110, 256-261.
H U M A N KIDNEY C A T H E P S I N
L.
Matjaz Kotnik, Tatjana Popovic, Vito Turk Department of Biochemistry, J. Stefan Institute, Jamova 39, 61000 Ljubljana, Yugoslavia
Introduction Among the lysosomal cysteine endopeptidases cathepsin L deserves particular attention because of some interesting properties. Compared to other lysosomal proteinases it has strong action against protein substrates (1, 2), shows broad specificity (3>
5) and is capable to inactivate glucose - 6 - phos-
phate dehydrogenase and aldolase (6). There have been two reports on the isolation of human cathepsin L (7, 8). Although purification procedures used were different, the isolated enzymes share some common properties. They have similar M r and pi, both degrade azocasein, but they differ in their activity against low molecular weight synthetic substrates. Isolation of cathepsin L is accompanied by some difficulties. The enzyme is rather unstable and tends to form tight complexes with its endogenous inhibitors (7). For this reason we tried to develop an improved purification procedure to obtain larger amounts of the purified enzyme.
Experimental Materials and methods Sephadex G-50 medium, Sephacryl S-200 superfine, CM-Sephadex C-50, lactalbumin, soya bean trypsin inhibitor, carbonic anhydrase, egg albumin, bovine serum albumin, phosphorilase and the FPLC system were supplied by Pharmacia, Sweden. Bz-D, L-Arg-NNap and DTE were from Sigma, USA, Z-Phe-Arg-NHMec from Bachem, Switzerland; Coomassie Brilliant
Blue G-250 from Bio Rad, FRG; Ep-475 and
leupeptin from Peptide Research Foundation, Japan, and iodoacetic acid, Triton X-100 and Brij 35 from Serva, FRG. Z-Phe-Phe-CHN2 was kindly given by Dr. E. Shaw, Friedrich Miescher Institute, Switzerland. Azocasein was prepared as described by Barrett and Kirschke (1). Pepstatin Sepharose was prepared as in
Cysteine Proteinases and their Inhibitors © 1 9 8 6 Walter d e Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y
44
(9). Human kidneys were obtained from the Institute of Pathology of Medical Faculty in Ljubljana. They were free from disease and stored at -20°C immediately
after autopsy until to be used.
Purification of human kidney cathepsin L All the purification steps were performed at 4°C with the exception of FPLC. 5 kg of frozen kidneys were thawed, cut into small pieces and homogenized in 1.5 volumes of extraction buffer (0.1 M Na acetate
buffer, pH 5.0 with 0.3 M
NaCl, 1 mM EDTA and 0.2$ Triton X-100). The homogenate was stirred overnight and then centrifuged (9200 g, 20 min). The supernatant was acidified to pH 4.2, left for an hour at room temperature, centrifuged (15000 g, 10 min) and brought back to pH 5.0. The proteins in the supernatant were precipitated by 20-70? (NHh)2 SOij saturation and the pellet after centrifugation (9200 g, 20 min) was suspended in buffer A (0.1 M Na acetate buffer pH 5.0 with 0.3 M NaCl and 1 mM EDTA). The suspension was dialyzed against the same buffer, centrifuged (15000 g, 15 min), filtered and concentrated. The concentrated sample and pepstatin Sepharose were equilibrated to pH 4.5 in buffer A, mixed together and allowed to be stirred for an hour. The unbound material (containing fractions active on azocasein and Z-Phe-Arg-NHMec) was washed with the same buffer. The bound cathepsin D was eluted with Tris/HCl buffer, pH 8.6. The pooled fractions active on azocasein and Z-Phe-Arg-NHMec were then chromatographed on a Sephadex G-50 column (6.5 x 127 cm) equilibrated by buffer A. Active fractions inhibited by 1 ¿iM Z-Phe-Phe-CHN2 were again combined, dialyzed against buffer B (20 mM Na acetate buffer, pH 5.0 with 1 mM EDTA) and applied to a CM-Sephadex C-50 column (3.3 x 40 cm). After thorough washing with buffer B bound proteins were eluted with a linear salt gradient to 1.0 M NaCl in buffer B (2000 ml total volume). Maximal activity on Z-Phe-Arg-NHMec was eluted at 500 mM NaCl. Active fractions were then chromatographed on a Sephacryl S-200 superfine column (3-7 x 114 cm), dialyzed against buffer B and applied to a mono S cation exchanger. A linear gradient to 0.5 M NaCl in buffer B (30 ml) was used. Two rechromatographies were necessary to obtain a single protein peak corresponding to cathepsin L activity.
45 Enzyme assays Enzyme assays and active site titrations were performed
essentially as descri-
bed by Barrett and Kirschke (1). Cathepsin L was routinely assayed with Z-PheArg- NHMec (5 ;jM) at 37°C and pH 5.5 or with 1% azocasein containing 3 M urea at
pH 5.5 and 37°C. Reactions were stopped by 1 mM iodoacetate and 0.3 M tri-
chloroacetic acid, respectively. Cathepsin B was assayed with Bz-D,L-Arg-NNap and cathepsin H with Leu-NNap as described in (10). Fluorimetric measurements were done on a Perkin - Elmer LS-3 fluorimeter ( X e x c = 370 nm, A e m = 460 nm) standardized by 0.1 juM 7-MCA. The molar concentrations of active cathepsin L were determined by titration with Ep-475. Papain was titrated by Ep-475 and the residual activity was measured with Bz-D,L-Arg-NNap as the substrate. The titrated papain was then used to determine the molarity of active chicken cystatin (11) using the same method. Determination of protein Protein concentrations were determined by the method of Bradford (12) with bo1i vine serum albumin as standard or by measurement
of A2go CA-j c m
= 10.5).
SDS-PAGE SDS-PAGE was performed as described by Laemmli (13) with phosphorilase B, bovine serum albumin, egg albumin, carbonic anhydrase, soya bean trypsin inhibitor and «C-lactalbumin as standards. Gels were stained for protein with Coommassie Brilliant Blue G 250. Determination of Kj,, for the substrate Z-Phe-Arg-NHMec
KJJJ
was determined using the method of Eisenthal and Cornish-Bowden (14). 2500
JJI of cathepsin L (5 pM) was activated with 250 JJI of 8 mM DTE (5 min, 25°C) and then 250 pi of Z-Phe-Arg-NHMec (5, 15, 20, 30 and 40 pM) was mixed in. Change of fluorescence was recorded and VQ were calculated. Determination of kabb o c o and K• -L using chicken cystatin as "inhibitor k a s s was determined by measuring residual cathepsin L activities after the reaction with chicken cystatin (times of reaction from 0 to 180 sec) under se-
46 cond order conditions (15). Initial velocities were calculated for the early part of the association process to eliminate the influence of the reverse reaction (16). k a s s was calculated from the replot of E -1 vs.time. Cathepsin L (50 ul, 0.54 nM) was activated for 3 min at 37°C with 50 jul of 240 mM DTE, diluted to 2900 ;J1 with 0.1? Brij 35 and then allowed to react with chicken cystatin (50 ¿il, 0.54 nM). After that 50 ul of 20 ;jM substrate was added and change of fluorescence was recorded. K^ value for the inhibition
of cathepsin L by chicken cystatin was determined
essentially as described by Pagano et al. (16). Apparent K^ values were calculated as described in (17). 25 Jul of cathepsin L (0.54 nM) was incubated for 25 min at 25°C with 25 Jul of chicken cystatin (0.06, 0.12, 0.24, 0.36, 0.48, 0.60, 0.72 and 0.84 nM). After activation with 25 iil of 8 mM DTE (5 min, 37°C) 25 JJ1 of Z-Phe-Arg-NHMec (10, 20, 40 and 60 ;jM) was added. After 10 min the reaction was stopped by adding 1 ml of 1 mM iodoacetate, reaction mixture diluted to 3 ml and fluorescence was measured. Blanks were prepared by adding the enzyme after stopping reagent. True K^ was determined from the replot of K
i app vs " S0"
Possible influence of cathepsin D
on cathepsin L activity
Cathepsin L (36 nM) and human cathepsin D (1.7 ;jM) were preincubated for 5 min at 37°C at different molar ratios (from 50 to 500 in favour of cathepsin D) and then the residual cathepsin L activity on Z-Phe-Arg-NHMec was measured. Results and discussion Purification of cathepsin L Approximately 18 mg of purified cathepsin L was obtained from 5 kg of human kidneys in a yield of about 8% as estimated by activity against azocasein in the presence of 3 M urea and the purification factor was about 1000. The introduction of affinity chromatography on pepstatin Sepharose (removal of cathepsin D) raised the final yield and activity of the purified enzyme. Gieselmann et al. (25) reported that cysteine proteinases appear to play a major role in processing of the intermediate form of cathepsin D (Mr = 47000) into the mature lysosomal form of the enzyme consisting of a larger and a smaller fragment with M p values of 31000 and 13000 - 14000, respectively. Treating cathepsin L with excess of cathepsin D we observed a different effect. Addition
47 of increasing amounts of cathepsin D reduced cathepsin L activity on Z-Phe-ArgNHMec (Fig. 1). High cathepsin D concentrations needed to achieve this effect indicate that this influence of cathepsin D on cathepsin L activity is presumably physiologically
irrelevant.
cath.D / c a t h . L molar
ratio
Fig. 1. Influence of increasing concentration of human cathepsin D on velocity of Z-Phe-Arg-NHMec cleavage by human cathepsin L. Cathepsin L was bound strongly to a mono S cation exchanger at pH 5.0 and three rechromatographies were sufficient to obtain electrophoretically pure material.
'lu *,.„ I»t
M N.CI 0.5
>0
20
JO Vtml>
Fig. 2. Fast protein liquid chromatography of cathepsin L: A
280'
A
h
366 ( y
drol
y
sis o f
NaCl gradient,
azocasein in presence of 3 M urea).
48 Properties of human kidney cathepsin L SDS-PAGE showed that cathepsin L was isolated in two forms: as an intact polypeptide chain (M^ = 32000) and as a mixture of two forms (32000, 25000;Fig. 3). Gel chromatography on Sephadex G-75 revealed that both protein peaks were enzymatically active. The electrophoretic pattern was the same under reducing and nonreducing conditions so that the 25000 fragment was possibly generated by the process of autolysis during the purification procedure. It could correspond to the heavy chain of human cathepsin L as described by Mason et al. (7) or to the heavy chain of the rat liver enzyme (18).
331 M(kDi) 94 67 45 30 20.1 14.4
1
2
3
4
5
Fig. 3- SDS-PAGE of cathepsin L 1: standards; 2, 3, 5: single chain form; 4: two chain form. Cathepsin L reacted with some protein inihibitors of cysteine proteinases isolated in our laboratory. Human stefin A (19), human cystatin C (20), chicken cystatin (11), higher molecular weight inhibitor from Sea Anemona (B. Lenarcic et al., this book), human stefin B (B. Lenarcic et al., this book) and human « L-leucine-2-naphthylamide
Fig. 2. E f f e c t s of c y s t e i n e p r o t e i n a s e inhibitors on the a c t i v i t i e s of cathepsin B and H purified f r o m r a t spleen. The a c t i v i t i e s of c a t h e p s i n s B and H a r e shown by open and closed circles, r e s p e c t i v e l y . The e n z y m e s in the r e s p e c t i v e assay b u f f e r s containing 10 mM c y s t e i n e and 4 mM EDTA w e r e p r e i n c u b a t e d at 37 °C for 5 min with each inhibitor at the c o n c e n t r a t i o n s indicated and then s u b j e c t e d to the r e s p e c tive assays. A c t i v i t i e s a r e expressed as % of those of controls t h a t w e r e d e t e r m i n e d by o m i t t i n g inhibitors.
82
Comparison
of
t h e m a j o r f o r m s o f c a t h e p s i n s B and H (B, f o r m
marked d i f f e r e n c e s between proteinase
atin, e l a s t a t i n a l ,
they strongly selectivity
revealed nearly
enzymes,
enzymes.
did not a l t e r
the a c t i v i t y of
of
chymost-
chloro-
c a t h e p s i n B. A l l
the same inhibitory
Especially,
the d i s c r i m i n a t i v e e f f e c t s o f
d i f f e r e n c e in the c a t a l y t i c s i t e r e g i o n s b e t w e e n leupeptin
is a t i g h t - b i n d i n g
active-site-directed
inhibitor
reversible
Since
t h e s e c o m p o u n d s on structures b e t w e e n
the
the t w o e n z y m e s ,
f o r c a t h e p s i n s B and H
sites
cathepsins these
l e u p e p t i n and E - 6 4 s u g g e s t
inhibitor o f
HgC^
r a t s p l e e n c a t h e p s i n H, but
as c a t h e p s i n B.
the d i f f e r e n c e s in t h r e e - d i m e n s i o n a l
inhi-
effects
inhibitors a r e due t o t h e i r a f f i n i t y f o r t h e s p e c i f i c i t y
t h e d i s c r e p a n c i e s in i n h i b i t o r y e f f e c t s o f
than
chlorome-
k e t o n e and t o s y l - l y s i n e
inhibition o f
i n h i b i t e d r a t l i v e r c a t h e p s i n H as w e l l
and p o t e n c y
B and H i n d i c a t e
that
and
l i v e r c a t h e p s i n H as t h e rat spleen e n z y m e . M e r c u r i c c o m p o u n d s such as
and p - c h l o r o m e r c u r i b e n z o a t e
of
chloromethly
m e r c u r i c compounds
leupeptin, antipain,
p-chloromercuribenzoate
k e t o n e , w h i c h all g a v e e s s e n t i a l l y c o m p l e t e
bitors tested except on rat
a f f e c t e d by
a c i d , E-64, H g C l g ,
thyl k e t o n e s such as t o s y l - p h e n y l a l a n i n e methyl
cysteine
In all c a s e s , c a t h e p s i n H w a s much less e f f e c t i v e
H w a s not so s t r o n g l y
iodoacetic
II) s h o w e d
t h e t w o in inhibition p r o f i l e s w i t h s o m e t y p i c a l
inhibitors ( F i g . 2).
c a t h e p s i n B. C a t h e p s i n
I ; H, f o r m
two the
b e c a u s e it is known
c a t h e p s i n B (8) and E - 6 4 is an
(9).
F i g . 3. E f f e c t s o f sodium s a l i c y l a t e , f l u f e n a m i c a c i d and i n d o m e t h a c i n on t h e a c t i v i t i e s o f c a t h e p s i n s B and H f r o m rat spleen. T h e p u r i f i e d c a t h e p s i n s B ( o ) and H ( • ) was p r e i n c u b a t e d at 25 ° C f o r 10 min w i t h e a c h a g e n t at the i n d i c a t e d m o l a r r a t i o s . T h e n , e a c h s a m p l e w a s s u b j e c t e d t o the r e s p e c t i v e e n z y m e assays using t h e m e t h y l c o u m a r y l a m i d e substrates. A c t i v i t i e s a r e e x p r e s s e d as % o f the r e s p e c t i v e c o n t r o l s that w e r e a s s a y e d in the a b s e n c e o f an a n t i - i n f l a m m a t o r y a g e n t .
83
In Vitro E f f e c t s of A n t i - I n f l a m m a t o r y
Agents
Figure 3 shows the experiments in which the molar ratio of each
anti-inflammatory
5
agent to cathepsins B (form I) or H (form II) varied from zero to 10 . The activities of cathepsins B and H w e r e measured by fluorimetric methods using c a r b o b e n z o x y - L phenylalanyl-L-arginine 4-methyl-7-coumarylamide ( Z - P h e - A r g - M C A ) (pH 6.0) and A r g M C A (pH 6.5) as the respective substrates (10), with a modification as described (11). It was found that f l u f e n a m i c acid and indomethacin g a v e an strong inhibition to cathepsin B only, and that sodium salicylate (SA) specifically activated cathepsin B when added to the e n z y m e solution in the presence of cysteine and E D T A . Cathepsin H was not a f f e c t e d by these agents when similarly treated at pH values between 4.5 and 8.0. Some other anti-inflammatory agents tested, such as prednisolone,
phenylbutazone,
acetaminophen and phenacetin, showed much less e f f e c t s on both enzymes (3).
The
inhibition of cathepsin B by f l u f e n a m i c acid and indomethacin was in a dose-dependent manner and occurred immediately upon their addition to the enzyme solution.
The
change of temperature at preincubation prior to assay had a rather small e f f e c t on their inhibitory potencies.
Also, no significant change in their inhibitory
potencies
f o r cathepsin B was observed by preincubation at pH values between 4.5 and 6.5. Since cathepsin B was unstable at pH values below 4.0 or above 7.5, such pHs w e r e not suitable f o r the inhibition experiments f o r cathepsin B. A f t e r establishing the conditions necessary for the inhibition of cathepsin B by flufenamic acid and indomethacin, the kinetic experiments w e r e performed in the absence and presence of each agents. The Lineweaver-Burk reciprocal plots showed that the inhibition of
cathepsin
B by these agents each was a non-competitive type and the Ki values w e r e 9 x 10 -4 M f o r f l u f e n a m i c acid and 1.4 x 10 M for indomethacin (Fig. 4). The Km value of -4 cathepsin B f o r Z - P h e - A r g - M C A was 2.86 x 10
M,
which is consisted with those
obtained from human liver cathepsin B (10) and porcine kidney cathepsin B (12). Since these two agents are thought to have a greater a f f i n i t y f o r protein lysyl
£-amino
groups than other groups (13), their inhibitory e f f e c t s on cathepsin B may be e x e r t e d through binding to the £-amino groups of the e n z y m e . The data given in Fig. 5 show the d i f f e r e n t e f f e c t s of S A and its analogues on rat spleen cathepsins B and H.
Of the three monohydroxy benzoates (HB), the ortho
isomer (SA) alone activated cathepsin B. The meta and para isomers showed little or no e f f e c t on the cathepsin B a c t i v i t y .
Also, acetylsalicylic acid (aspirin) did not
a f f e c t the cathepsin B a c t i v i t y . Cathepsin H was not a f f e c t e d when similarily with SA and its analogues. sins B and H.
The similar results w e r e obtained with rat liver
treated
cathep-
The s e l e c t i v i t y and activating potency of SA for cathepsin B is due
largely to its high binding capacity for the e n z y m e molecule, because the presence of
84
Fig. 4. Lineweaver-Burk reciprocal plots of the cathepsin B a c t i v i t y at d i f f e r e n t substrate concentrations, (^athepsin B was preincubated at for 10 min at pH 6.0 with or without 7.8 x 10 M flufenamic acid and 2.8 x 10 M indomethacin. Then the residual activities w e r e measured with d i f f e r e n t concentrations of Z - P h e - A r g - M C A .
Cathepsin B V77nm Cathepsin H COONa
COONa 200-
OH (SA) o-HB
H2B>H1.
Cathepsin I was less active than cat-
hepsin B, exhibiting slight activity with histone H4 as substrate H2B, H2A, H3).
(H4>H1,
Cathepsin D showed slight activity on three histones, H2A,
H2B, H3.
Table 3.
pH
Histone Digestion by Lung Lysosomal Proteases.
Histone
Cathepsin L
Cathepsin B k
Cathepsin I
cat/Km'(min"1
mM
Cathepsin D
LysCpase B
~1)
HI
1334 + 287
76 + 7
27 + 3
0
0
H3
861 + 134
0
21 + 3
39 + 9
0
H2B
1126 + 134
161 + 17
30 + 3
55 + 9
0
H2A
951 + 134
0
28 + 3
79 + 28
0
H4
545 + 134
239 + 36
54 + 5
12 + 4
0
HI
1379 + 269
1056 + 269
161 + 23
0
0
H3
1351 + 303
372 + 64
136 + 23
0
0
70 + 8
H2B
327 +
87
228 + 64
0
0
H2A
177 +
87
96 + 64
73 ± 8
0
0
H4
187 ± 142
244 + 135
66 + 5
0
0
The slopes of the lines (in Fig. 5b) are equal to the pseudo first order rate constants (=k . / K *[E]. Dividing this value by [E] yielded the second order C3t f ffl rate constant, k c a t / K m values for cathepsin I at pH 6.0.
The
k
c a t
/ K m values
for the degredation of the histone fractions by the other proteases were obtained in the same way.
108
At pH 5.5, cathepsin L had high catalytic efficiency with histories HI and H3 and showed much less (but still significant) activities on the other three histones (HI, H 3 » H 2 B , H2A, H4). each of the
histones
Cathepsin B was as active as cathepsin L on
except H3
(H1»H3, H4, H2B, H2A).
Cathepsin I, in
general, was less active than cathepsin B (HI, H3>H2B, H2A, H4).
Cathepsin D
had no significant activity at this pH and lysosomal carboxypeptidase B had no activity at either pH.
Discussion There is good agreement between many of the results obtained with the two different methods used to assay histone digestion. L
For example, with each
method, (a) cathpesin^was the most active enzyme at pH 3.5; (b) at pH 5.5, again, cathepsin L was the most active histone hydrolase, followed by cathepsin B and then cathepsin I; (c) cathepsin D was somewehat active at the more acid pH, and not at pH 5.5;
(d) lysosomal
carboxypeptidase B was inactive at
either pH. However, there were some differences.
For example, the k c a t / / K m values ob-
tained by the two methods for cathepsins L were different.
The reasons are
not
in the methods
fully understood, but may be due to basic differences
involved.
Measurement of arginine liberation involved long incubation times
producing largely digested material and many small peptides. gel
electrophoresis,
the
histone
disappearance
actually
However, with
measured resulted
from initial cleavages during short incubation times with the production of major peptides. It is of interest that each one of the five histone fractions can readily be degraded by cathepsin L at pH 5.5 or 3.5. enzyme (or by other lysosomal
Whether histone digestion by this
enzymes) is physiologically significant, re-
mains to be demonstrated.
Abstract Five
lysosomal
lysosomal
proteases,
Cathepsins
B, D,
I (BANA
Hydrolase) and L and
carboxypeptidase B, have been obtained in apparently homogeneous
form from rabbit lung.
These proteases have been tested for their abilities
109
to
degrade
methods:
purified
(1)
calf
thymus
colorimetric
histories,
determination
arginine-containing material
utilizing
of
the
two
release
different of
assay
acid-soluble
and (2) determination of the rate of disappear-
ance of individual histones by electrophoretic separation and quantitation of the remaining histone substrate at several digestion times.
The optimum pH was determined for the digestion of histone substrate by each protease.
A single optimum pH was observed for cathepsin B (5.5) and cathepsin
D
However, double pH optima were observed for cathepsin I (3.5 and
(2.8).
6.0),
cathepsin
L (3.5 and 5.5) and
lysosomal
carboxypeptidase
B (3.5 and
5.5).
Cathepsin L had the highest specific activity (nanomoles of equivalent arginine released/min/mg protein) at pH 5.5, followed by cathepsins B and I.
Cathepsin
D and lysosomal carboxypepidate B had little or no activity at this pH
At pH
3.5, cathepsin L again had the highest specific activity followed by cathepsins D, I and B.
Lysosomal carboxypeptidase B was relatively inactive.
Cathepsins L and I bound most tightly to calf thymus histones (as determined by K m
values) while cathepsin D binding was the least effective.
active protease
(as judged by k
^/k
The most
values) was cathepsin L, followed by
cathepsins B, I and D, in that order.
The ability of each proteolytic enzyme to degrade the individual histones was determined by the electrophoretic
method mentioned
above.
At pH 5.5,
the
most active enzyme was cathepsin L which actively degraded all the histones (in the order HI, H 3 » H 2 B , H2A and H4. L
on
each
of
the
histones
active than cathepsin B.
except
Cathepsin B was as active as cathepsin
H3.
Cathepsin
I, in general, was
less
Cathepsin D had no significant activity at this pH.
At pH 3.5, cathepsin L was again the most active enzyme, readily
digesting
all the histones (HI, H2B, H2A, H3>H4), followed by cathepsin B which digested only
histones
H4>H2B>H1.
Cathepsin
D and cathepsin I exhibited
relatively
slight activities at this pH, and lysosomal carboxypeptidase B had no significant activity at either pH.
Supported
by
NIH
Grant
HL16920
and
by
a Wellcome
Research
Travel
Grant.
110
References 1.
Bartley, J. and Chalkley, R. 1970. J. Biol. Chem. 245, 4286-4292.
2.
Brandt,
3.
Eickbush, T.H., Watson, D.K. and Moudrianakis, E.N. 1976. Cell 9, 785-792.
4.
Commerford, S.L. , Carsten, A. L. and Cronkite, Acad. Sei. U.S.A. 79, 1163-1165.
5.
Pehrson, J.R. and Cole, R.D. 1982. Biochemistry 21, 456-460.
6.
Djondjurov, L.P., Yancheva, N.V. and Ivanova, E.'C. 1983. 22, 4095-4102.
Biochemistry,
7.
Davies, P., 428-440.
Biochem.
8.
Heinrich, P.C., Raydt, G., Puschendorf, B. and Jusic, M.. 1976. Eur. J. Biochem. 62, 37-43.
9.
Suhar, A. and Marks, N.
W.F.,
Böhm,
F.
Krakauer,
and Von Holt,
K.
and
C. 1975.
Weissman,
G.
FEBS
E.P.
1972.
Lett.
1982.
Anal.
51,
88-93.
Proc.
Natl.
45,
1979. Eur. J. Biochem. 101, 23-30.
10.
Kirschke, H., Langner, J., Weideranders, B., Ansorge, S. and Bohley, P. 1977. Eur. J. Biochem. 74, 293-301.
11.
Panyim, S. , 4206-4215.
12.
Oliver, D. , Sommer, K. , Panyim, S. , Spiker, S. , and Chalkley, R. 1972. Biochem. J. 129, 349-353.
13.
Singh, H. and Kalnitsky, G. 1978. J. Biol. Chem. 253, 4319-4326.
14.
Singh, H. and Kalnitsky, G. 1980. J. Biol. Chem. 255, 369-374.
15.
Lones, M. , Chatterjee, R. , Singh, Biochem. Biophys. 221, 64-78.
16.
Benuck, M. , Grynbaum, A. and Marks, N. 1977. Brain. Res. 143, 181-185.
17.
Barrett, A. 1972. Anal. Biochem. 47, 280-293.
18.
Charney, J. and Tomarelli, R. 1947. J. Biol. Chem. 171, 501-505.
19.
Taylor, S. , Ninjoor, V. , Dowd, D. and Tappel, A. 1974. Anal. 60, 153-162.
20.
Panjim, S. and Chalkley, R. 1969. Arch. Biochem. Biophys. 130, 337-346.
21.
Hardison, R. and Chalkley, R. 1978. In: Methods in Cell Biology (ed. Stein, G., Stein, J. and Kleinsmith, L.) 17, 235-251.
Bilek,
0.
and
Chalkley,
H.
R.
1971.
and
J.
Biol.
Kalnitsky,
G.
Chem.
1983.
Footnote 1.
Chatterjee, R. , Lones, M. , and Kalnitsky, G. , unpublished data.
246,
Arch.
Biochem.
P R O P E R T I E S O F A C Y S T E I N E P R O T E I N A S E FROM H U M A N
A.D.
THYROIDS
Dunn
D e p a r t m e n t of M e d i c i n e , U n i v e r s i t y of V i r g i n i a M e d i c a l Charlottesville, Virginia 22908
School,
Introduction The final p r o c e s s i n g of t h y r o i d h o r m o n e s p r i o r to their e n t r y the b l o o d s t r e a m i n v o l v e s the r e l e a s e of the b i o l o g i c a l l y iodoamino acids thyroxine
(T 4 ) and t r i - i o d o t h y r o n i n e
large s t o r a g e p r o t e i n t h y r o g l o b u l i n b e l i e v e d to o c c u r m a i n l y
(Tg).
in l y s o s o m e s
This p r o c e s s W e have
active
(Tj) from
(1), but little
is a v a i l a b l e as to the e n z y m e s i n v o l v e d .
of p e p t i d e
intermediates
(2,3).
is
previously
C B a c c o u n t e d for less than 1/2 of
f r a c t i o n of these r a b b i t t h y r o i d s .
enriched
An a d d i t i o n a l e n z y m e ( s )
this c l a s s w a s p a r t i a l l y p u r i f i e d from t h y r o i d s of this The p h y s i o l o g i c a l
is s u g g e s t e d by the finding
their a c t i v i t y v a r i e s w i t h t h y r o i d state
(4).
of
species
i m p o r t a n c e of c y s t e i n e p r o t e i n a s e s
p r o c e s s i n g of t h y r o i d h o r m o n e s
and
Tg ^ n v i t r o to a v a r i e t y
the c y s t e i n e p r o t e i n a s e a c t i v i t y found in a l y s o s o m a l
(3).
the
information
p u r i f i e d c a t h e p s i n s D a n d B (CD a n d CB) from r a b b i t t h y r o i d s found them to be c a p a b l e of h y d r o l y z i n g
into
in the that
In the p r e s e n t
s t u d i e s we h a v e p u r i f i e d a n d p a r t i a l l y c h a r a c t e r i z e d a c y s t e i n e proteinase
in h u m a n t h y r o i d s a n d have e x a m i n e d
its a c t i o n o n T g .
We t e n t a t i v e l y d e s i g n a t e this e n z y m e Tg h y d r o l a s e
(TgH).
Methods T h y r o i d s w e r e o b t a i n e d at s u r g e r y from p a t i e n t s w i t h disease.
s o l u b l e e x t r a c t w a s o b t a i n e d by c e n t r i f u g a t i o n . a c t i v i t y w a s m e a s u r e d using substrate.
Graves'
The g l a n d s w e r e f r o z e n , t h a w e d , a n d h o m o g e n i z e d .
[ 1 2 5 I ] Tg
Tg
i_n v i t r o l a b e l e d r a b b i t
hydrolase 1 2s [ I] Tg as
(30-50 ug) a n d s a m p l e w e r e i n c u b a t e d
ul of a m e d i u m c o n t a i n i n g
A
in 70
12 m M G S H , 1 m M EDTA, 1 uM p e p s t a t i n ,
Cysteine Proteinases and their Inhibitors © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany
112 and either 0.14 M sodium formate buffer, pH 3.5, or 0.14 M sodium acetate buffer, pH 5.0. 37°.
Incubations were for 15-120 m i n u t e s at
Activity was assessed by the percent of
[ 1 2 5 I ] Tg degraded
to peptides of less than 100-kDa as evaluated by polyacrylamide gel electrophoresis
(PAGE) in SDS.
One unit of Tg hydrolase
activity is defined as the amount of enzyme required to digest 1 ug of Tg to peptides of less than 100-kDa in one m i n .
Under the
conditions of the assay, activity was linear with time and proportional to enzyme concentration from 5 to 25% hydrolysis of 125 [
I] Tg.
CD activity was measured in a similar assay at pH 3.5
with leupeptin (10 uM) replacing p e p s t a t i n . employed in other enzyme assays
Standard m e t h o d s were
(5,6,7).
Results TgH was purified from human thyroid extracts in a series of chromatographic steps including gel filtration on Bio-Gel A.5M, DEAE ion exchange, hydroxyapatite adsorption, and finally gel filtration o n Sephacryl 200.
The enzyme was stabilized during
purification with mercuric chloride (0.5 mM) and EDTA (1 m M ) .
The
purifed enzyme preparation on SDS-PAGE contained a major protein band of
14-kDa
On PAGE at p H 4.5 the enzyme preparation showed two
protein bands which coincided well with enzyme activity evaluated on an adjacent gel (Fig. 2).
_
A
B
Fig. 1: SDS-PAGE of purified TgH, 12% gels stained with silver. A. unreduced, B. reduced. Molecular m a s s e s were estimated from accompanying standards. —
30 K
—18K —14K
113
Purified TgH was optimally active in the Tg hydrolase assay at pH 3.5-4.0.
In contrast, purified CB obtained from the same thyroids
was most active in this assay at pH 4.5-5.0. V
max
value
for
t
The apparent K m and
9 with CB, TgH, and CD are shown in Table 1.
had a higher affinity for Tg than did either CB or CD.
TgH
Based on
Vmax /Km values this enzyme utilized Tg 7-10 times more efficiently J 3 •* than either of the other two cathepsins.
Table 1.
Kinetic parameters for Thyroid proteinases
pH
TgH
3.5
0.73
0.016
0.022
CB
5.0
2.96
0.009
0.003
CD
3.5
0.018
0.002
Enzyme
max. _. umol min~ mg
Vmax /K .m 1 min" mg
m uM
10.8
Km and Vmax , were calculated from rplots of S _ 1 versus V - *, co
o k•D
È
n3
o
O) o
H
40 20 -J
1
1
10
L
20
slice number Fig. 2: PAGE at pH 4.5 of purified TgH, 4.5% gels. One gel was sliced and assayed for TgH activity, the duplicate was stained for protein with Coomassie Blue.
114
Purified TgH was more sensitive to inhibition by leupeptin than was CB (Table 2).
TgH was also very sensitive to inhibition by
two peptidyl diazomethyl ketones (Table 2).
Its sensitivity to
Z - p h e - p h e - C H N 2 was similar to cathepsin L described in other tissues
(8).
Table 2.
Concentration of Compounds Causing 50% Inhibition of Thyroid Cysteine
Proteinases (mol" 1 )
Inhibitor Concentration Inhibitor
TgH
CB
Leupeptin
6 x io" 9
10" 7
Z-Phe-AlaCHN2
5 x 10" 8
6 x 10"7
Z-Phe-PheCHN-
7
2 x 10"
10" 4
Enzymes were incubated at pH 3.5 (TgH) and 5.0 (CB) for 5 m i n u t e s at room temperature prior to the introduction of [125-1] Tg. Subsequent incubations were for 60 minutes at 37 C.
TgH showed little or no activity against substrates used to identify a variety of cysteine proteinases including CB (BANA and Z Arg-Arg-MeOBNAP), cathepsin H (Arg-BNAP), cathepsin N or cathepsin L (azocasein and Z-Phe-Arg-NMec)
(collagen),
(Table 3).
The
specific activity of this enzyme with the latter substrate was approximately rat liver (6).
1/10 that calculated for purified cathepsin L from CB had an activity profile similar to that found
for this enzyme in other tissues
(6).
The specificity of TgH was investigated with the oxidized 3 chain of insulin and with a synthetic hexapeptide
(Fig. 3).
Cleavage
sites on the B chain of insulin clustered in the center of the peptide, the major site occurring at Glu ( E ) ^ - A l a
(A)
site at Tyr (Y)^g-Leu ( L ) ^ and minor sites at Ala
(A)^-Leu
and Leu ( L ) 1 5 ~ T y r
(""O^g-
a second
Major cleavage sites on the hexapeptide
occurred at Trp (W)-Met (M) and Arg (R)-Phe (F). site occurred at Leu (L)-Trp (W).
An additional
The cleavage pattern seen by
115
TgH with these substrates differs from those described for other cysteine proteinases
Table 3.
(6,8,9).
Action of Cyste ine Proteinases on Various Substrates Enzyme Activity - units/mg protein
Substrate
TgH
PH
CB
BANA
6.0
0.005 (1)
Z-Arg-Arg-•MeOBNAP
6.0
0.28
(1)
Arg-BNAP
6.0
ND
(1)
0.005
Z-Phe-Arg-•NMec
5.5
5.4
(1)
6.9
Azocasein
6.0
ND
(2)
395
Azocasein + urea
5.0
ND
(2)
154
Collagen, insoluble
3.5
ND
(3)
3.3 13.8
0.41
One unit is defined as the amount of enzyme required to: (1) release 1 umol of substrate in one minute, (2) degrade 1 ug of azocasein to peptides soluble in trichloroacetic acid per minute, or (3) to release 1 umol of hydroxyproline in one minute. The specific activity (units/mg protein) on the standard TgH assay of TgH at pH 3.5 was 22,500 and that of CB at pH 5.0 was 3,307. ND = not detectable.
*
(a)
T
10
(b)
*
F-V-N-Q-H-L-C-G-S-H-L-V-E-A-L-Y-L-V-C-G-E-R-G-F-F-Y-T-P-K-A 15
20
25
30
I 1
L-W-M-R-F-A
Fig. 3: Cleavage sites by TgH in (a) oxidized p chain of insulin, (b) synthetic hexapeptide. Heavy arrows denote major cleavage sites, light arrows denote secondary sites, open arrows denote minor sites. *C was in the form of cysteic acid.
116 The products of Tg digestion by TgH were examined under conditions of limited hydrolysis to obtain information on favored cleavage 125 sites within this substrate. In this experiment 30% of [ I] Tg was degraded to peptides of < 100-kDa. Digestion products as 12 5 evaluated by the distribution of I on SDS-PAGE were ru 30-50-kDa (Fig. 4).
These were resolved into at least three peptide bands 125 i - t The a content of these peptides 125 I T showed little increase compared to intact Tg but they were ~ 3
on stained gels (Fig. 4).
enriched (Table 4). with trypsin.
The 30-50-kDa fragments of Tg were digested
After separation by HPLC two T^-containing
(peaks A and C, tig. 5 and Table 4) and a single site (peak B, Fig. 5 and Table 4) were SO K D a
sites
^-containing
identified.
30 KDa
15
s 11leucylamido(4 guanidino)butane; NMec, 4^nethyl-7-coumarylamide; Tos, tosyl-; Z, benzyloxycarbonvl.
Acknowledgement We gratefully ackowledge the technical assistance of Mrs. R. Schwitte and Mrs. M. Koenen. This work was supported by the 'Ministerium für Wissenschaft und Forschung des Landes Nordrhein-Westfalen', Düsseldorf, by the 'Bundesministerium für Jugend, Familie und Gesundheit', Bonn, by a grant (SFB 113) fron the 'Deutsche Forschungsgemeinschaft', Bonn and by a NATO research grant.
References 1. Göll,D.E., J.D.Shannon, T.Edmunds, S.K.Sathe, W.C.Kleese, P.A.Nagainis. 1983. In: Calcium Binding Proteins (DeBernards,B., G.L.Sottocasa, G.Sandri, E.Carafoli, A.N.Taylor, T.C.Vanaman, R.J.P.Williams,eds.) Elsevier Science Publ. Amsterdam pp. 19-35. 2. Brush,J.S. 1971. Diabetes 20, 151-155. 3. Shroyer,L.A., P.T.Varandani. 1985. Arch.Biochem.Biophys. 236, 205-219. 4. Okitani,A., T.Nishimura, H.Kato. 1981. Eur.J.Biochem. ^15, 269-274. 5. Liao,J.C.R., J.F.Lenney. 1984. Bicchem.Biophys.Res.Canmu . T24, 909-916. 6. Dahlmann,B., L.Kuehn, H.Reinauer. 1983. FEBS-Lett. J60, 243-247. 7. Dahlmann,B. 1985. Biochem.Soc.Trans. JjS' 1021-1023. 8. Dahlmann,B., L.Kuehn, M.Rutschmann, H.Reinauer. 1985. Biochem.J. 228, 161-170.
146 9.
Dahlmann,B., M.Rutschmann, L.Kuehn, H.Reinauer. 1985. Biochan.J. 228, 171-177.
10. Green,G.D.J., E.Shaw. 1981. J.Biol.Chem. 256, 1923-1928. 11. Barrett,A.J., A.A.Kembhavi, M.A.Brown, H.Kirschke, C.G.Knight, M.Tamai, H.Hanada. 1982. Biochem.J. 201_, 189-198. 12. Kirschke,H., E.Shaw. 1981. Biochan.Biophys.Res.Canmun. 101, 454-458. 13. Desautels,M., A.L.Goldberg. 1982. Proc.Natl.Acad.Sei.USA. 79, 1869-1873. 14. Tanaka,K., L.Waxman, A.L.Goldberg. 1983. J.Cell Biol. 96, 1580-1585. 15. Dahlmann,B., H. Reinauer. 1981. In: Streptozotocin. Fundamentals and Therapy (Agarwal,M.K., ed.) Elsevier, Amsterdam pp. 129-140. 16. Etlinger,J.D., H.McMullen, F.R.Rieder, A.Ibrahim, R.A.Janeczko, S.Mamorstein. 1985. In: Intracellular Protein Catabolism (Khairallah,E.A., J.S.Bond, J.W.C.Bird, eds.) Alan R. Liss, Inc., New York pp 47-60. 17. DeMartino,G.N. 1983. J.Mol.Cell.Cardiol. 1j>, 17-29. 18. Ismail,F., W.Gevers. 1983. Biochim.Biophys.Acta
742, 399-408.
19. Chowdhary,B.K., G.D.Smith, T.J.Peters. 1985. Biochim.Biophys.Acta 840, 180-186. 20. Rivett,A.J. 1985. J.Biol.Chem. 260, 300-305.
MULTIPLE FORMS, STRUCTURE AND SPECIFICITY OF CLOSTRIPAIN
B. Keil and A.-M. Gilles
Institut Pasteur, 28 rue du dr. Roux, 75015 Paris, France
Introduction The SH-proteinase clostripain is interesting from several points: its activity progressively increases with the opening of several disulphide bridges, its highly active form is composed of two chains held together by non-covalent bonds and its narrow specificity makes out of it a valuable tool in protein sequence determination. Ot-Clostripain, Highly Active Form of the Enzyme
As will be discussed below, the preparation of a homogeneous clostripain is a rather tricky affair. The anaerobic Clostridium histolyticum produces a mixture of extracellular proteolytic enzymes, which undergo proteolytic degradations during the relatively long time of culture; one of the enzymes present, clostripain, can assume various forms created by opening or reforming several disulphide bridges of the molecule, which can lead to different active or irreversibly
inactiva-
ted forms. As starting material for the purification of clostripain was usually used either a culture filtrate of C. histolyticum
Cysteine Proteinases and their Inhibitors © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany
148
(1) or commercially available preparations of Clostridium collagenase (2,3,52). In both cases, values of specific esterolytic activity varied according to the procedure, up to 125 U/mg on BAEE)(3). This still was not considered as the upper limit: Porter and Mitchell
(4) suggested the existence of two
chromatographic conformers in apparently homogeneous active clostripain which were supposed to be due to the formation of two separate and equally stable disulphide pairings. From a fresh culture medium of C. histolyticum, we succeeded to separate a highly active form and an inactive form of clostripain by hydrophobic chromatography on a Sepharose C^NI^ column (5). Apart the difference in activity, these two forms had the same molecular weight and amino acid composition. For this highly active form of the enzyme (specific activity 550-600 U/mg) we have proposed the name of OC-clostripain. When commercially available collagenase preparations were used as starting material for the same purification, the inactive form was largely predominant and the heterogeneous profile of activity of the active fraction indicated that it was composed of several active forms. The study of these multiple forms revealed that they differ in the content in free-SH groups. At least one accessible sulfhydryl group is indispensable for the appearance of activity; in the most active preparations practically all the cysteine groups are free. On the other hand, one free sulfhydryl group exists in both active and inactive forms of clostripain, with the difference that in the inactive enzyme it can be revealed only under de-
naturing conditions. The comparison of the commercial and fre shly prepared enzyme preparations thus suggested that the mul tiple forms are the result of modifications which probably oc cur under conditions of industrial treatment of C. histolyticum culture filtrates. The complex character of clostripain preparations was demonstrated independently by the studies on the inactivation of the enzyme by active site directed inhibitors, the chloromethylketones. The most efficient of this series proved to be N -p-nitrobenzyloxycarbony1-L-arginine
chloromethylketone
(p-NC^-ZACK)(51): it removes the activity completely in less than 2 min at room temperature. The enzyme can be protec ted against this irreversible inactivation by the competitive inhibitor benzamidine. However, this very efficient arginine chloromethylketone is rather unstable; for practical experimental reasons, it was replaced in quantitative studies by the corresponding lysine derivative, tosyl-L-lysine chloromet hylketone (TLCK). In an early study (3), clostripain was found to incorporate TLCK in an approximative molar ratio 1:4 instead of the expec ted eguimolar ratio. Because the enzyme preparation used for this active site titration was chemically homogeneous, the authors concluded that a large fraction of the enzyme was in an inactive state. This early conclusion on the multiplicity of forms present in any clostripain preparations was confirmed by the titration of our highly active
£>, 1311-1314. 20.Hanada, K., Tamai, M., Yamagishi, M, Ohmura, S., Sawada, J., Tanaka, I. 1978. Agric. Biol. Chem. A2, 523-528. 21.Barrett, A.J., Kembhavi, A.A., Brown, M.A., Kirschke, H., Knight, C.G., Tamai, M., Hanada, K. 1982. Biochem. J. 201, 189-198. 22.Shih, W.K., Hash, J.H. 1971. J. Biol. Chem. 246, 9941006. 23.Drickamer, K. 1981. J. Biol. Chem. 256, 5827-5839. 24.Verheij, H.M., Westerman, J., Sternby, B., Dehaas, G.H. 1983. Biochim. Biophys. Acta 747, 93-99. 25.Shimomura, H., Kanai, Y., Sanada, K. 1983. J. Biochem. 93,857-863. 26.Fuglistaller, P., Suter, F., Zuber, H. 1983. Z. Physiol. Chem. 364, 691-712. 27.Dianoux, A.C., Tsugita, A., Klein, G., Vignais, P.V. 1982. FEES Letters 140, 223-228. 28.Moonen, P., Akeroyd, R., Westerman, J., Puijk, W.C., Smits, P., Wirtz, K.W. 1980. Eur. J. Biochem. 106, 279-290. 29.Haniu, M., Armes, L.G., Tanaka, M., Yasunobu, K.T. 1982. Biochim. Biophys. Res. Commun. 105, 859-894. 30.Svendsen, I.B., Hansen, S.I., Holm, J., Lyngbye, J. 1984. Carlsberg Res. Commun. 49, 123-131. 31.Sidler, W., Gysi, J., Isker, E., Zuber, H. 1981. Z. Physiol. Chem. 362, 611-628. 32.Rask, L., Anundi, H., Peterson, P.A. 1979. FEBS Letters 104, 558-562. 33.Mitchell, W.M., Harrington, W.F. 1971. In: The Enzymes (Boyer, P.D. ed.). Academic Press, New York & London, vol.3, pp.699-719. 34.Hoog, J.O., Jornvall, H., Holmgren, A., Carlguist, M., Persson, M. 1983. Eur. J. Biochem. 136, 223-232. 35.Haniu, M., Tanaka, M., Yasunobu, K.T., Gunsalus, I.C. 1982. J. Biol. Chem. 257' 12657-12663. 36.Gagnon, J., Christie, D.L. 1983. Biochem. J. 209, 51-60. 37.Christie, D.L., Gagnon, J. 1983. Biochem. J. 209, 61-70.
162 38.Manjula, B.N., Mische, S.M., Fischetti, V.A. 1983. Proc. Natl. Acad. Sei USA 80, 5475-5479. 39.Jackson, K.W., Tang, J. 1982. Biochemistry 21, 6620-6625. 40.Mitchell, W.M. 1966. Diss. John Hopkins University. 41.Wouters-Tyrou, D., Martin-Ponthieu, A., Briand, G., Sautiere, P., Biserte, G. 1982. Eur. J. Biochem. 124, 489-498. 42.Amons, R., Pluijms, W., Roobol, K., Moller, W. 1983. FEBS Letters 153, 37-42. 43.De Caro, J., Boudouard, M., Bonicel, J., Guidoni, A., Desnuelle, P., Rovery, M. 1981. Biochim. Biophys. Acta 671, 129-138. 44.Mitchell, W.M. 1968. Science 162, 374-378. 45.Saitoh, E., Isemura, S., Sanada, K. 1983. J. Biochem. 93, 495-502. 46.Isemura, S., Saitoh, E. , Sanada, K. 1982. J. Biochem. 91, 2067-2075. 47.Saitoh, E., Isemura, S., Sanada, K. 1983. J. Biochem. 93, 883-888. 48.Ishaque, A., Hofmann, T., Eylar, E.H. 1982. J. Biol. Chem. 257, 592-595. 49. Pfletschinger, J., Braunitzer, G. 1980. Z. Physiol. Chem. 301, 925-931. 50. Byf ield, P.G.H., Zuber, H. 1972. FEBS Letters 2J3, 36-40. 51.Siffert, 0., Emod, I., Keil, B. 1976. FEBS Letters 66, 114-119. 52.Emod, I., Keil, B. 1977. FEBS Letters 77, 51-56.
ENZYMATIC AND
IMMUNOLQGICAL
PROTEINASE
PROCOAGULANT
B. C a s a l i ,
C. Gambacorti
N.
Semeraro», M.B.
Istituto
di
Istituto Nazionale and U n i v e r s i t y
There abnormal
per
and S.G.
la C u r a dei
established
disease
tumor
(1-3).
cells and within
facilitate
arrest within
organs
them from
anticoagulants
that m i g h t
to
the
have
been
initiate
shown
that
coagulation
Address:
Università'
di
tumor
for
tumor
system
(4-7).
cells
cancer
di
within
facilitate growth
(7-11).
their
a s well
as
Some growth
(12-17).
from m a l i g n a n t
(13,19)
and
around
nodules
that m a y
by d i r e c t l y
Istituto
Bari, Bari,
of
USA
between
-fibrin d e p o s i t i o n
and humans
a substance,
Denver,
to r e d u c e m e t a s t a t i c
coagulation
of
* Permanent
CO.,
a substance
identification initiated
Center,
metastatic
immune
animals
identify
Italy,
-formation o c c u r s
a phenomenon
and
Tumori, Milano,
relationship
susceptible
Fossati,
"Mario Negri"
aggregation
the h o s t
experimental
Efforts
Fibrin
G.
Gordon
of C o l o r a d o M e d i c a i
the m i c r o c i r c u l a t i o n ,
in b o t h
Passerini, A. Falanga,
Farmacologiche
is a well
Fibrin may
protect
MELANOMA.
intra- and extra-vascular
malignant primary
IN H U M A N
Donati
Ricerche
I D E N T IFI C A T I O N O F A C Y S T E I N E
has
led
to
procoagulant activating
Patologia
cells the (CP),
Factor
Generale,
Italy.
Cysteine Proteinases and their Inhibitors © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany
X
164
(20-24).
This
protein
characterized and
o-f
human
molecular that
-from
it
rich
inhibited
in
serine
by
mercury, It
particular,
CP
a c t i v i t y
the
in
distinguished procoagulant
X)
i solated
within
it
of
o-f
is
IXa the
not
-from
the
c e l l s
including
in
sarcoma
serum and
normal
the
associated
with
Thus,
factor
metastatic
is
-factor
different
(23). activation
a
variety
me 1 a n o m a , addition,
it
and
extracts
human
tumors.
It
not
been
cell
was
of
c e l l s
tissues.
procoagulant tumors
and
interest
expressed
human
and
by
melanoma
(27,23,35),
malignant to
c e l l s tissue
isolated and
of
tissue
lines
whether from
benign
in
is
cell
determine
1unq
has
found
However,
of
of
Lew i s
medium has
than
serine
culture
several
was
proteinase
tissue
with
In
In
nonenzymatic
(direct
B16
and
chemical
free
normal
it
the
cascade
(24,29,34).
and
KCN
i=
easily
o-f
association
that
by
cysteine
known
a c t i v i t y
4.8
no
-factor.
two
that
coagulation
has
activated
by
other
o-f
proteinase
lipoprotein
mal i gnan t JW
point
tissue X
S 50 o CJ c: 5 25 CL 0
1
2 Enzyme (ytl)
3
Fig. 5. Papain inactivation assay with rising amounts (O.1-3.0(11) of enzyme. Partly purified material from a peak fraction of a Sephadex chromatogram. (The apparent final papain activity of 3-4$ depends to a large degree on the accuracy of the assay blank and may, therefore, approach zero percent.) less effective than the 3 other compounds. The possibility that this latter papain inhibitor is identical with the aforementioned enzyme inhibitor cannot be ruled out as yet; this awaits further experimental clarification.(The well-known toxin "coprin" from Copr. atramentarius has a papain-inhibiting capacity too (9) although only after hydrolysis in vivo, in a mammalian organism.) Regarding the papain inactivation proper, Figs. 2 and 3 show the time course of and the effect of various amounts of the partially purified Coprinus enzyme on the remaining activity of papain. There is, within the boundaries of first order reaction kinetics, proportionality between incubation time (or amount of enzyme respectively) and inactivation of the substrate enzyme. The optimum pH is between and 5« Specificity of the enzyme In addition to papain, some other cysteine proteinases have been tested for inactivation. Bovine spleen cathepsin B and lysosomal carboxypeptidase B as well as ficin were inhibited to almost the
215
same degree as papain, whereas bromelain - as known from a number of protein inhibitors - appeared to be less susceptible to inactivation by the Coprinus enzyme. The enzyme is practically not active towards serine proteinases as trypsin or chymotrypsin, or towards pepsin. However, the fungus contains another protein which inactivates at least chymotrypsin and possibly trypsin as well. Properties of the enzyme The enzyme is destroyed by heating to 80° for 5 min and severely impeded when heated to 60°. Nevertheless, its protein nature could not be clearly proven by e.g. digestion with trypsin or pronase; either enzyme caused some loss of inactivating activity but did not destroy it. The enzyme is stable at least between pH 4- and 8; its activity is influenced by certain buffer salts, e.g. formate or citrate ions which lead to a decrease of its activity. It has been kept in 100 mM acetate buffer - with or without EDTA - in the frozen state for almost two years. On the other hand, cooled but unfrozen acetate buffer, when kept in the icebox for a longer period of time, even when in a concentrated form, seemed to bring about invisible microbial growth which led to a partial inactivation of the enzyme when such a buffer was added to an inactivation assay. Boiling of the acetate buffer did not eliminate this effect but rather increased it, pointing perhaps to a l.m.w. microbial metabolite as the cause and, at the same time, underlining the sensitivity of the enzyme to certain agents. When using gel chromatography as a means of purification, the enzyme appears in the breakthrough fractions together with a fungal polysaccharide. This seems to have some protective properties since fractions of this kind keep much better than those from a more purified stage, although they may be prone to microbial growth. EDTA and sodium azide. A large number of experiments was carried out in order to investigate the influence of EDTA - alltogether with erratic results. In about one half of the assays, EDTA in concentrations of 0.1mM and higher caused a distinct improvement of the inactivating activity, whereas in the other half no effect was seen. Possibly, the accidental presence of [heavy] metal ions
216
.7
—•-» .25
.5
Inhibitor
i
i
1.0
2.5
5.0
conc. (JJM)
Fig.4. Inhibition of the inactivating enzyme by FeCl3 or ascorbic acid. An appropriate amount of enzyme was preincubated with the inhibitor for 15 min in a volume of 300|il, followed by an incubation with papain and, thereafter, by the papain assay with L-BAPA. may be the cause, giving at the same time a hint to the presence of thiol groups in the enzyme. - Sodium azide consistently produced a' certain loss of activity, beginning at concentrations as low as 10|im and rendering the enzyme completely inactive at 10mM azide. ^ -, • T, + + + -n ++ »» ++ ^ + + + ^ + + + TT + + Heavy metal ions, such as Fe , Fe , Mn , Co , Cr , Hg , Zn ++ , Ni ++ , Cu ++ , proved to be inhibitors of the Coprinus enzyme, on the average with effective concentrations as low as 10|j.M. As already mentioned, particularly Fe + + + , but probably also Fe ++ , proved to be very powerful. Here, concentrations as low as 1p.M FeCl, were able to counteract the enzyme for circa 50$ (Fig.4). Since the normal iron content in Copr. atramentarius according to Schmitt et al.(8) and confirmed by analyses with the bathophenanthrolin method in our investigations is in the range of 0.1mM, the enzyme should, in fact, normally be present in an inactive
217
form and, therefore,a crude homogenate supernatant be without inactivating effect. Since a normal Coprinus supernatant does have a papain-inactivating effect, this must be ascribed in the first place to the aforementioned l.m.w. inhibitor, an assumption that could be proved by heating a crude supernatant: its inhibiting capacity decreased only slightly. Influence of dithioerythritol. As the inactivation assay was normally conducted, it is circa 1.25 m M i n dithioerythritol (from the a activation of papain) and its pH is below 5« more neutral pH, this thiol is clearly of deleterious influence on the enzyme, particularly if applied in a preincubation in the absence of papain, even at concentrations of only 10^.M; at pH 5 or "below, this occured only erratically. However, under the usual conditions of the enzyme assay (pH 4.7) dithioerythritol can even be favourable, be it by a more complete activation of papain or by activation of the inactivating enzyme itself (see below). These results, too, were not always reproducible, the outcome depending on the enzyme preparation or column fraction, respectively, and perhaps also on the age of the papain. With activated papain but freed of dithioerythritol by passage through Sephadex, the enzyme effects were always considerably smaller than in a milieu containing a thiol. No inactivation of papain was observed when using an assay system without a thiol and using papain without previous activation: When, after preincubating papain plus enzyme under these conditions and subsequently inhibiting the enzyme with 10^iM PeCl 5 , the papain was activated with dithioerythritol,a fully active papain could be recovered. This means that the enzyme either needs an active papain or an activation or preservation of its own thiol groups (as known from many other enzymes) or both. However, the inactivating enzyme does not seem to belong to the cysteine proteinases as it cannot be bound to mercurated Sepharose. Experiments to clarify these observations are under way. As to a loss of activity of the Coprinus enzyme, the effect was not limited to dithioerythritol at neutral pH but was also given by e.g. ascorbic acid, already at concentrations of 0.5p,M and at an acid pH (Pig.4) as well as by NADH S or NADPE, . In this latter case, the concentration-dependent response was very complex and requires further investigation. - It may be mentioned that papain is also inhibited by higher concentrations (1mM) of NAD(P)H 2 .
218
No experiments have been carried, out so far to investigate the possible reduction of constitutive disulfide bonds and thus leading to inactivation of the enzyme as e.g. described for ribonuclease (10) and other proteins. It cannot be excluded that this proteinase inactivating enzyme has already been described under another name or with another specificity.
Acknowledgement This research was supported by the German Fonds of the Chemical Industry.
References 1. Gatt, S. 1969. Methods Enzymol.
152.
2. L e w y , G.A. and Conchie, J. 1966. Methods Enzymol. 8, 571. 3. Cory, J.G. and Frieden, E. 1967- Biochemistry 6, 116. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. 1951. J.Biol.Chem. 265. 5. Ashwell, G. 1957. Methods Enzymol. ¿, 80. 6. Landers, J.W. and Zak, B. 1958. Am.J.Clin.Pathol. 2$, 590. 7. Meisch, H.U., Schmitt, J.A. and Reinle, W. 1977. Z.Naturforsch. 52c, 172. 8. Schmitt, J.A., Meisch, H.U. and Reinle, W. 1977. Z.Naturforsch. 52c, 712. 9. Wiseman, J.S. and Abeles, R.H. 1979. Biochemistry _18, 427. 10.White, F.S. 1960. J.Biol.Chem. 235, 383.
DISTRIBUTIONS AND LOCALIZATIONS OF LYSOSOMAL CYSTEINE PROTEINASES AND CYSTATINS
N. Katunuma, E. Kominami Department of Enzyme Chemistry, Institute for Enzyme Research, School of Medicine, The University of Tokushima, Tokushima 770 , Japan
Introduction Lysosomal cysteine proteinases (cathepsin B, H and L) have been purified from various tissues of many mammals and their enzymological properties including substrate specificity have been demonstrated.
Recent studies disclosed the amino acid sequences (1) and
the carbohydrate structures (2,3) of cathepsin B and H.
However,
there are still quite few investigations on functions of each cysteine proteinase in vivo.
Although it is no doubt that cysteine
proteinases play a important role in degration both of intracellular proteins and of endocytosed exogenous proteins, basic knowledge such as tissue distributions of three cathepsins and their cellular localizations is scarce. On the other hand, informations on low molecular weight inhibitors specific for cysteine proteinases, cystatins have been accumulating recently.
Two intracellular types, cystatin-a and cystatin-g and
three secretory types, cystatin-y, cystatin S and chicken cystatin have been identified up to now (4-7).
Secretory types cystatins
have intramolecular sulfhydryl bridges, while intracellular types have not.
Cystatin a contains no cysteine residue, but cystatin-g
contains two free cysteinyl residues, one (cys-3) of which is involved in the formation of a mixed disulfide with glutathione.
We
showed the possibility that the activity of cystatin-g is regulated by formation of a protein mixed disulfide or by reduction of the mixed disulfide (8).
Our classifications of cysteine proteinase
inhibitors from molecular evolutional aspects and biological aspects are summarized in Fig. 1 and 2, respectively.
Cysteine Proteinases and their Inhibitors © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany
220 Kininogen
Serum
E g g White
Cystatin S
Cystatin y
Cystatin a
Cystatin 0
Cystatin
Calpastatin
Calpastatin
(-S-S-Cystatin)
Fig. 1.
Classification of cysteine proteinase inhibitors from molecular evolutional aspects
r Cystatin a . Cystatin
•< Cystatin ¡5
. Intracellular type
0.5
0.001
0.56 ± 1 . 1
Macrophages had very high concentrations of cathepsin B and H but had much lower level of cathepsin L than those of cathepsin B and H. The levels of cathepsin B and H in lymphocytes and neutrophils were less than 10% of those of macrophages. Neutrophils contained a high concentration of cystatin-a, whereas other cells tested contain low concentration (11). In fact, Brzin et al. (13) purified cystatin-a (human stefin) from human leucocytes. The level of cystatin-g was highest in macrophages followed by neutrophils and lymphocytes. Thus, phagocytic cells, especially macrophages contain high amounts of cathepsin B and H. Furthermore, the level of cathepsin B in macrophages elicited by injection of sodium caseinate increases 6 times that in resident macrophages, indicating that cathepsin B play a important role in digestion of exogenously injected proteins. Cathepsin H, L and cystatin-g did not show much increase in elicited macrophages. Localizations of Cathepsins (B, H and L) and Cystatins (a and g) Liver three cathepsins shown different distribution patterns (14). Staining of cathepsin B was strong in the periportal sinusoidal cells, possibly in Kupfer cells and weaker in panlobular hepatocytes. Staining of cathepsin H was strong in panlobular hepatocytes, especially in the periphery of cytoplasm. Staining
224 of cathepsin L was very strong in centrilobular hepatocytes. Staining of cystatin-g was very strong in Kupfer cells and quite weaker in hepatocytes. Low level of cystatin g in hepatocytes was also recognized by measuring its level in isolated hepatocytes. Staining of cystatin-a was background level. Thus, hepatocytes contain very little cystatin-g. It seems, therefore, unlikely that cystatins regulate activities of lysosomal cysteine proteinases in hepatocytes. Kidney Stainings of three cathepsins were all prominent in the proximal portion of convoluted tubules. The staining granules were localized preferentially in the basal cytoplasm. Stainings were very slight in the rest of the tubules, including the loop of Henle, distal convolution, collecting tubules. No staining was observed in glomeruli. Staining of cystatin-g was found not only in the proximal portion of convoluted tubules but also in the loop of Henle and collecting tubules. Distal convolution tubules was only faintly stained. "Amounts of cysteine proteinases present in proximal tubules may contribute to active intralysosomal digestion of reabsorbed proteins and peptides. Lymphatic Tissues The most intensive stainings of cathepsin B, H, L and cystatin (3 were all found in the macrophages and reticular cells in lymphatic tissues (spleen, lymph nodes and thymus). Staining of lymphocytes in those tissues were negative. Nerve Tissues Strong granular staining of cathepsin B and L was found in the large nerve cells of cerebral cortex (pyramydal cells) and of cellebeller cortex (Prkinje cells). Small nerve cells and various types of glia cells were only faintly stained. But the stainings of cathepsin H and cystatin-g were limited to pericytes along the capillaries and nerve cells and glia cells were not stained. Heart and Skeletal Muscle Stainings of cathepsin B, H and cystatin-g were observed in infiltrative cells, chiefly macrophages scattered in connective tissues rather than muscle cells. Physiological Roles The levels of three cathepsins varied not only in different tissues but also in individual cells in each tissue, indicating that lysosomes are heterogenous in the levels of cathepsins. Three cathepsins may function cooperatively in certain cells such as tubuler epithelial cells in kidney and macrophages in lymphatic tissues,but play different roles in other cells such as nerve cells and pancreatic
225 islet cells.
Distributions of cystatin-a in tissues and free cells
are quite limited. and neutrophils.
It is found only in the squamous cell epithelia Cystatin-a may provide protection against cysteine
proteinases contained in food and bioattackers
(bacteria, insects) .
Neutrophils contained both cystatin-a and $, but the physiological role of inhibitors in those cells are unknown.
Cystatin-f5 is
localized at the same cells as lysosomal cathepsins in several tissues such as kidney and lymphatic tissues.
The levels of cathep-
sins varied markedly in those tissues, whereas the levels of cystatin-g are quite similar and much lower than the sums of three cathepsins.
Cystatin-g in hepatocytes and large nerve cells in
cerebral and cerebellar cortex is absent , while
Cystatin-g is
found to distribute widely in cells of reticuloendothelial system scattered in connective tissues: Kupfer cells in liver, macrophages in spleen, thymus and lymphnodes, and peritoneal macrophagse contain high levels of cystatin-g.
The role of cystatin-g in phagocytic
function of macrophages is a problem to be dissolved.
Present
results suggest that the physiological role of cystatin-6 is not so simple.
The fact (15) that cystatins and cysteine proteinases are
located in the different cellular compartments should also be evaluated. Role of Lysosomal Cysteine Proteinases in Pathological Conditions Lysosomal proteinases have been implicated in inflammatory process and in certain diseases such as muscular dystrophy and various aspects of cancer including the invasion of host tissue and metastasis.
We showed (16) that increases in the levels of
cathepsin B and L were marked in skeletal muscle of dystrophic hamsters, and demonstrated immunohistochemically that those increases are due to the presence is invading phagocytes, chiefly macrophages. Inflammatory macrophages contain much higher levels of cysteine proteinases than skeletal muscle (Table 1, 2). For examining relation between macrophage invasion and muscle degeneration, we provoked acute muscle breakdown in rats by a single intramuscular injection of a anti-malaria drug, plasmocid.
Six
hours after treatment, the muscle fibers began to decrease, and the degeneration was most marked 48 h after injection.
About 10 times
increase in cathepsin B and L activities was found in the injured muscle. 1
Immunohistochemical examinations with anti-cathepsin B and
H Fab -peroxidase conjugates showed that the increased cathepsin B
226 and L activities originate from phagocytes invading between and inside of the muscle fibers.
Coinjection of cycloheximide with
plasmocid markedly inhibited the increase in lysosomal enzymes and degradation of muscle proteins (17).
Infiltration of phagocytes to
the injured muscle was delayed by injection of cycloheximide. Experiments on same direction with a local anesthetic, bupivacaine were performed by Ishiura et al. (18,19).
Macrophage cysteine
proteinases may operate as scavenger for cleaning the degenerated muscle fibers and the following regeneration. Distal myopathy with rimmed vacuole is a unique form of myofibrillar degeneration without associating invasion of phagocytes.
It has
been known that rimmed vacuole is positive histochemically for acid phosphatase.
We demonstrated immunohistochemically that abnormal
increases of cathepsin B and H in this disease were due to the presence at the site of rimmed vacuole and its earlier lesions within the skeletal myofibers.
However, the presence of cystatin a and B
at the lesions was not shown.
Abnormal increases in these intra-
myofibrillar proteinases may involve in development of myofibrillar destruction rather than clean the degenerated myofibers, although the cause and mechanism of increases are unknown.
Inhibition of
lysosomal cysteine proteinases in this disease may prohibit the progression of myofibrillar degeneration.
Acknowledgement This work was supported by Grant-in-Aid for Scientific Research (No. 59065007) from the Ministry of Education, Science and Culture of Japan.
We thank for Dr. Ii for his help with immunohistochemical
staining.
We also wish to thank Ms. E. Inai for expert secretarial
assistance.
References 1. Takio, T., T. Towatari, N. Katunuma, E.C. Teller, K. Titani. 1983. Proc. Natl. Acad. Sci. USA 8_0, 3666. 2. Taniguchi, T., T. Mizuochi, T. Towatari, N. Katunuma, A. Kobata. 1985. J. Biochem. (Tokyo) 97, 973.
227 3. Takahashi, T., P.G. Schmidt, J. Tang. 1984. J. Biol. Chem. 259, 6059. 4. Katunuma, N., E. Kominami. 1985. Curr. Top. Ce1lui Regul. 27, 345. 5. Katunuma, N., E. Kominami. 1985. In: Intracellular Protein Catabolism (E.A. Khairallah, J.S. Bond and J.W.C. Bird, eds.). Alan R. Liss, New York, p. 71. 6. Barrett, A.J. 1985. In: Intracellular Protein Catabolism (E.A. Khairallah, J.S. Bond and J.W.C. Bird, eds.). Alan R. Liss, New York, p. 105. 7. Turk, V., J. Brzin, B. Lenarcic, P. Locnikar, T. Popuvic, A. Ritonja, J. Babnik, W. Bode, W. Machleidt. 1985. In: Intracellular Protein Catabolism (E.A. Khairallah, J.S. Bond and J.W.C. Bird, eds.). Alan R. Liss, New York, p. 91. 8. Wakamatsu, N., E. Kominami, K. Takio, N. Katunuma. 1984. J. Biol. Chem. 259, 13832. 9. Kominami, E., T. Tsukahara, Y. Bando, N. Katunuma. 1985. J. Biochem. (Tokyo) 98^, 87. 10. Kominami, E., Y. Bando, N. Wakamatsu, N. Katunuma. 1985. J. Biochem. (Tokyo) 1437. 11. Kominami, E., T. Tsukahara, K. Ii, K. Hizawa, N. Katunuma. 1984. Biochem. Biophys. Res. Commun. 123, 816. 12. Ishikawa, E., M. Imagawa, S. Hashida, S. Yoshitake, Y. Hamaguchi, T. Ueno. 1983. J. Immunoassay 4, 209. 13. Brzin, J., M. Kopitar, V. Turk, W. Machleidt. 1983. HoppeSeyler's Z. Physiol. Chem. 364, 373. 14. Ii, K., K. Hizawa, E. Kominami, Y. Bando, N. Katunuma. 1985. J. Histochem. Cytochem. in press. 15. Kominami, E., N. Wakamatsu, N. Katunuma. 1982. J. Biol. Chem. 257, 14648. 16. Kominami, E., Y. Bando, K. Ii, K. Hizawa, N. Katunuma. 1984. J. Biochem. (Tokyo) 96_, 1841. 17. Kominami, E., N. Katunuma. 1983. In: Ann. Rep. Clin. Res. Mus. Dystro. (in Japanese)(K. Miyoshi, ed.). p. 195. 18. Ishiura, S., I. Nonaka, H. Nakase, K. Tsuchiya, S. Okada, H. Sugita. 1983. J. Biochem. (Tokyo) 9A_, 311. 19. Ishiura, S., I. Nonaka, T. Fujita, H. Sugita. 1983. J. Biochem. (Tokyo) 94, 1631.
Reprint from Cysteine Proteinases and their Inhibitors Editor: V. Turk © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany
FUNCTIONAL SHARES OF CATHEPSINS B, H AND L IN AUTOPHAGY AND HETEROPHAGY
E. Kominami, T. Ohshita, N. Katunuma Department of Enzyme Chemistry, Institute for Enzyme Research, School of Medicine, The University of Tokushima, Tokushima 770, Japan
Introduction During their break down, most intrinsic proteins are sequestered in autophagosomes and packaged in lysosomes by autophagosome-lysosome fusion, while exogenous protiens invaginated in the plasma membrane are incorporated into endosomes, which later fuse with pre-existing lysosomes.
The proteins incorporated into lysosomes are mainly
digested by cysteine proteinases, because leupeptin and E-64, an inhibitor of cysteine proteinase, inhibit degradation of endogenous (1,2) and exogenous (3) proteins. The metabolic activity of hepatic lobules varies in relation with the direction of blood flow, the distributions of various enzymes being different in periportal and centrilobular cells.
Previously,
we reported (4) differences in the locations of cathepsins B, H and L in rat liver; we found cathepsin B and H throughout the lobules, but cathepsin L in centrilobular regions.
These findings suggested
different physiological roles of the three cathepsins.
Which are
more important in autophagy and heterophagy periportal cells or centrilobular cells?
Asialoglycoproteins are taken up by the plasma
membranes of hepatocytes facing sinusoids.
If lysosomes fuse with
autophagosomes or heterophagosomes when they become in juxtaposition, the lysosomes involved in the two processes may be different.
To
examine this possibility, we studied the possible heterogeneity of lysosomes from the aspect of their functions.
Cysteine Proteinases and their Inhibitors © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany
230
Results and Discussion Experimental Design When mitochondrial-lysosomal fractions are centrifuged on a Percoll density gradient a bimodal density distribution of lysosomal markers is observed, most markers being recovered in fractions of lower density (5).
Administration of leupeptin to rats induces the
accumulation of numerous autophagic vacuoles in the liver and the migration of most lysosomal enzymes to the fraction of heavier density in a Percoll gradient (1,5).
Namely, inhibition of intra-
lysosomal proteolysis by the injection of leupeptin leads to the accumulation of autolysosomes containing both undegraded sequestered intracellular proteins and lysosomal enzymes.
In fact, we detected
several cytosolic enzymes in isolated autolysosomal fractions (5,6). Using this system, we tested which lysosomal cysteine proteinases participate in intralysosomal degradation of endogenous proteins and of endocytosed asialoglycoprotein.
Cathepsin B, H and L were assayed
with enzyme immunoassay systems for each enzyme (7). Cysteine Proteinases Involved in Autolysosomes In animals, autophagy and subsequent intralysosomal proteolysis in the liver are enhanced by deprivation of amino acids and insulin, and these enhancements are reversed by administration of amino acids and insulin (8,9).
Acceleration and repression of autophagy in vivo
can be induced by starvation of animals and refeeding after starvation, respectively.
In this work rats were starved for 48 h and
then refed for 12 h.
Leupeptin was injected into animals 1 h before
their sacrifice, and the mitochondrial-lysosomal fraction of their liver was prepared.
This fraction was then subjected to Percoll
density gradient centrifugation (52.6%).
The typical distribution
patterns of lactic dehydrogenase and cathepsin B, H and L in various fractions of the livers of control (dotted line) and leupeptintreated rats (solid line) are shown in Fig. 1.
Lactic dehydrogenase,
which is a cytosolic enzyme was detected in the autolysosomal fraction of starved, but not refed rats, indicating that autophagic sequestration of cytosolic proteins is markedly inhibited by refeeding.
Cathepsin B, H and L were recovered in the "light lysosomes"
of both starved and refed animals without injection of leupeptin, but after administration of leupeptin to starved animals, most of the cathepsin B and H and part of the cathepsin L were found in the "heavy lysosomes", indicating that the three cathepsins are all
231 Faatad
Rafad
Leupeptin
Aj O
Control
•
Leupept in
l.ioS
0.1 0.4
0
Po
d b
ex: 3
§10
I
— S
10 Frac t ion No
IS
20
5
10
15 20
Fig. 1. Distributions in a Percoll density gradient (52.5%) of lactic dehydrogenase (LDH), acid phosphatase (ACP), and cathepsin B, H and L of crude heavy lysosomal fractions from control ( o ) and leupeptin-treated ( • ) rats. Rats were starved for 48 h (starved) and then refed for 12 h (refed).
232 contained in autolysosomes. In refed animals after administration of leupeptin shift of cathepsin B and H to fractions of heavier density was also observed, but scarcely any of the cathepsin L shifted to heavier fractions. The components in these heavier fractions that are rich in cathepsin B and H may be heterolysosomes. This Possibility was confirmed by experiments with asialoglycoprotein, as described later in this paper. Localizations of Lysosomal Cysteine Proteinases in The Livers of Starved and Refed Rats To demonstrate these differences in the movements of cathepsin B, H and L in starved and refed animals by an other means, we examined the localizations of cathepsin B, H and L imiminohistochemically. Photomicrographs showed granules stained for cathepsin B. Staining was intense in pericanalicular regions and weak in the pancytoplasm of hepatocytes of starved rats, while it was seen only in a linear arrangement in the periphery of hepatocytes in pericanalicular regions of refed rats (Fig. 2).
Fig. 2. Immunohistochemical localizations of cathepsin B in the livers of starved (A) and refed (B) rats
233 Similar results were obtained on the distributions of cathepsin H and cathepsin L in hepatocytes distant from the center of lobules. Morphological changes in the distributions of cysteine proteinases were observed not only on refeeding but also on administration of cycloheximide and insulin. These treatments are known to inhibit hepatocellular autophagy by repressing the formation of autophagosomes. The results suggest that the granules containing cathepsins seen in the periphery of hepatocytes in the pericanalicular region were residual bodies, and that the fine granules in the cytoplasm of hepatocytes in starved rats were autolysosomes. Cathepsins Involved in Heterolysosomes Desialylated glycoproteins are known to be removed rapidly from the blood plasma of mammals, and there removal has been shown to occur exclusively in the liver and to be mediated by a carbohydrate recognition system. The ligand bound to the plasma membrane is rapidly internalized, transported within membrane-bound structures to lysosomes and subsequently degraded. However, the types of lysosomes involved in degradation of endocytosed, proteins are unknown. We examined the distributions of exogenously injected asialofetuin and cathepsins by Percoll density gradient centrifugation. FITC-labelled asialofetuin (3 mg/100 g body weight) injected into a tail vein was rapidly removed from the circulation with a half time of about 18 min. Concommittantly, FITC-labelled asialofetuin by accumulated in the liver, reaching a maximum after 60 min of 70% of the amount injected. Postnuclear supernatants from the livers of control and leupeptintreated rats were prepared 10, 20 and 30 min after injection of asialofetuin and subjected to Percoll density gradient centrifugation (40%) . Leupeptin was injected 30 min before asialofetuin in each case, since it inhibits digestion of endocytosed proteins such as asialofetuin (3). Ten minutes after injection of the ligand, fluorescence was found between the fractions of microsomal and mitochondrial markers, with only a trace in the lysosomal fraction (Fig. 3). These results indicate that asialofetuin was present almost entirely in endocytic vesicles 10 min after its injection and that little had been transferred to lysosomes. As shown in Fig. 3, the fluorescence in the lysosomal fraction increased with time after injection of the ligand.
234 O • A 1 15
30
Succinic I,VI reductase ti-6-Päse 5 -Nuc reot idase
15
! 1 10
e
1 05 4-»
20
-
10
10
-
5
C
a 00
0
0
.laHt»!»»»«» 10
15
20
15
10
o Jr •it
Ü 0
10
15
10
15
20
« 15-
10
5 3
5
IA
> -J co z IH £ CC
EH
p
z
*
*
«
>
UJ
s» IH o U. CO u
>•
IH > u o u. IJL C/1 CO u u
CL
J
2 2 û a < o E- E- E- J Si ^ O O a co cc co a>
0)
CO
2
b 3 SZ
l-H fc a U
1
CÛ AI
£
e 2
g
CO CT)
s C 2
2
5
1 1 1 1 1 1
co
*
>
a. a. a»
*
o
£
> > > >
a
CQ CL O
1
i j i i co i i M i i £ i i O i i J < t a. p i a a i o o i EH i i z CO i > i EH co t 0. -i i >- Ei IH S. i Si E i Si O I w O I * C/1 u. a. a.
J J J >• J UJ UJ UJ UJ > Q Q Q Q w Cl X 2 X < * z < E- UJ o
CO
W >>
1 1 1 1 1 1 1 t 1
1 1 1 1 1 1 u. U- U. o >- O
£ i>
T
1/> 1 > o Ä 3 x: 5
s 2
E- CL w
D o O o co co u- > > E-
î
*
Si cc cc cc > M > < > > > > > Si Si z o o CE er
(Ti io CTILO m CD CO rv CQ
0) C/l
o sc
z
Si < 2T X UJ CL CL cy UJ z Q X X u. UH CL w Ü u w p o J HJ z Q < Ü o CO co co 1 1 1 1
cys
J u. u. si > Si > o ¡x w p >• IK > U. Si si e UJ si U4 O Z z Z z H >- H < Si si si si UJ UJ UJ UJ UJ UJ UJ UJ J J j O o o o CL co IX co Si ce Si si
>•
>*
> >
u. EU J ESi H
UJ
>-
CE
CD
CO
S:
b
CO co CD
CO
2 S
W >» Ü £
ü
380 d i f f e r e n t b e c a u s e they i n t r o d u c e d a l a r g e r n u m b e r of g a p s 13)
(n = 12 -
into the s t e f i n - l i k e i n h i b i t o r s t h a n we d i d (n = 0 - 1).
Note
h o w e v e r that these d i s c r e p a n c i e s do n o t a f f e c t the c a l c a l u t i o n s the
r e l a t e d n e s s of the p r o t e i n s b y the R E L A T E p r o g r a m
(vide
on
infra)
as this l a t t e r p r o g r a m d o e s n o t a l l o w for gap i n t r o d u c t i o n at all. The c o n c l u s i o n that the k i n i n o g e n h e a v y c h a i n is c o m p o s e d of cystatin-like data
from
segment
the
has
RELATE program.
significant
stefin-like inhibitors, bitors,
three
r a t h e r t h a n s t e f i n - l i k e c o p i e s is c o r r o b o r a t e d b y the
T a b l e 2.
The s c o r e s i n d i c a t e that
s e q u e n c e h o m o l o g y to 3 of
the
the
4
known
a n d all of the 4 k n o w n c y s t a t i n - l i k e
U n l i k e the A 3 s e g m e n t ,
the B 3 s e g m e n t
A3
inhi-
exhibited
s e q u e n c e h o m o l o g y e x c l u s i v e l y to the c y s t a t i n - l i k e i n h i b i t o r s ,
but
w a s t o t a l l y u n r e l a t e d to the s t e f i n - t y p e of i n h i b i t o r s .
two
of the four c y s t a t i n - l i k e
inhibitors,
b u t n o t a single
Also,
stefin-like
i n h i b i t o r h a v e s i g n i f i c a n t s e q u e n c e h o m o l o g i e s to e a c h of the
three
A B r e p e a t s , cf. Table 3. F r o m this w e c o n c l u d e that the h e a v y
chain
of
k i n i n o g e n s is e s t a b l i s h e d b y three c y s t a t i n - l i k e b l o c k s
rather
b y t h r i c e r e p e a t e d s t e f i n - l i k e u n i t s as s c h e m a t i c a l l y
demon-
than
s t r a t e d in Fig. 8.
hu ste
hu CPI
ra li
ra ep
TPI
TPI
h u cys
h u SAP
bo CPI
ch CPI
A3
6.11
3.00
1.29
4.12
6.96
6.09
10.41
5.90
B3
-0.91
-0.43
-1.68
-0.77
1.79
5.39
5.63
3.85
h o m o l o g i e s b e t w e e n the h u m a n k i n i n o g e n heavy Table 2. S e q u e n c e c h a i n s e g m e n t s A 3 a n d B3, stefin-like inhibitors and cystatin-like inhibitors. Segment comparison s c o r e s w e r e c a l c u l a t e d b y the RELATE program (16) u s i n g a s e g m e n t l e n g t h of 25 (15) amino a c i d s f o r the A (B) repeats. Scores ^ 3.0 SD u n i t s i n d i c a t i n g s i g n i f i c a n t homology a m o n g two s e q u e n c e s are s h a d e d . The a c r o n y m s f r o m Fig. 7 are u s e d .
A m o n g the v a r i o u s A B r e p e a t s , A 3 B 3 h a s the m o s t p r o n o u n c e d to
low-Mr
CPIs
(mean score 5.73),
r e l a t e d (mean s c o r e s 2.72 a n d 2 . 3 0 ) ,
while A2B2 and A1B1 Table 3.
homology are
less
This suggests
that
381
the A3B3 domain which precedes the kinin segment in mammalian kininogens has been best conserved during the evolution of mammalian CPIs (vide infra).
hu ste hu CPI
B
ra ep ra li hu cys hu SAP bo CPI ch cys TPI
X
TPI
A1B1
0.39
2.64
1.96
3.14
1.56
0.70
3.15
4.89
2.30
A2B2
1.38
1.04
0.83
2.52
4.32
1.52
5.14
5.02
2.72
A3B3
6.01
3.19
1.80
4.00
6.26
8.37
10.25
5.99
5.73
Table 3. Sequence homologies between the internal repeats A1B1, A2B2, and A3B3 of the human kininogen heavy chain, stefin-like inhibitors and cystatin-like inhibitors. Segment comparison scores were calculated by the RELATE program (16) using a segment length of 25 amino acids. Scores i 3.0 SD units indicating significant homology among two sequences are shaded. For acronyms, cf. Fig. 7. x, mean comparison scores.
stefin
stefin
stefin
.—• .• i | kininogen heavy chain cystatin
cystatin
cystatin
Fig. 8. The human kininogen heavy chain is built by three cystatinlike copies. The human kininogen heavy chain can be dissected into three tandemly repeated AB units each of which corresponding to a single cystatin-like copy (AB unit) rather than a stefin-like copy (A unit). Supportive evidence that the kininogen heavy chain is formed by three cystatin-like blocks comes from the hydropathy profiles of cystatin, repeat A3B3, and stefin: the profile of cystatin matches
382 almost perfectly with that of A3B3, while the corresponding profile of stefin fits only moderately, Fig. 9.
+10 H
hu cys JI JJ p i ' T|PI|||f P|
o-10-
x
; .
HP'
. iJliL
L . If'fl^
-20-
a> x>
A3B3
o Z 0fo §• -10-
a 11.L
i j
p
1
. 1
Mk .JJ r iif
i i t . l .mi..
r r n f f
] l i p i i "
hu ste I
-10-
, P^'llljl
¡Ijlll
, 1
1
"I'l
u ^r
B |IJ
J|
-20-
25
50
75
sequence number
I
100
125
Fig. 9. The hydropathy profiles of the kininogen heavy chain repeat A3B3 and cystatin are similar. The overall hydropathy using a sliding window of 9 amino acids was calculated (17). Hu cys, human cystatin; A3B3, internal repeat A3B3 of the human kininogen heavy chain; hu ste, human stefin. Abszissa: numbers indicate relative positions of the residues in the amino acid sequences. Gaps are introduced as in Fig.7. Kininogens as multi-headed inhibitors. In
view
of the three cystatin blocks establishing
heavy chain one might expect that the kininogens are inhibitors. potential have
kininogen
triple-headed
However, inspection of the sequence portions holding a reactive site indicates that only repeats A2B2 and
conserved the consensus sequence Q-V-V-A-G (cf.
troduction"), sequence (Fig. of
the
A3B3
section "In-
while the A1B1 repeat has almost entirely lost
this
10). It is tempting to hypothesize that at most two
the three internal repeats,
i.e.
A2B2 and A3B3,
expose func-
tionally active inhibitor sites. Only recently, this conclusion has
383
been
c o n f i r m e d in an experimental
study by Salvesen and
coworkers
(13). AI
40
I T E A T K J T V G S D I T F Y S F K
A2
164
V K R A Q Rl>
05 O
o
•C o
8 8
b
oí
2
0-' £ o
m >,
o
O
n o>
s œ CM m 00 s sr 8 tO
01 H 00
O! tri
S
o r-
s tT> r-H CM
(M «T
rH 00 tO
? 8 Q
00
V ë
OJ •p w
H
00 t-t
CM 00 -
c
c
•f*
2 3c
J
1
c
5
ou O ci
«
m
tft
«i
00 00 CM
l-t w
tO ID 00
O
Si
r-t CM Iii
ci
tO l/l
tîo tp
CM "ï M'
8 cm'
m
tn o Iii
CTI f> »H CM oó œ
^
O ci 8 (O
en
á CM O O CM
8 T' fo
oo O
T
^ s
«
i i i
00
û
ru ru CM Oí
S
in to
$
n
cr i
fe
o H
cr _)
Ï« * z o ^ vO ** m M O o < G-D >• > cr
t4 06
< M
z a M o O tt ÎH (14 H C S5 Z
&
H* H
§
§ J
(A > >
cr h-)
> oí Vi Vi >H
S
s»
z M
co J H p^ M s w n a* hj Pu U1 pu >
cr
1 à « J i CO i i 1 i 1 i i 1 i 1 1 o oi 1
fe >
at
U
ss >
a s
"1
u.
*
u z
m
X
h
fe
PH Z H es •
f» cr
tt
ei UH co M
Ui
fe
> Q E] I »
cr z
1
•H (d t» « o
G M •H X U
_)
> M
X ta KJ
fe où Ü z o > p >
ÏM O Vf) ^ ÎM eJ M Z cr H u Ü < < > > > >
cr H
>*
cr Vi >*
cr
Uì
>
>
s «
0 u HJ U M W o 1 1 1 1 ÎH ï« H S * U z Z H H
fe
tí
M &
G
-H Îo M CJ C
Q
fe
w z H oí w p O g " S " M s» -< W PS o > > >
M ÎH
>
M H
»i
i i
$>
rsj «J « «1 4) a¡
•-I «J « ai
a X
cr
«
>
O se i 1
fe
w
U
w
fe fe fe w
H U U
>i
cr
M fe H h
1
¡j 4J
S
C •H U CO C
1
31
ï« •H
Ui «J 3
m -p Ol rH G -H i >i C - r í ^ Ol o u ai 4-1 ai O g-HXi M oi ai : Id fi -P a a « o n >h a; e a. > • a) h g 10 M -H M 2 •rH •H M fi o ai 3 C H H 0 E < a> 0) -H 4J En O -P ra ra fi fi O •H -P -H IO Id -P U M » .H fi id X) • c a— « ' M -H •H ai fi » fi Cl Q) M -P — rH (N 4J i 0 0) fl) C ^ -H (NT3 O -P -H Xi CN ai Q< O -H Q. 10 O* 1 « s.: o M -P D)=0 . • P U H 0 MH io ai ra J i 10 -H O 10 O u >I-P I a) fi -H 01 o id T) a o fi n c CM !U 4J n oi c io ; ai io T3 : M T) o -a o g e ja C H « G T3 10 10 10 C 3 io xi ^ g c ai ai fi 3 d C H 3 H Ä 3 » n io ai o -P CPMH c o • E t f Ä il ai o 3 -H Ol -P H ra M MHT3nJ •P fi o< io o m ai c 10 a i a i a i m a i a i - p i d •P ai , S ra fi O c c CP ai rH ai id o ai oí o ra 3 rH 01 rH ai » fi ai O" - o o i j i j ä n ^ O _ o a i e g Ä C ^ E n o C l i o G » -H o p H 4J g UFI M ai -H • ai fi ai ai • xi .H » 3 crai O1 tí +i +) +J x> oi >i -H io — « • H a K ' H B — tjij3 io 0173 1033 OC-pfM C ß U C C H -H HI O « -H H -HO AGNHIOAIAIGAÏC o O13-H0fi-P0ra-H id a i f i c - r H E - i O f i i O N tP-P Vl u o •H C A N -H m Xh Cl c io ai g • M oi ai •rH ra fi g -p o g ai o -H o o rH •p tí X a fi C O c o s o ai C -P •rH U +) -H MH O -H -P 0 -H 10 fi fi o 01 3 a i - P f i r o r a m - P 3 J > i •rH m io -p ai -P o 1 io H ^ io fi ra ^ o ^ Eh 10 O c . . -P m o o -P ai ai ai 0,-P "H a> u _ >i g "rH ai m 3 u ai 01 -H 1-* 0 _ -' O J3 •p G T) C M ftiH ai U *rH o o -H ai ifl u s; > fi (H -H 01 tjl-p o Cl H . G a en ai ai io id -p -P • r H ni M h c ai m M -H — o rH M H -P xi f> ai • ai o tn io »•H œ a< "d &1 01•H ai -H H -H C fi ai ra IH M •H c rH ai io fi fi o c -p -p 10 o & - P -H • >1 O I
399 found as the most conservative amino acid sequence through LMWand HMW-TPIs (Figs. 3 and 5), and was predicted to be a possible reactive site of the inhibitors (39). 5. The above pentapeptide sequence was positioned in each of two tandemly repeating sequences consisting of 122 amino acid residues in the heavy chain (Fig. 4) . When the middle parts of the repeating sequences surrounding the proposed reactive site were compared with the corresponding regions of human cystatin (24, 31), chicken cystatin (29) , rat liver TPI (26), human stefin (28), and rat epidermal TPI (32), the repeats 1 and 2 showed 20-34% homologies with cystatin type inhibitors and 14-23% homologies with stefin type inhibitors (Fig. 5). It is assumed from the above data (4 and 5) that a 2 TPI and LMW-TPIs would have the same ancestor gene and that the kininogen precursor gene and cystatin type gene recombinated before or after triplicated elongation (42, 43) of the cystatin type gene. The two repeats retained most fundamental structures as a cystatin type, but the other part of the gene coding for amino terminal side peptide was considerably modified. Thus, it is quite reasonable that LMW-kininogen and also HMW-kininogen, which has an identical heavy chain (40, 41, 44), are included in the same superfamily of cystatin type proteinase inhibitors.
LMW-Kininogen and HMW-Kininogen as Thiol Proteinase Inhibitors Some questions which arise here are whether there is any difference between the 012TPI and LMW-kininogen, what the situation of ofiTPI is, and which proteins have stronger inhibitory activity, CITPIs or kininogens. To resolve these questions, LMW- and HMW-kininogens were purified from human plasma, and their properties as thiol proteinase inhibitors were investigated. Human calpains I and II and urinary kallikrein were also prepared for this experiment. Purifications of LMW- and HMW-kininogens, calpains I and II, and kallikrein. LMW-kininogen was isolated from fresh citrated human plasma by a procedure emlpoying DEAE-Sephadex A-50, ammonium sulfate precipitation, DEAE-Sephacel, Red Sepharose, hydroxylapatite, and Butyl-Toyopearl (manuscript in preparation). HMW-kininogen was purified from fresh citrated human plasma by two step column chro-
400
LMW-Kininogen (pg) Fig. 6. Inhibitory activity of LMW-kininogen. Inhibitory activity of LMW-kininogen toward calpain I (-#-) , calpain II (~0~) , and papain (-Ar) was measured with casein as substrate. The caseinolytic activities of these enzymes were adjusted to give approximately 0.26 UV absorbance at 280 nm in the assay system employed for this experiment.
matographies on DEAE-Sephadex A-50 (45) and Zn-chelate Sepharose 4B (46). All purification procedures were performed at room temperature to avoid cold activation in the presence of proteinase inhibitors (47). The purified proteins gave a single band on SDS polyacrylamide disc gel electrophoresis in either the presence or absence of reducing reagent. Calpain I was purified to apparent homogeneity from the cytosol fraction of human erythrocytes by the method of Hatanaka et al. (48). Calpain II was purified from human kidneys by a procedure involving column chromatographies on DEAE Cellulose, Sephacryl S-300, DEAE Bio-Gel A, and Red Sepharose (manuscript in preparation) . The specific activities of the purified calpain I and calpain II were 120 units and 117 units per mg of protein, respectively. Kallikrein was purified from pooled fresh urine from healthy subjectes by the method of Ole-MoiYoi et al. (49). The purified urinary kallikrein migrated as a single band on SDS polyacrylamide
401
HMW-kininogen ( j j g ) Fig. 7. Inhibitory activity of HMW-kininogen (50). Inhibitory activity of HMW-kininogen toward calpain I (-•-) , calpain II and (-O-) , papain ( - A - ) , trypsin (-+-) , chyraotrypsin thermolysin (-A-) was measured with casein as substrate. The caseinolytic activities of these enzymes were adjusted to give approximately 0.3 UV absorbance at 280 nm in the assay system employed for this experiment.
disc gel electrophoresis, and its specific activity was 0.58 units per jil of solution. Inhibitory activities of LMW-kininogen and HMW-kininogen. The inhibitory activity of LMW-kininogen was examined employing calpain I, calpain II and papain as target proteinases. As shown in Fig. 6, LMW-kininogen strongly inhibits papain, whereas the kininogen shows rather weak inhibitory activity to calpain I and calpain II. This result was not compatible with our previous data about «^TPI and OI2TPI (11), in which calpain (CANP) was more susceptible to both OlTPIs than papain. This difference in inhibitory activities between LMW-kininogen and 02TPI is examined more precisely in the last section. The inhibitory activity of HMW-kininogen was estimated by using calpain I, calpain II, papain, trypsin, chymotrypsin, and thermolysin. In this experiment, calpain II was most susceptible to HMW-kininogen followed by papain and calpain I (Fig. 7) (50). Serine proteinases, trypsin and chymotrypsin, and metalloproteinase, thermolysin are not inhibited, indicating that
402
1 min
LMW KG
t 1.0 nM
0 5 nM
t Sample
r
2.0 nM
azTPh t 0.5 nM
t I.OnM
a2TPI2
t Sample
t 2.0 nM
L0.5 nM - std.KD-
t 1.0 nM
t Sample
f
2.0 nM -std.KD-
Fig. 8. Kallidin releasing capacity of LMW-kininogen, (*2TPII and 0(2 TP 12. LMW-kininogen, OI2TPI1 and W2TPI2 (final concentration, 10.4 fig/ml) were each incubated at 37°C for 30 min with human urinary kallikrein (17.4 units/ml) in 2.2 ml of 20 mM Tris-HCl buffer, pH 7.5. Samples of 100 jil were taken out and used for bioassay of kinin, which was liberated during incubation. Kinin activity was assayed using rat uteri isolated from virgin rats, weighing 185-220 g, which had received a subcutaneous injection of 10 pi estradiol benzoate/100 g body weight 24 h before sacrifice.
403
HMW-KG
Jt
0.5 nM
o^TPI
J
t
O.SnM
I
t
I.OnM
10 nM
Sample
V
t
Sample
std.KD
f
2.0nM
20nM
sld.KD-
Fig. 9. Kallidin releasing capacity of HMW-kininogen and «^TPI. HMW-kininogen and OtiTPI (final concentration, 22.0 fig/ml) were each incubated at 37°C for 30 min with human urinary kallikrein (17.4 units/ml) in 2.2 ml of 20 mM Tris-HCl buffer, pH 7.5. The following procedure was the same as described in Fig. 8.
HMW-kininogen is a group specific inhibitor to thiol proteinases. A cytosolic inhibitor specific to calpains, namely calpastatin, has been reported to inhibit calpain II stronger than calpain I (51). HMW-kininogen showed the same character as calpastatin as an inhibitor for calpains. Potential capacity to release kinin.
The kinin releasing ability
404
>
u m u.
o
.c c
2 Incubation
3 0 time
Fig. 10. Inhibition of papain activity by HMW- and LMW-kininogens and their derivatives (50) . In this experiment, activated papain without any added reducing reagent was used to avoid the effect of the reagent on disulfide bonds in the kininogen molecules. Prior to the experiment, papain was activated with 1.4 M /9-ME for 10 min at room temperature, and the residual /S-ME was removed by passing the reaction mixture through a PD-10 column twice. All the buffers used were bubbled with N2 gas for 60 min. The activated papain and kininogen or its derivatives were preincubated in a cuvette placed in a spectrophotometer. The reaction was started by adding the substrate of papain, Bz-L-Arg-pNA, and the increase in absorbance at 410 nm was monitored for 3 min. (A). Inhibitions by HMW-kininogen and its derivatives. Rates of hydrolysis of Bz-L-Arg-pNA by papain: without any additives (p), and in the presence of reduced and S-carboxymethylated (SMC-) heavy chain, light chain and HMW-kininogen (a, b, c), 1.4 M /8-ME reduced HMW-kininogen (d) , 0.14 M /6-ME reduced heavy chain and HMW-kininogen (e, f), kinin-free HMW-kininogen (g), native HMW-kininogen (h), kinin- and fragment 1-2-free HMW-kininogen (i) and native heavy chain (j). (B). Inhibitions by LMW-kininogen and its derivatives. Rates of hydrolysis of Bz-L-Arg-pNA by papain: without any additives (p), and in the presence of reduced and S-carboxymethylated/'
405
of LMW- and HMW-kininogens and Ofi and (*2TPIs was examined by means of rat uterus contraction by the method of Shimuta et al. (52). One sample each of LMW-kininogen, O^TPIi and O2TPI2 was incubated with kallikrein, and the aliquots were applied to the muscle contraction experiment (Fig. 8) . The incubation mixture of LMW-kininogen showed a typical contraction, whereas those of ot2TPIi and ^ T P I j induced no contraction. The same experiment with HMW-kininogen and OtiTPI showed that «^TPI has no potential ability to release kallidin, whereas HMW-kininogen induced a typical muscle contraction (Fig. 9). The inability of a^TPI, of2TPIi and a 2 TPl2 to release kallidin was further confirmed by the use of 10 times more concentrated samples. Clotting activity. Clotting activity of (*iTPI was also examined by the two-stage assay for actin-activated partial thromboplastin time using HMW-kininogen deficient plasma (53). However, no activity was found in H1
T)
ni
rH m e Ol u tJl-rH 0 £1 G a •rH n ) VH C •H Di
M s g œ
-d 0) C •rH
e Vi a) -M en 0 ) IV - a •H +> a i -.H >H 0) •H ï •P U] U
>i+> >H 10 IH o -p -rH Ö l • i l C • H •rH r H S: • a 0 G e G • H • H 10 X I SI •P G T J ai G 0 (0 - p H • H ai C eu • H
0 U
G
o • •H •P (0 o e m CD — •C U] O e n ai >
.—.
i •a ta vi
£I
M CQ
•H •P
I-H o I—I 10 > p—i en -rH h
-H M ai • H T)
407
o) +>
e
•H O M
CD 5
in
• a >n c 10 > i rH
u»
C H
Cl (0 tn U
O
w
O -H e s:
o Z
•h
a (0
•H ^ M t7> O Ò m
o Ö in
o
CN
ò in
in in
o -4
a> in
I
EË *O S a) c •H •H g
O M V 0) +J ai ai
•H -A
•H ai >
i Z o i/i
1 o t/1
z t_> i/i
LMW-KG
tre al
e
-C
Z
V X
ci
>c
ÏC
1 $ Z
«,-T* ai a . il— I 1
M Z
2
>
ï s
n
•H 0)
O
•tree LMW-TPI,)
Z
O
LMW-KG
H
tlJ Z c
I ?
¡5
c.
s u
«
1 ° 2
z
-M •H XI 0> . •H e »H Ä c
H -o
o c
•H C (tí •H XI 73 j a 4J
ç Z n u .Cl/1
a. l/l-
i/iS
S
S
r co 1
S
r f ¡/i m
E u i/iH
I -
C
c î
X i/i-
¡2"
?
] r—i
r LO 1 t/>
G
?
r i/i i i/i
m i i/i
L
L
L
1
-
.C M
l/l
t/1
•P Si 01 -rH
S
ao
00
z u i/H
c ai « -a 0 +> c ra (tí a ai (0 M m u 3 c M -M o (U U •H e 3 4J 1 ^
r i/i i/i j .
^ (0
C S rH I
3 -M U C •• ai
ai c
O
M C
H
U -H
G ai -H
Z u i/H
X i/>-
u
X l/l-
X l/l —
i i/i-
eu
•H
^
>1
•a -p m • n (0 ja § m
•C
E
. -H
.
^ i « rH « r H
• -H
•
en M o i
•H a)
T i fa
408 nase inhibitor activity of HMW- and LMW-kininogens, several derivatives of the kininogen molecules were prepared (50). In this study, 1.24 n moles of papain (16.3 units/mg) and 0.45 n moles of each sample were used for the assay of inhibitory activity. The active site of papain was titrated with E-64. In this system, native HMW-kininogen and LMW-kininogen inhibited 52% and 50% of the papain activity (Fig. 10). These inhibition capacities correspond to 1.4 molecules of papain per kininogen molecule. The inhibitory activities, binding ratios, and the schematic representations of all the samples are summarized in Figs. 11 and 12. As shown in the figures, 1.4 M yfl-ME reduced and carboxymethylated (SCM-) heavy and light chains and SCM-HMW- and LMW-kininogens did not show any detectable inhibitory activities, while the partially reduced (0.14 M ifl-ME treated) heavy chain and HMW- and LMW-kininogens retained 30%, 33.6% and 50% inhibitions, which corresponded to 0.8, 0.9 and 1.4 molecules of papain per kininogen molecule, respectively. This result indicates that tertiary structure supported by intra-molecular disulfide bonds is necessary for the inhibitory activity of the kininogens. Derivatives of HWM- and LMW-kininogens generated by digestion with kallikrein generally exhibited higher inhibitory activities (55% — 62%) than the native kininogens (50% — 52%) with the exception of kinin-free HMW-kininogen (42%). The highest inhibition capacity (74%) with a full binding ratio (2.0) was observed in the heavy chain which was free of kinin, fragment 1-2 and light chain. This stoichiometry (2.0 papain molecules bound to one molecule of the heavy chain) agreed well with the predicted number of reactive sites located in the heavy chain of ra ID >1 (d "H co O 4-1 ID G 4-> O 4J -H A) IO 3 1 H Ä 4 J O 4-1 RA C >. O M G O RA C O •HO -D V4 -H -A CD M 4-1 D) - P OJT4 a ID G TRI H O O -H >1 . O A-RAX AI RA 4-> -O •D CR ID A) -H C G G -P A, ID -H > , •OH > , » M C AI X A W O > O C V H XI M >D M +J M RA C A) ID ID ID 10 S-4 4 J O T3 L-L G -H H O AI N 4 J EN O 4-1 G A
ra
m
w
e applied
and renal
column.The
to be 1 1,000\ig/l In
Inhibition.
Cyitatin
by EL1SA
thiol ZOOvl
proteaiei
Imu^^lency C level
: uie obtained
Inhibitor
oi total Into o& thli the
ierum
normally
¿-torn a
a H.P.L.C.
gel
ierum
determined
uiai
chromatogramme
ihouied
: 0($ papain
M.U.
cloie
6ound
at
to
enzymatic
SO,000
13,000
Valtoni.
Valtoni,
Inhibitory
activity
No enzymatic
although
Cyitatin
wai
obierved
Inhibition
hai
C antibody
In
been ELZSA
at
512
rzcogniiz Cyitatin C " antigzn" oh thz iamz iitz. wz In ordzr to invzitigatz thii lack o^ activity at 13, 000 Valtom applizd thz iame. proczdurz to analyze thz urinz oh a patiznt with, a proximal tubulopathy having a Cyitatin C urinary Izvzl zquivalznt to thz izrum Izvzl oh patiznt dzicribzd above.Two iitzi with znzymatic papain inhibition havz bzzn locatzd, including onz at [Fig. 4) . 1 3, 000 daltom It appzan inhibitory
thzrzhore that izrum Cyitatin activity than that oh urinary
C hai a Izaer Cyitatin C.
papain
VISCUSION Our rziulti ihouii that izrurn Cyitatin C, an alkalinz LMW protein normally prziznt in izvzrat human u.idi ii zlzvatzd in patiznti with SLE. iimilar To our knowlzdgz only onz othzr tzam claimi to havz hound >iziulti in patiznti with autoimmunz diizaizi[7). Howzvzr, Biz in and coll.(7) do not ipzcihy ih thzir patiznti wzrz in rznal iniuhhicizncy, although thz Izvzli oh Cyitatin C reportzd in thzir work Izad onz to bzlizvz that thzy indzzd wzrz in kidnzy imuhhicizncy at thz timz oh thz itudy.Wz alio havz hound izr a Izvzli oh up to twzlvz timz-i thz normal valuzi (mzan 6) but all thziz patiznti wzrz in chronic rznal imuhh^^ncy. Sincz Cyitatin C cataboliim ii mainly rznal and iti izra Izvzl with thz glomzrular ¿titration ratz (GFR) raiizd varizi invznzly izrum Izvzli diicoverzd in thziz patiznti izzm to bz thz comzquzncz oh impairzd GFR rathzr than an incrzaiz in iti iynthziii{12). Morzovzr our rziulti conczrning thz high corrzlation Cyitatin C- 82 microglobulin in patiznti with rznal -tniu^-cc-tenct/ conhirm thii hypothziii. Nzvzrthzlza wz havz alio dztzctzd iera Izvzli 1.8 h°Z-d highzr than normal valuzi in patiznti with SLE having normal GFR; thzrzhorz raiizd Izvzli in thziz patiznti might bz a rziult oh hypzrproduction oh Cyitatin C. Rzlativz diaociation in thz correlation bztwzzn Cyitatin C- B2 microglobulin (0.66) in our Lupui patiznti with normal GFR allowi ui to concludz that thz mztaboliim oh thziz two protzim in
513 pa.t-Le.nti with The leaioni pieiently
Lupui
unknown.
with. Lupui
level
13,000 Valton
Beioiz
whzieai
in ioui diiiziznt
Cyitatin ; SLOW
patient, ieia
(unlea
ioim OK linked
diicoveied
ioui amino
One iynthetic
acidi
having
SLE hai recently
which
in uiine
C might
papain C) may be
activity
expieaed
on the iui^ace by laige
mediatoii
(20) . Although
than
acid iequencei that
119).The
oi immune
iyitem
in
ai
patienti
hypothazi oh Fc
leceptoii
oi a blockadz (I.C) oi
diicuaed
coiielation
iitz.
phagocytoiel18].
complexei
ii highly
hai
teiminal
two
celli
we
hai been utilized oi
aie a leduction
a direct
between
oi
theiz
unknown
between
IC
in the Cyitatin
C
l.C.
In concluiion pieient
we piopoie
in ieia
oi a tetia phagocytic
that
oi patienti
peptide
FAST Cyitatin
with
LVS-PRO-PRO-ARG,
capacity.
in
in a
the FAST ioim
at the NHz
on phagocytic
paiameten
and
ioim piziznt
be pieient
the exiitence. oi coiielatiom
we have iound
uiinz
tubulopathy
piotein) .
oi phagocytic
quantitiei
ieiologic
ihould
ii an activatoi
direct
between
iiom
be the main
except
been lepoited thii
leceptoii
and
have, at the
a FAST and a SLOW ioim
the iamz izquzncz
hand an impaiiement
to explain
any
(Cyitatin
we had obizivzd
(LVS-PRO-PRO-ARG)
peptide
iuggzitzd
tubulopathy activitiei
inhibitory
¿oim amino
On the othei
and diUzient
papain
a>ie identical
o{ TUFS1N
liteiatuie,
including
to anothzi
an inhibitoi with
patient
did.
the piotzin
FAST and SLOW
that they
iiom a
did not exhibit
C wai diicovzizd
whe.fi.eai FAST Cyitatin
Whzn wz compaied
pioximal
C, diHeient
kidney.
SLE a'le
comm.)
ilom SLE patienti
polymziiizd
loit
ioimi,
in the with
tela
poit globulin
the diUzizncz
Cyitatin
with
the uiinz that
C hai a lowei
C [Pen.
Thii ¿act explaim izia
; the izia
we have ihown
(17). FAST Cyitatin SLOW
zxcluiively in patienti
oi Cyitatin
iitz
activity,
thii,
pieient
iiom a patient
and uline
inhibition
C incieaie
Ace.on.dlng to oui iindingi,
ion. an equivalent iamz
doe.-6 not occui
i01 a Cyitatin
C ii the main
SLE, lead to a laiied which
in tuin leaem
holm
pioduction the
Flgui t 3 H.P.L.C. elution pattern of S.L.E serum Four molecular markers were used : Immunoglobulin 6 (M W 150 OOO D), Bovine Serum Albumin (M.W : 68 000 D), Light Chains of Immunoglobulin 6 (M.W: 25 000 D) and Beta 2 Microglobulin (M W: 11 600 0). Solid bars correspond to the papain enzymatic Inhibition zone Arrow indicate the positive Cystatin C EllSA determination.
515
Figure 4 H P L C elution pattern of urine with proximal tubular dysfunction Three molecular markers were used
Immunoglobulin 6 (M.W
150.000
D), Bovine Serum Albumin (M.W : 68 000 D), and Beta 2 Microglobulin (MW
11 600 D)
Solid bars correspond to the papain enzymatic InWbttlon zones
516 REFERENCES 1.
BuUeA,
E.A., F.l/. Flynn.
2. Clauen,
1961.
J .
4. Cejka,
J.,
5. Collé,
A., R. Guinet,
6. Hochwald,
Pathol.
172
J . R . CoAgAove. 1961. Can. J . Biochem.
L.E. Fle.iAch.man. 7973. AAch. Biochem.
G.M., G.J.
U,
Soc. Exp. B-toi. Med. _J07, 770
PAOC.
3. Mac PheAAon, C.F.C.,
1961 . J . Clin.
It. LecleAcq, ThoA.beeke.
BiophyA.
V. Manuel. 1976. Clin.
1963. Aich.
Biochem.
PhyAiol. 157_, 68
Chim. Acta.. 67_,93
101, 325
7. BzAin, J . , T. Popovic, V. TuAk, U. BoAchaAt, W. Machleidt. BiophyA. ReA. Commun. IIS, 103 8. BaAAet, A.3., 120, 631
U.E. Va\>ieA, A. GAubb. 19S4. Biochem.
39,7567
1984.
Biochem.
BiophyA. Reó. Commun.
9. Manuel, y., J . P . RevillaAd. 1970. in : PAoteinA in noAmal and pathological uAine. Y. Manuel. J . P . RevillaAd, J . P . 8 e t h u e l , edi. New VoAk BaAel KaAgeA p. 153 10. Cohen, V.H., H. FeineA, 623
0. JenAAon, B. Frangiane.
19S3. J . Exp. Med. 15S,
7 7. GAubb, A., 0. JeviAAon, G. Gudmyi&Aon, A. AAnaAon, H. LöfibeAg, J . Malu. N. Engl. J. Med. 31±, 1547 12. Simomen, 97
0.,
A. GAubb, H. ThyAAel. 1985. Scand.
J . Clin.
Lab. InveAt.
1984. 45,
13. Tan, E.M., A.S. Cohen, J . F . File*, A.T. MaAi, P . J . McShane, N.F. Roth&ielA, J.G. SchalleA, N. Talal, R . J . WincheAtZA. 1982. AAthAitiA, Rheum. 25_, 11 14. BaAAett, A . J . 1981. Methodi Enzymol. 80_, 535 15. LambAé, C., K.N. KaAtuAi. 1979. J . Immunol. Methods. 16. TheophilopouloA, 57, 169
A.N., C.B. WilAon, F . J . Vixon.
17. Tonnelle, C., A. Colli, ReA. Commun. 86_, 613
26_, 61
7976. J . Clin.
M. Fougefieau, V. Manuel. 7979. Biochem.
Invent. BiophyA.
18. NajjaA, V.A. 7979. In : The AeticulaendotheliaZ AyAtem : a compAehenAive tAeatiAe [SbaAAa A . J . , StAauAA R.R., edA.l 1loi II, BiochemiAtAy the Reticuloendothelial SyAtem, Plenum PAeAA, New yoAk. 79. HambuAgeA, M.I., 1982. AAthAitLi,
J . T. Lawley, Rheum. 25, 7
R.P. KimbeAly, P.H. Plötz,
20. PaAAiA, T.M., R.P. KimbeAly, R.P. Inman, J.S. C.L. ChA-Utlan. New yoAk and Atlanta GeoAgia. 526 27. Lloyd,
W., P.H. SchuA. 1981. Medicine.
60_, 208
M.M. FAank.
McVougal, A. GibobAky, 1982. Ann. Int. Med. 97,
DIFFERENTIAL ACTIONS OF HUMAN CYSTATIN C ON DIFFERENT FUNCTIONS OF GRANULOCYTES
J.L. SANSOT, J.J. BOURQARIT, C. BLANC, Y. MANUEL , A. COLLE INSERM U 139, Hôpital Henri Mondor, 94010. Créteil Cedex, France,
J. LEUNG-TACK , INSERM U 119,27 bdLei-Roure, 13009 Marseille, FRANCE.
J.L. MEGE Laboratoire d'immunologie, Hôpital Sainte Marguerite, B.P. 29, 13277 Marseille Cedex 09, l-rance.
INTRODUCTION.
Post gamma globulin is an alkaline "Low Molecular WeighV'protein (13,260 daltons) firstly described in 1961 as a constituent of cerebrospinal fluid and of urine from patients with tubular disorders (1-3). It has also been found in several fluids of normal humans : saliva, serum, urine, ascitic and seminal fluids (4,5). Kutt ( 6 ) suggested that post gammaglobulin might be a bradykinin activating enzyme, but other experiments have failed to show any enzymatic activity
(7,8). Recently, a serum cysteine protease inhibitor was shown to have
a N terminal amino acid sequence matching that of post gamma globulin which suggested that these proteins were identical (9). Barret (10) showed that post gamma globulin is a cysteine protease inhibitor and named it cystatin C. Tonnelle (11), by sequence analysis of the fifty N terminal amino acids of post gamma globulin, has evidenced several forms with different N terminal amino acids and named them "native slow", "slow", "intermediate" and "fast" forms respectively. So far cystatin C has been completly sequenced (12) and corresponded to the "native slow" form sequence determined in this previous work. A spontaneous degradation was evoked to account for different N terminal amino acids (13). In fact, these various stuctural forms could derive from the "native slow" form of the protein, through release of N-terminal peptides following
Cysteine Proteinases and their Inhibitors © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany
518
exo or endopeptidase attack. More precisely the tetrapeptide L Y S - P R 0 - P R 0 - A R 6 was shown to be present in both the "native slow" and "slow" forms and not in the "intermediate" and "fast" forms ( 1 3 ) . Since such a peptide was described by many authors as an antagonist of Tufstin ( 1 4 , 1 5 ) , which is a natural stimulator of phagocytosis, we have investigated the possible effects of the different forms of post gamma globulin (particularly the "slow" and "fast" forms)
on phagocytic and chemotactic activities
of human
polymorphonuclear
leukocytes ( P M N L ) . Although post gamma globulin is identical to cystatin C we use the former term since only post gamma globulin has been described under different forms.
MATERIALS AND METHODS.
Human pnst gamma glnhnlin (nystatin C) The protein was isolated from urine of patients with renal failure as described by Tonnelle ( 1 1 , 1 3 ) . The purity was assessed by electrophoresis In 5i? polyacrylamlde-agarose gel ( 16) and by Immunoelectrophoresis ( 17) developed with polyvalent antisera against total human serum and monospecific antiserum against human post gamma globulin. Ouchterlony's double immunodiffusion method ( 18) was also used, developed with monospecific antisera against post gamma globulin, beta 2 microglobulin, Fab, Fc, kappa or lambda chain
of
Immunoglobulin 0 .
Phagocytic activity of polymorphonuclear leukocytes from normal suhjacts Phagocytic activity was assayed as described by Bongrand ( 19). We used two suspensions prepared following two procedures : - Ficoll purified cells, which yielded 9 5 $ PMNL and 51? mononuclear cells. - Dextran T 5 0 0 ( Z % ) purified cells which yielded 7055 PMNL and 3051 mononuclear cells. Briefly, 10 6 PMNL were suspended in 0.3 ml of test medium with different concentrations of post gammaglobulin ( 0 to 8 3 ug/ml) and 0.4 mgof opsonized zymosan. Incubation proceeded for 6 0 min. at 3 7 ' C , the mixtures were centrifuged and supernatants were assayed for optical density with a Beckman spectrophotometer. Cell pellets were deposited on microscopic slides and stained with Wright Oiemsa reagents. Ferricytochrome C reduction was monitored by measuring the absorption peaks at 5 2 0
and 5 4 9 nm. Superoxyde production was
determined by:
(deltaO.D.) x 14.29 = n.molesuperoxyde anion / 10 6 cells.
Phagocytosis was quantified as described by Bongrand (19). Fraction of cells having ingested at least one particle (P.F.) and the mean number of ingested particles per cell containing at least one particle (P./P.) were determined.
Chemotaxis assay Cells, processed as in the phagocytosis assay, were tested for their chemotactic activity (20). Cells ( 5 x 10 5 ) incubated with post gamma globulin were introduced into the upper part of a Boyden chamber and tested against 2% Bovine Serum Albumin (B.S.A.) and 2% B.S.A. + 205K zymosan activated human plasma. After 1 hour incubation at 37'C the cells that were present at the lower surface of the filter were counted and the migration index was determined as the number of cells per field. The results were obtained by reading 10 fields on the filters in duplicate experiments.
RESULTS.
The post gamma globulin samples showed one single band in polyecrylamide-agarose gel electrophoresis. When a Ficoll purified cells suspension (95!? PMNL) was used, the fast post gammaglobulin form activated superoxyde anion release, at concentrations ranging from 1 to 63 ug/ml, with an optimum effect for a 3 ug/ml concentration. The slow form had a dual effect depending on its concentration. Superoxyde anion release was depressed by concentrations below 25 ug/ml and enhanced by higher concentrations (Table la). The two post gamma form action curves (Figure I) allowed to determine the difference between these two actions. The resulting curve (Figure II) admited a maximum which, in these experiments, was close to 3 ug/ml of post gamma globulin. When Dextran T 500 purified cells, that is containing 30!? mononuclear cells (macrophages or lymphocytes), were used, superoxyde anion release was increased whatever the post gamma form used to treat the suspension (Table lb).
Results concerning the phagocytic activity are expressed in Table II. When Ficoll purified cells were treated with the slow form, the percentage of phagocytosing cells (P.F.) and the
520
CONCENTRATION (U* / •!>
FAST FORM
SLOW FORM
SUPEROXYDE ANION RELEASE (mole / 10 cell»)
RECOVERY ( X >
0
2 23
100
» 16
2 74
121
3 14
13»
1.3
3 55
158
1
2 67
117
0
1 21
100
83
1 84
152
«1
1 21
100
20
1
83
3.3
0 72
60
0.3
0 80
67
TABLE la : FICOLt PURIFIED CELLS.
CONCENTRATION (Bg / »1)
FAST FORM
SLOW FORM
SUPEROXYDE ANION RELEASE (mole / 10 celle)
RECOVERY ( X )
0
0.63
100
83
2.04
327
16
1.41
223
0
0.63
100
41
1.61
270
8.3
1.32
215
1.6
1.41
223
TABLE lb : PEXTEAN T 500 PURIFIED CELLS.
TABLE I. FFRRICYTOCHROMF C. RFDUCT ION BY POLYMQRPHONUCl EAR LEUKOCYTES. Two suspensions prepared following two different procedures were used: Ficoll purified cells and Dextran purified cells (see materials and methods). Polymorphonuclear leukocytes were incubated with ferricytochrome C and opsonized zymosan. Results are expressed as nanomoles of enzyme reduced per 10 6 cells. In each case, mean values were calculated from results obtained from different donors. Results were expressed as percentage recovery of ferricytochrome C reduction activity.
number of phagocytized particles per cells (P./P.) were decreased for concentrations below 40 ug/ml and increased for higher concentrations. Comparable results were obtained with two different slow post gamma globulin preparations. Results of phagocytosis in the presence of the fast form were less clear. When 3 0 $ mononuclear cells were present in the cell suspension (Dextran T 5 0 0 purified cells) the results were complex and required further investigations (data not shown).
521
CONCENTRATION (ug / Bl)
P.F.
RECOVERY ( I )
0
3«
100
1.20
100
83
50
147
1.80
150
41
26
83
0.90
75
20
29
86
1.09
»0
3.3
27
60
0.9
75
0.3
23
68
0.65
55
0
13
100
0.23
100
25
6
47
0.07
31
SLOW FORM M M
SLOW FORM N°2
P./P.
RECOVERY ( I )
TABLE 2. PHAGOCYTIC ACTIVITY OF POI VMORPHOM« FAR I FUKOCYTFS Polymorphonuclear leukocytes purified on Flcoll were incubated with opsonized zymosan. Fraction of cells having ingested at least one particle (parameter P.F.) and the mean number of Ingested particles per cell containing at least one particle (parameter P./P.) were determined. For each serie of measurements the mean value was calculated in duplicate. Results are expressed as percentage of recovery from control.
WITHOUT POST GAMIA
PMNL 1
22
PMNL 2 B.S.A. 21 ACTIVATED PLASMA (201)
WITH POST GAMMA
RECOVERY
(10 u s / . l )
( t )
20
91
43
32
75
PMNL 1
44
17
39
PMNL 2
618
243
40
B.S.A. 21
FICOLL PURIFIED CELLS
WITHOUT POST GAMMA
WITH POST GAMMA
RECOVERY
(10 ug/ml)
( I )
B.S.A. 21 + ACTIVATED PLASMA (201)
116
137
116
DEXTRAN T500 PURIFIED CELLS
TABLE 3. CHFMOTAXIS Polymorphonuclear leukocytes, processed as In the ferr ¡cytochrome C reduction assay, were incubated with post gamma globulin and introduced into the upper part of the Boyden chamber. Results were obtained by reading 10 fields on the filter in duplicate. Results are also expressed as percentage of recovery from control.
In chemotaxis assay the slow form at 10 ng/ml had an inhibitory effect, particularly in orientated migration {2% B.S.A. + 20% zymosan activated human plasma) (Table III). As previously observed during superoxyde anion release assays, the presence of 3 3 8 mononuclear cells beside PMNL led to an opposite action of the slow post gamma form which became activator.
DISCUSSION.
In biological fluids the slow and the fast forms of post gamma globulin are the most commun. It is clear from our results that the slow and fast forms have a dual effect on various polymorphonuclear leukocytes functions. An inhibitory effect on superoxyde anion release, phagocytosis and chemotaxis was associated with low concentrations of the slow post gamma globulin form. Conversely high concentrations (beyond 2 5 ng/ml) increased phagocytic activities. The activation effects produced by the fast form, which can be elevated, can be explained by contact phenomena either simple or due to the presence of specific receptors on the polymorphonuclear leukocytes membrane.
recovery ( % )
*
CONCENTRATION ( 2-5 % CC>2 f o r E-64-c. that
3 hr a t
The c o n t r o l
37 °C in t h e p r e s e n c e or a b s e n c e o f 30 JIM o f
buffer
c o n s i s t e d o f t h e same s o l u t i o n
1 mM o f EGTA was s u b s t i t u t e d f o r c a l c i u m On e l e c t r o n m i c r o s c o p y ,
clearly
25 jig/ml
5 mM of g l u c o s e and 0 . 5 mM of c y c l o h e x i m i d e under 95 %
lost their
preservation
t h e muscles
Z-band s t r u c t u r e
in c o n t r o l m u s c l e .
except
ion.
incubated with calcium
in s t r o n g c o n t r a s t
to
ion
their
The f a c t t h a t t h e Z-band was
removed when i n c u b a t e d w i t h c a l c i u m - i o n o p h o r e
in t h e p r e s e n c e
of
c a l c i u m i o n s t r o n g l y s u g g e s t e d t h a t CANP in muscle c e l l p l a y e d an initial
role
in d e g r a d a t i o n o f t h e m y o f i b r i l s .
The d e c r e a s e
in a -
actinin content
in muscle was i n v e s t i g a t e d u s i n g SDS g e l
electrophoresis
s i n c e t h e p r o t e i n was an i m p o r t a n t c o n s t i t u e n t
Z-band and was h a r d l y h y d r o l y z e d by p r o t e i n a s e s . was e x t r a c t e d from m y o f i b r i l s
When a - a c t i n i n
and f r a c t i o n a t e d by ammonium
sulfate,
t h e r e was a marked d e c r e a s e o f a - a c t i n i n
in c a l c i u m t r e a t e d
The r e l e a s e
into the incubation
o f a - a c t i n i n from m y o f i b r i l s
was o b s e r v e d by means o f SDS g e l e l e c t r o p h o r e s i s diffusion.
The a d d i t i o n
r e l e a s e of
of
muscle. medium
and immuno
30 pM o f E - 6 4 - c c l e a r l y s u p p r e s s e d
a - a c t i n i n from t h e muscle c e l l
i n EDL ( T a b l e 1 ) .
d e g r a d a t i v e a c t i o n o f CANP to t h e m y o f i b r i l s
Table
1.
effect
in
CaCl 2 CaCl2, Muscles
Wet wei ght
and p r o b a b l y t o have a
from i n t a c t
± 0.012
0.098 ± 0.014
soleus
Released
muscle.
a-actinin
(pg/3h)
(g) 0.097
E-64-c
55.3
± 5.2
33.1 ± 7.4
(p < 0 . 0 0 1 )
(n=5) were i n c u b a t e d w i t h A23187 and C a C ^ in t h e
or a b s e n c e o f E - 6 4 - c
the
vivo.
The r e l e a s e o f a - a c t i n i n
Addit i o n s
the
These
d a t a s t r o n g l y s u g g e s t e d t h a t t h e i n h i b i t o r was a b l e t o s u p p r e s s beneficial
of
(30 pM) as d e s c r i b e d i n t h e t e x t .
a - a c t i n i n was d e t e r m i n e d e l e c t r o p h o r e t i c a l l y .
presence
The
(Mean ± S . D . )
released
636 Inhibitory An o r a l
activity
drug i s
as muscular intestine
and a b s o r p t i o n
desirable
dystrophy.
strongly
for
absorbability
The low a b s o r b a b i l i t y
soon a f t e r
also c,
E-64-c but
inhibit
activity
proteinases peptidase,
of
EST and E - 6 4 - c
hamsters. t a k e n out cysteine
of
proteinase After
e a c h compound, at
be
hydrolyzed
against
less
cathepsin
H.
While
extent
and c a t h e p s i n
of
B,
could
than t h a t of
proteinases
E-64-
even
at
non-cysteine
elastase,
amino-
D, and n o n - p r o t e o l y t i c
indicated that
H and
B and
EST
o f them showed any e f f e c t
chymotrypsin,
of c y s t e i n e
against
purified cathepsin
cathepsin
orally
dehydrogenase EST was a
thiol
and
strictly
in analogy with
an o r a l
blood,
the
a d m i n i s t e r e d EST and E - 6 4 - c
administration
heart,
quadriceps
indicated times.
proteinases,
(shown as c a t h e p s i n
the
of
E-64-c,
activity
femoris
and l i v e r
of c a t h e p s i n s
0.5
of
of
on
B and L a s a w h o l e
as a s u b s t r a t e .
hr a f t e r
of
were
the e f f e c t
was m e a s u r e d by t h e u s e
15)
in every t i s s u e
mostly within
the
Syrian
100 mg/kg body w e i g h t
As an i n d i c a t o r
B & L hereafter)
B & L activities
EST were i n h i b i t e d
on
a c t i v i t y were examined u s i n g normal
succinyl-L-Tyr-L-Met-naphthylamide cathepsin
of
E-64-c.
The e f f e c t s cysteine
activity
Neither
These r e s u l t s
but weaker than
to
activities
glyceraldehyde-3-phosphate
inhibitor
E-64-c,
was r e a d i l y
1 mM on t h e a c t i v i t y
collagenase
hexokinase.
slight
in terms
it
against
such as t r y p s i n ,
enzymes s u c h a s specific
2.
of
of
rat
was
since
enzymes w i t h f a r
a s shown i n F i g .
the concentration
from
such
absorbed.
inhibitory
these
of E-64-c
t o improve E - 6 4 - c
the ethylester
showed s t r o n g
less
disease
in vivo i n h i b i t i o n
p r o t e i n a s e were examined using
papain. papain,
We t r i e d
and f o u n d EST,
The i n v i t r o cysteine
its
as a prodrug o f E - 6 4 - c
into E-64-c
EST
long term use in c h r o n i c
suggested that
when i t was g i v e n o r a l l y . applicable
of
hamsters
of
The treated
administration.
with
637
Inhibitor C o n c e n t r a t i o n (M) Fig. 2. Inhibitory activities of EST and E-64-c against cysteine proteinases in vitro. After preincubation for 5 min with EST (filled) or E-64-c (open), the remaining activities of cathepsin B ( # , O )# cathepsin H (A . A
) o r papain ( • , Q
) was measured according to the method
of Barrett et al. 9) using Z-Phe-Arg-NMec, Arg-NMec and Z-Phe-ArgNMec as substrates, respectively. The inhibition continued at least for 3 hr and then disappeared gradually within 24 hr (Fig. 3). liver and slightest in heart.
The inhibition was highest in
It seems likely that the intensities
of inhibitory activity corresponded intimately with the amounts of drug in tissue cells.
On the contrary, orally administered E-64-c
was far less effective than EST.
Neither EST nor E-64-c had any
effect on cathepsin D and acid phosphatase activity in the heart, skeletal muscle and liver of hamsters.
These results suggest that
both inhibitors affect only cysteine proteinases in vivo as well as in vitro.
638
Fig. 3. Changes in cathepsin B&L activity in hamsters after oral administration of EST or E-64-c. Syrian hamsters were orally given 100 mg/kg body weight of EST (
) or E-64-c(
).
At the time indicated, hamsters (n=4) were
sacrificed and heart(O)» removed.
quadriceps femoris ( # ) and l i v e r ( A ) were
The cathepsin B&L activities in these homogenates were
measured by the use of succinyl-L-Tyr-L-Met-NA as a substrate 15). EST was found as E-64-c in the plasma of all animals when it was given orally and was not detected as original form ( 1000. On the other hand, equ. 3 i s v a l i d only i f the substrate present in the b i o l o g i cal medium does not s i g n i f i c a n t l y compete with the i n h i b i t o r for the binding of the
enzyme,
= Michaelis
i.e.
i f [S°] «
constant).
If
Km
([S°] = in
significant
vivo
substrate
concentration,
competition occurs, the delay time of
i n h i b i t i o n in presence of substrate, d ' ( t ) i s given by eq. 4 :
d 1 (t)
5(1 + [S°]/K )/k [I°] m ass
(4)
Extent of substrate h y d r o l y s i s during the delay time of i n h i b i t i o n In v i v o ,
proteinases
act in a medium containing both substrate(s)
and i n h i b i -
t o r ( s ) . Even i f the substrate does not s i g n i f i c a n t l y compete with the i n h i b i t o r for
the binding of enzyme, there w i l l
be some substrate h y d r o l y s i s during the
delay time of i n h i b i t i o n . The f r a c t i o n of substrate turned over during d(t)
is
given by :
[ P°° ] _ k cat [S°]1 L
or
where :
[ ET
d(t) (5)
K m
[P Q°] = [S°]
k
cat Km
[E"], '
1 \
s
(6)
s
[ P 0 0 ] = concentration of product formed (substrate turned over) during d(t) [S°l
= in vivo substrate concentration
[E°]
= in vivo proteinase concentration
k
=
cat
K m
c a t a l y t i c constant (turnover number)
= Michaelis constant
The above equations
assume that [ P ° ° ] i s
neglectible with respect
t h i s assumption i s not made, the r e l a t i o n s h i p s become too complex.
to
tS°].
If
696 The fraction of substrate hydrolyzed during the delay time of inhibition is thus positively and
correlated
with
the
efficiency
of
the
enzyme-substrate
interaction
of the enzyme-inhibitor
negatively correlated with the efficiency
interac-
tion.
Pseudoirreversible behavior of reversible
inhibitors
In
is
case
of
equilibrium
reversible
inhibition,
there
mixture
enzyme
inhibitor.
concentrations
of
of
and
inhibitor
and
if [ 1 ] / [ E ° ] > 10 and [I°]/K i present
enzyme
present
in
an
It
may
be
is only 0 . 1 % or
shown
that
less free
enzyme
(e.g. if [ E°] = 0 . 1 PM, [ I°] =1JJM and K..=lnM). Under these
at equilibrium
conditions,
there
free
Let [I°] and [E°] be the in vivo
proteinase.
1000,
>
0
always
reversible
inhibitors
yiel
almost
irreversible
inhibition
(i.e.
there is virtually no free enzyme present in a mixture of enzyme and inhibitor). From
[I°l/K. i
>.
1000 we
of complex formation »
aet -
k
ass
[ 1 ° ] » k.. , i.e. first-order diss
first-order
rate
constant
rate constant of complex dissociation.
The
delay time of inhibition concept may therefore be applied here. On
the
other
hand,
inhibitor
complex
fulfilled
: [S°] /K
significant
is
unlikely [I°J
>
substrate-induced since
the
dissociation
following
condition
of
the
would
enzyme-
have to be
1000.
Possible Biological Functions of Proteinase
Inhibitors
Prevention of proteolysis
A number of proteinase living
organisms
inhibitors must have been designed by Nature to protect
against
the
deleterious
action
of
sites where they do not normally play a physiological lar release of
lysosomal
proteinases).
mise the breakdown of endogenous therefore
be
"fast-acting"
function
Such inhibitors must
substrates
irreversible
proteinases
or
liberated
(e.g.
at
intravascu-
act so as to mini-
by the released enzymes. They must pseudoirreversible
inhibitors
i.e.
their delay time of inhibition should be "as low as possible". It is difficult to predict how low d(t)
should be in order to allow proteinase
inhibitors to efficiently prevent proteolysis in vivo because d(t) is not the
697 sole factor that governs the extent of substrate tion process {sec eq. 5), i.e. if [E°] . cant
substrate hydrolysis even
if d(t)
with extreme values of [E°] and k compatible
with
significant
k
cat
hydrolysis
is
during the inhibi-
"large" there may be signifi-
is "small". Calculations based on eq. 5
,/K
Cat
/Km
III
prevention
suggest that the highest of
proteolysis
is
limit of d(t)
about
1 second
if
[E°] and k
./K are unknow. For instance, if [E°] is a high as 1 pM and cat m c i ./K as large as 10 M~ s , no more than 2 t of substrate will be hydrolyzed 3 cat m if d(t) = 1 second.
k
It is however may
still
[E°]. k 1 nM, k
noteworthy that an inhibitor with a d(t) as high as
significantly ,/K
Cat
= 10"
m
4
i
i
during
1 pM,
k
the
1000 seconds
inhibition
,/K = 10 in
2
Cat
process
M'V
1
if
or [ E°] =
M s .
if d(t) > 1 second, [E°] and k c a t / K m must be
the inhibitor
serves to prevent or to
regulate
proteolysi s.
Extracellular regulation of proteolysis
Proteolysis
plays
important
process
obviously
protein
proteinase
requires
physiological efficient
inhibitors.
As
a
functions
in living organisms.
This
regulation
which may be brought about
matter
fact,
of
inhibitors
are
by
usually
detected at sites where proteinases occur. The mechanisms by which they regulate proteolysis may depend upon the site where proteolysis takes place, i.e. extra or intracellular. Extracellular prothrombin, action the
on
proteinases occur mostly as inactive precursors or zymogens plasminogen).
their
target
coagulation
target subtile
Upon activation, these
zymogens must have a
substrate. For instance, when thrombin
cascade,
it
must
fibrinogen
but
not
all.
mechanism
once
a certain
cleave Its
some molecules
action
must
of
therefore
amount of fibrinogen
is generated
its be
(e.g.
limited by
physiological
depressed
has been converted
by
a
into
fibrin. Was feel that irreversible or pseudoirreversible proteinase inhibitors may serve such regulatory breakdown
during
regulatory
role
functions. the of
delay
Eq. 5 and 6 which describe the time
inhibitors
of
inhibition,
althoug
they
may
are
be based
extent used on
to the
of substrate discuss
the
assumption
that[P < » ] « [S°]. In the preceding paragraph we have already arrived at the
698 tentative conclusion that for
d(t) > 1 second, substantial substrate hydrolysis
may occur if [E°] and k _ . / K are large enough. In other words, to be a possible cat m candidate
for
a
regulatory
function,
i.e.
to
allow
substantial
substrate
breakdown before full inhibition, a protein proteinase inhibitor should have two characteristics
(i) it should behave as an irreversible or a pseudoirreversible
inhibitor (ii) its d(t) should be greater than one second. The magnitude of d(t) depends of course upon the values of tE°] and kcat,/K m which must be determined to ascertain this regulatory function. Since
extracellular
regulation
of
proteolysis
and
prevention
of
proteolysis
proceed through identical mechanisms, it is not unlikely that the same inhibitor acts as a protecting agent against a given proteinase and regulates the activity of another proteinase.
Such a dual function would provide a biological meaning
to the poor specificity of proteinase inhibitors. Eq. 6 shows that regulation of proteolysis may take place [I°]and k a s s .
Large variations
in the concentration
through variations in
of some
proteinase
inhi-
bitors have been detected in inflammation. These variations might serve pecular regulatory functions. Alternatively, k g s s may undergo variations due to changes in
the
physical-chemical
properties
of
the
medium
in which
the
inhibitory
reaction takes place or to binding of the inhibitor or the enzyme with soluble or insoluble components
(cofactors) present in the biological medium. In parti-
cular, the product of substrate hydrolysis might increase k efficient feed-back control of the proteolytic process.
ass
so providing an
Intracellular regulation of proteolysis Intracellular proteinases control.
proteinases
which might
occur
only
serve digestive functions,
Similarly,
structures
probably
as
active
probably
enzymes.
Lysosomal
do not require
proteolytic enzymes bound to membranes or other
not require
regulation
special cellular
by inhibitors. Proteolysis by soluble
cytoplasmic proteinases must however be controlled by proteinase inhibitors. We have imagined that such intracellular inhibitors might act both as "amplifiers" and depressants of proteinase activity. The
possible
relationsh ip proteinase
amplifier between
inhibitor
role
total
of
and
proteinase free
inhibitors
proteinase
in
is
presence
suggested of
a
by
the
reversible
(see the figure). For the sake of clarity we have chosen
the following example : the total inhibitor concentration is constant and equal
699 to 100 nM, the total proteinase concentration varies from 0 to 200 nM and the K^ varies from 1000 nM to 1 nM. The f i g u r e shows that as K^ decreases, the curves r e l a t i n g free to total enzyme concentration become more and more concaved. The concentration
of
variations
the
amplifier
of
free total
enzyme
becomes
therefore
enzyme concentration,
more
i.e.
and more
sensitive
the i n h i b i t o r
of proteinase a c t i v i t y and i t s amplifying capacity
is
acts
to
as an
related
to
i t s K.j value.
FREE
PROTEINASE
( nM )
UJ
Figure Speculative role of proteinase i n h i b i t o r s as amplifiers of proteinase a c t i v i t y . The figure shows theoretical curves r e l a t i n g free to total proteinase concentration in the absence and in the presence of a constant 100 nM concentration of a r e v e r s i b l e i n h i b i t o r whose K. varies from 1000 mM to 1 nM. Note that the abscissa and the ordinate scales are not i d e n t i c a l . The arrow indicates the equivalence between enzyme and i n h i b i t o r i . e . total enzyme concentration = total i n h i b i t o r concentration.
700 How can such a theoretical cell
synthesizes
observation have a practical relevance ? Suppose the
an i n h i b i t o r
with a 1 nN t
t r a t i o n of 100 nM. Suppose that at by
the
cell,
say
ca.
nM.
2
up to an i n t r a c e l l u l a r
concen-
time t-| "very l i t t l e " proteinase i s required
To
have
2 nM free
proteinase,
the
cell
will
synthesize 70 nM enzyme (68 nM w i l l be bound to the i n h i b i t o r ) . Suppose now that at
time
t^
20-times more proteinase
is
rapidly
required
(ca.
40 nM).To have
40 nM free proteinase, the cell w i l l just have to synthesize the same amount of enzyme as before, Thus,
the
available
cell
i.e.
is
the total
able
proteinase
to
concentration w i l l
rapidly
without
satisfy
having
to
the
just
need of
increase
its
have to be 140 nM. large
increases
rate of proteinase
of bio-
synthesis. The amplification i s most pronounced near the equivalence of enzyme and i n h i b i t o r . As already pointed out, the magnitude of the amplification depends upon K^. If
we
use
a K^
of
concentration w i l l
0.1
nM
in
the
preceding
example,
the
free
proteinase
increase by a factor of 175 i f the total proteinase concen-
t r a t i o n increases from 70 to 140 nM ! As mentioned in a preceding section, the potency
of a r e v e r s i b l e i n h i b i t o r
is
best defined by the dimensionless number [I°]/K... In our foregoing examples [I°] was 100 nM and K i was
1 nM or 0.1 nM i . e . [ 1 ° w a s
therefore the capacity to increase the concentration
Biosynthesis depress
of
the amplification power just by increasing
of the i n h i b i t o r .
course required to allow e f f i c i e n t inhibitor
proteinase
Concomitant synthesis of proteinase i s of
amplification.
without
activity :
function of i n t r a c e l l u l a r
100 or 1000. The cell has
this
concomitant might
be
synthesis the
peoteinase i n h i b i t o r s .
of
proteinase
complementary
will
regulatory
I f the enzyme-inhibitor system
i s in "amplification conditions"
([E°]= ! [I 0 1 ; [I°]
small
r e s u l t in a large decrease of enzyme a c t i v i t y .
increase in i n h i b i t o r w i l l
100), i t i s obvious that a
Hence, rapid enzyme i n a c t i v a t i o n may be achieved.
Proteinase i n h i b i t o r s
as proteinase r e s e r v o i r s
In normal conditions most e x t r a c e l l u l a r proteinases occur as inactive precursors (e.g.
prothrombin,
plasminogen).
p r o t e o l y s i s to "release"
These "proteinase r e s e r v o i r s "
require
limited
active enzyme. P r o t e i n a s e - i n h i b i t o r complexes might be
considered as alternative types of proteinase r e s e r v o i r s . Suppose a cell has to synthesize a proteinase that plays an e x t r a c e l l u l a r function and that should be
701 excreted
in
an
inactive
form.
If
the
cell
concomitantly
synthesizes
a
t i g h t - b i n d i n g i n h i b i t o r of the proteinase, an enzyme-inhibitor complex will form intracellularly
and t h i s
complex may be excreted and transported
at
specific
s i t e s . Free proteinase may be released e x t r a c e l l u l a r l y by different mechanisms. Firstly,
active proteinase may spontaneously escape the complex. This i s often
observed
in
Secondly,
vitro
and the
phenomenon has been named "temporary
i f the p r o t e i n a s e - i n h i b i t o r complex i s r e v e r s i b l e ,
extracellularly
inhibition".
i t may dissociate
under a variety of conditions. For instance, i f the e x t r a c e l l u -
lar concentration of complex i s lower than the i n t r a c e l l u l a r one, d i l u t i o n - i n d u ced
dissociation
(extracellular)
of
> IC
the
complex
occurs.
(intracellular).
Dissociation
Besides,
t i g h t - b i n d i n g target substrate, the latter w i l l the complex. A l t e r n a t i v e l y ,
the i n h i b i t o r
ligands present in the e x t r a c e l l u l a r
if
also
occurs
if
K^
the proteinase encounters a
also favor the d i s s o c i a t i o n of
may react with soluble or
insoluble
space and t h i s may also r e s u l t in complex
dissociation. The advantage of the above hypothetical
reservoir
systems over the
classical
zymogen r e s e r v o i r s l i e s in the fact that they do not require proteolytic
activa-
t i o n . A cascade activation system in which each step i s catalyzed by a p r o t e i nase generated from a zymogen one step before, may require an i n i t i a l
proteinase
which
It
does
unlikely
not
that
have this
to
be generated
initial
by
proteinase
proteolytic is
activation.
"reservoired"
by
a
is
not
proteinase
inhibitor.
Prediction of In Vivo Potency of Drugs The delay time of
inhibiton
of and the pseudoirreversible i n h i b i t i o n
concepts
may help f i n d i n g the therapeutic concentration of drugs or at least the i n i t i a l dose of drug to be administred in the course of animal
studies.
I f the drug i s an i r r e v e r s i b l e i n h i b i t o r whose k g s s i s known we may rewrite eq. 3 in the following way :
[drug] = 5/k
abb
. d(t)
(7)
702 Since d(t) should be higher or equal to 1 second i f [E°], k c a t and Km are unknown, i t follows that :
[drug] >
5. k
-1
. s
(8)
I f the druq i s a r e v e r s i b l e i n h i b i t o r with known K. and k values, conditions ° i ass for pseudoirreversible i n h i b i t i o n y i e l d the minimal concentration of drug to be used :
[ drug] >
1000
K
(9)
i
I t has then to be checked whether t h i s concentration i s large enough to s a t i s f y eq. 8. I f not, higher amounts must be used. The above r e l a t i o n s h i p s
do of course not take into account the p a r t i t i o n ,
the
elimination or the destruction of the drug in the organism. They may however be helpful
to the pharmacologist
for f i n d i n g
the f i r s t dose of drug to be admi-
nistrated to an animal or to the chemist for knowing the threshold of the kinet i c constants of a potential drug.
Discussion Our
previous
studies
were exclusively
focused on the
"safety
guard"
role
of
proteinase i n h i b i t o r s (1,2). I t i s obvious that t h i s cannot be the sole function of i n h i b i t o r s and therefore inhibitors.
: physiological requires
p r o t e o l y s i s must take place within certain l i m i t s
subtle
regulation
which
is
likely
to be operated by
None of the two regulatory mechanisms we propose r e s t s on experi-
mental data : they are based on sound kinetic knowledge and on some imagination. The
same
is
true
for
devoted to experimental
the proteinase r e s e r v o i r work on cysteine
concept. We f e l t
proteinases
that
and i n h i b i t o r s
a book
may have
some space l e f t for speculation. We also feel that some of the ideas put forward in t h i s paper may be amenable to experimentation. Our a r t i c l e might therefore be a b i t more than useless fantasy.
703 References 1. Bieth, J.G. 1980. B u l l . Europ. Physiopath. Resp. Jj> ( s u p p l . ) , 183. 2. Bieth, J.G. 1984. Biochem. Med. 32, 387.
A CURVE-FITTING APPROACH TO THE DETERMINATION OF KINETIC CONSTANTS OF PROTEINASE
INHIBITORS.
Werner Machleidt Institut für Physiologische Chemie, Physikalische Biochemie und der Universität München, Goethestr. 33, D-8000 München 2, FRG.
Zellbiologie
Irmgard Machleidt, Werner Mliller-Esterl Abteilung für Klinische Chemie und Klinische Biochemie in der Chirurgischen Klinik Innenstadt der Universität München, Nussbaumstr. 20, D-8000 München 2, FRG. Joze Brzin, Matjac Kotnik, Tatjana Popovic, Vito Turk Department of Biochemistry, J. Stefan Institute, Jamova 39, YU-61000 Ljubljana, Yugoslavia.
Introduction
Due to progress in peptide synthesis, a great substrates
has
become
available
variety
of
fluorogenic
sensitivity of fluorimetric assays enables the detection of proteinases
minute
amounts
concentrations,
it
virtually
turnover
of
substrate
low
complex
formation
and is
inhibitor
concentration.
If
the
to
information
stoichioinetry
of
defined, only one of the latter concentrations has to be
known. This is the rationale of our curve-fitting approach to the
determination
kinetic constants from each single inhibition assay. Our attempts to realize
this approach led to the development of an on-line computer system metric
enzyme
of
for
fluori-
assays which proved equally valuable for all kinds of conventio-
nal kinetic measurements. This preliminary report tries to evaluate tial
enough
to calculate the association and dissociation rate constants for a given
pair of total enzyme
of
pro-
constant. According to theory, a single product concen-
tration curve recorded in such an experiment should contain all the needed
the
the approach to enzyme-inhibitor equilibrium can be follo-
wed in an continuous assay keeping the consider
of
and proteinase inhibitors. Moreover, new procedures for the experi-
mental determination of kinetic constants can be envisaged. Selecting per
peptide
for specific assays of proteinases. The high
the
poten-
the new method using computer simulations and experimental data obtai-
ned with cysteine proteinases and their
inhibitors.
Cysteine Proteinases and their Inhibitors © 1986 Walter de Gruyter & Co., Berlin • New York - Printed in Germany
706 Materials and Methods
Experimental Enzymes
and inhibitors, Human cathepsin L (M. Kotnik et al., this volume) and H
(T. Popovic, unpublished), chicken cystatin (1) and kininogen
(2,3)
human
high-Mr
and
low-Mr
were isolated and characterized as described. Approximate con-
centrations of the enzymes were calculated from their absorbance at 280 nm, inhibitors
were
the
precisely quantitated by amino-acid analysis. As all the algo-
rithms used for
the
precise
concentration,
enzyme
calculation
of no
kinetic
constants
were
independent
of
attempt was made in this study to determine
the enzyme concentration by active-site titration. Instrumentation.
Two
different
systems were used for the fluorimetric assays.
One system was a filter fluorimeter FFM 32 from with
Labotron
(D-8192,
Geretsried)
its analog output connected to a computing integrator SP 4100 from Spectra
Physics (D-6100 Darmstadt). This integrator designed for cations
comprises
printer-plotter. Data were stored with drive.
chromatography
appli-
a small computer programmable in BASIC and a high-resolution a
Kerr
Minifile
4100
D
floppy
disk
The filter fluorimeter contains a mercury lamp coated with a fluorophore
emitting a broad band around 360 nm which is isolated by a filter.
The
emitted
light
is passed through a < 415 nm cut-off filter. Assays were performed in 10 x
10 mm
rectangular
cells
placed
in
a
self-constructed
thermostatted
cell-
holder. The second system used was a spectrofluorimeter Eching)
SFM
25
from
Kontron
computer equipped with Interface I and two microdrives. Graphics were on
a
(D-8057
with its RS 232 C digital output connected to a Sinclair Spectrum+ home
monochrome
displayed
video monitor and copied from screen using a JUKI 5520 matrix
printer connected via an extra parallel interface. 10 x 10 mm rectangular were
cells
used in a thermostatted cell holder. Data werde collected at inonochromator
settings of 380 nm for excitation and 460 strings
nm
for
emisssion.
Output
of
data
was controlled either by the built-in time function of the spectrofluo-
rimeter or externally by the Sinclair computer. Assays.
The
fluorimeter
was
calibrated with 200 nM aminomethylcoumarin
dard. Cathepsin L was assayed with cathepsin
H
with
the
fluorogenic
substrate
Arg-NMec essentially as described (4). Typically, substrates
were 10 /uM in a total volume of 3.0 ml 0.3 M sodium acetate buffer catheps in
5.5
for 1.5
mM EDTA and 0.05 % BRIJ-35. After addition of 0.03 ml of freshly prepared 10
mM
solution
and
pH
0.1 M phosphat© buffer pH 6.5 for cathspsin H, containing
DTE
L
stan-
Z-Phe-Arg-NMec,
and
0.03
ml of substrate solution (1 mM in dimethyl
0.03 ml of enzyme solution (freshly diluted from more concentrated tions simultaneously frozen in small aliquots
sulfoxide), stock
solu-
to be used only once) was added
707 and
mixed
by
reverting
the cell sealed with parafilm. Activation by 1 mM DTE
(final concentration) was allowed to proceed until a constant slope ved
added in approx. 0.03 ml and mixed. When working inhibitors
was
obser-
after 10-20 min). Then the freshly diluted inhibitor solution was
(usually
(kininogen),
the
enzymes
were
with
thiol-sensitive
preincubated
enzyme solution diluted to 0.1 mM DTE in the assay. The
in
assays
protein
1 mM DTE and the were
performed
at 30 °C for cathepsin H and 25 °C for cathepsin L.
Theory The interaction of enzyme and inhibitor is described by the
differential
equa-
tion d(E)/dt = kdiss (EI) - kass (E) (I) where: kass
(1)
= association rate constant
kdiss = dissociation rate constant (E)
= concentration of free enzyme
(I)
= concentration of free
(EI)
= concentration of enzyme-inhibitor
inhibitor complex
Replacing (EI) = (Et) - (E) and (I) = (It) - (EI) = (It) - (Et) + (E) and rearranging yields d(E)/dt = -kass (E) 2 - (kdiss + kass (It) - kass (Et)) (E) - kdiss (Et) where: (Et) (It) As
(2)
= total enzyme concentration = total inhibitor concentration
long as the fraction of substrate turned over is small compared to the total
substrate concentration, the catalytic constant, kcat,
of
the
enzyme
may
be
regarded virtually constant, hence v = kcat (E) or (E) = v/kcat
(3)
and vo = kcat (Et) where: v
or
(Et) = vo/kcat
(4)
= reaction rate at enzyme concentration (E)
vo
= initial reaction rate without inhibitor when (E) = (Et)
Substituting (3) and (4) into (2) yields dv/dt = a v 2 + b v + c
(5)
where:
(6)
a
= -kass/kcat
b
=
kass ((Et) - (It)) - kdiss
(7)
c
=
kdiss vo
(8)
Entering (It) and vo as known parameters, kass, kdiss and (Et) can
be
calcula-
ted solving the equations (4)-(8), and Ki = kdiss/kass. If (Et) is entered as a known parameter, (It) can be calculated. Both (It) can be obtained when kcat is known.
(Et)
and
708 How
can the coefficients a, b, and c be determined from experimental data?
integrated form of equ. (5) cannot be solved directly and has to the
experimentally
determined
product
concentrations
by
fitted
to
iterative computer
analysis (5). Following an opposite approach, we tried to fit the
be
The
equation
(5)
to
dv/dt and v values derived from the experimental data. Once the equation is
fitted, the kinetic constants can be calculated directly from
the
coefficients
a, b, and c. The problem was to find a way to derive dv/dt as a function of v from the fluorescence
intensities
measured
in
the
fluorirneter.
noisy
For this purpose the
fluorimeter was connected on-line to a computer collecting and storing the (5-20
samples/min)
for
subsequent
tails). After calibration with a converted
into
calculations
standard,
the
fluorescence
intensities
the
are
product concentrations prior to storage. The evaluation of sto-
red data was performed off-line using visualization by high-resolution on
data
(see Methods section for de-
screen.
Two
different
graphics
methods were used for the calculation of v and
dv/dt from the stored product concentrations: Moving-tangent.
Tangents
to
the product concentration curve are calculated by
least-squares fitting over a moving window of 5-100 data samples. rates
v
as
a
function
tangents. The rate curve is already displayed during on-line and
helps
rate,
curve
are
collection
coefficients
graphic
from
which
the
vo, and the equilibrium rate of the inhibited reaction, vi, are calculated
correla-
can be used to control the validity of the assignments
1 b). This method of slope determination is more usual
sample
selected
calculated by least-squares fitting of straight lines. The tion
reaction
to follow the time course of inhibition (Fig. 1 a). According to the
rate curve those portions of the product intitial
The
of time are obtained as the slopes of the consecutive
evaluation
of
recorder
sensitive
and
(Fig.
accurate
then
tracings. The second derivative of the
product concentration curve, dv/dt, is obtained by moving a
tangent
along
the
rate curve (Fig. 2). Polynomial curve-fitting. A polynomial the
product
concentrations,
(mostly with an order of 5) is fitted
(P) = c^ n" + c^ n
+ c^ n
+ c^ n
to
+ c-J n + CQ
((P) = product concentration, n = number of data sample), v is calculated as 4 3 2 the first derivative, v = 5 Cj n H e , n t 3 Cj n + 2 c^ n + c^, and dv/dt as the second derivative, dv/dt = 20 Cg n the
quadratic
equation
(5)
to
+ 12 c^ n^ + 6 c^ n + 2
yields the coefficients a, b and c. The individual stages of cess
cFitting
the obtained pairs of data v(n) and the
dv/dt(n)
fitting
pro-
are visualized on the screen facilitating the optimal selection of fitting
ranges (Fig. 3).
709
(a)
VO=
1 r [Pl(nM) 500
IS
11
•
ns=
20
590
d'2015'"''
:
f
..
/
X \
\
/).. \
/ +Inhibitor
600
60
time(min)
120
[P](nM) | ,-500
(b)
da0s c
/
/ .f
HP]
70 455
10. o 5 0.4-7'
110 54 0 J
I
I 60
i
time(min)
0.9-599 0,9958 500 1S0
Fig. 1. Screen copies of the graphic representation of a t y p i c a l i n h i b i t i o n experiment as displayed during o n - l i n e data c o l l e c t i o n , (a) shows the product concentration curve, ( P ) , and the rate curve, d(P)/dt = v, obtained by the moving-tangent method over a window of 20 data samples (sampling rate 5/min). The rate curve shows the a c t i v a t i o n of the enzyme (cathepsin L) by DTE u n t i l a constant i n i t i a l r a t e , vo, i s reached. After addition of the i n h i b i t o r (low-Mr k i n i n o g e n ) , the reaction rate approaches the constant value corresponding to enzyme-inhibitor e q u i l i b r i u m , v i . The rate curve reacts s e n s i t i v e l y to irregular noise which i s not detectable in the product curve. By inspection of the rate curve two portions of the product curve were selected for precise calculat i o n of vo and v i by l e a s t - s q u a r e s f i t t i n g of s t r a i g h t l i n e s ( b ) . A vo of 10.35 nM rnin-1 was calculated from samples 70-110 ( c o r r e l a t i o n c o e f f i c i e n t 0.9999) and a vi of 0.47 nM min-1 from samples 455-540 ( c o r r e l a t i o n c o e f f i c i e n t 0.9958).
710
[P] (n M) bflfl d s
w
i t i l £L
10
n 5 1 56 56
/ i 3
ns= 100
10 950 94-5 94-5
10
94-5
/ 200
0
1000
50 time(min) dv/dt
0
100
Fig. 2. M o v i n g - t a n g e n t fit of a simulated inhibition e x p e r i m e n t w i t h o u t noise (see Table 1 for nominal constants and c o n c e n t r a t i o n s ) . {P) = Product concentration curve, v = r e a c t i o n rate c a l c u l a t e d by fitting a tangent to (P) over a moving w i n d o w of 100 data samples (represented as v/vo on the ordinate), dv/dt(v) = second d e r i v a t i v e of the product curve as a function of v o b t a i n e d by fitting a tangent to the rate curve over a moving w i n d o w of 10 data samples (represented on the abscissa).
Results
Fitting of simulated data The
algorithms
described
above
were
tested
concentration data c a l c u l a t e d according to equation kdiss,
(1) for given sets of
data
without noise, the results of both the tangent and the values
(Table
1),
that
algorithms are c o r r e c t . For precise fitting of the p o l y n o m i a l ,
individual
coefficients
of the polynomial
the
and the c o r r e l a t i o n
are calculated by the computer p r o g r a m , a graphic r e p r e s e n t a t i o n of rences
between
experimental
and
fitted
reacts
of
coefficient the
diffe-
data provides the best c r i t e r i o n for
v e r y sensitively to m i s f i t t e d p o l y n o m i a l s , and w i t h some
it is possible to select the best portion of the primary polynomial lation of its first and second d e r i v a t i v e s .
the
however,
errors
selection of the fitting range (cf. Fig. 3). The parabola described by (5)
kass,
polynomial
indicating
only a limited portion of the product curve can be used. Though the
product
concentrations.
approach were close to the theoretical derived
simulated
(Et) and (It) and adding different percentages of random noise (0.1, 0.5
and 1.0 %) to the c a l c u l a t e d Fitting
using
equation experience
for
calcu-
711 (a)
[P](nM> 600
d f =1000 v o =10
ns=10
fx = 1 0 0
5Si»2a,(
- /-x, ,dv/dt(v) / \cr*
r~r
/200
_J
0
[P](nM) 1 r- 6 0 0 dsinaa
\
^¿dv/dt(v)
-// / "¿0 0 I
n S = 10 6 S
1
100
d f =1000 V Q = 10 f X = 10 0 1 b00 «0 550
(b)
/P'l
V I
•1000
I 1_ 50 time(min) dv/dt
dP/*
I
I I 50 time(min) dv/dt
I
J
1000 I I 100
Fig. 3. Polynomial curve-fitting of simulated inhibition data without noise (nominal constants and concentrations see Table 1). (P 1 ) = product concentration calculated from the fitted polynomial, dP = percent deviation of original product concentration from the concentration calculated from the polynomial (full scale of the ordinate +/- 0.5 %), v = reaction rate calculated as the first derivative of the polynomial (represented as v/vo on the ordinate), dv/dt(v) = second derivate of the polynomial as a function of v (represented on the abscissa). A polynomial of an order of 5 was fitted to each second sample in the range between samples 1-1000 (a), and 1-600 (b). As can be seen from the dP curve, the smaller range (b) is fitted with higher precision. To improve the results, both ends of the fitting range of the polynomial are skipped when the parabola corresponding to equation (5) is fitted to the dv/dt and v data calculated from the polynomial. A range of sample 125-860 was used in (a) and of 60-550 in (b). The resulting kinetic constants and concentrations are shown in Table 1 as P(a) and P(b).
712
Table 1. Kinetic constants and concentrations determined from simulated inhibition experiments using the polynomial (P) and the moving-tangent (T) fit. P(a) and P(b) are the results from the fits shown in Fig. 3 a and b (10 samples/min, vo = 10 nM min-1, (It) = 200 pM) Noise
Inactivation
Ki
kass
kdiss
kcat
(Et)
per tnin
(pM)
(M'V1)
(s" 1 )
(s" 1 )
(pM)
20.,5
3..99 X 10 b
8.,19 X 10" b
3..81
43..8
P(a)
20..1
4..04 X 10
6
8,.13 X 10" 5
3..33
50 .1
P(b)
19..8
4..01 X 10 6
7..95 X lO" 5
3..30
50,.4
T
19..9
3,.94 X 10 6
7,.86 X 10" 5
3,.68
45,.3
P
19.,6
4..02 X 10
5
5
3..24
51,.4
T
18..3
4.00 X 10 6
7..34 X 10" 5
3,.17
52 .5
P
19.,9
4..01 X 10
6
5
3..32
50,.3
T
18..0
4,.25 X 10 6
7,.67 X 10" 5
2,.16
77 .0
P
20..8
3..87 X 10
6
5
4..61
36,.2
T
17..8
4,.24 X 10 6
7 .53 X 10" 5
4,.25
39 .2
P
17..0
4..25 X 10
6
7..23 X 10" 5
3,.69
45,.0
T
15..2
4,.54 X 10 6
6,.92 X 10" 5
4,.13
40 .3
P
14..9
4..51 X 10
6
6..73 X 10" 5
4,.10
40,.7
T
13..2
4,.81 X 10 6
6,.34 X lO' 5
4,.42
37 .7
P
13.,2
4..76 X 10
6
5 6..29 X 10"
4..54
36,.7
T
20..0
4..00 X 10 b
8..00 X 10" b
3..33
50,.0
0
0
0.2 %
0
0.5 %
0
1.0 %
0
0
0.3 %
0
0.6 %
0
0.9 %
Nominal values
Introduction
7..88 X 10'
7..95 X 10"
8..05 X 10"
of random noise up to 1 % (starting with a product concentration of
200 nM) decreases the accuracy of the results suitable
fitting
and
makes
the
selection
of
a
range for the polynomial more difficult. Whereas the greatest
deviation of kass is 6.25 % (1 % noise), the values of (Et) and more
Method
kcat
are
much
sensitive to noise showing deviations up to 50 %. The Ki values calculated
from kass and kdiss are within a range of +/- 10 % of the theoretical
values.
713
In order to test the effect of deviations from the "ideal" inhibition varying
degree
of
irreversible inactivation of the enzyme has been
in some of the simulated product concentration curves. As expected, fitting
procedures
resulted
in
However,
the
than
for
the
for an assumed irreversible enzyme inactivation of
even
by not more than 35 % from the nominal
and
Ki
values.
data
Building-up on the results of the computer simulations, the cedures
curve-
"ideal"
0.9 %/min (!), the kass and kdiss values deviated by not more than 21 %
Fitting of experimental
curve-fitting
pro-
were tested with experimental data collected in inhibition studies with
cysteine proteinases and their protein inhibitors. Though basically superior the
polynomial,
because
to
the moving-tangent approach was found to be hardly practicable
with experimental data from both the filter fluorimeter and meter,
a
apparently higher kass and lower Ki and kdiss
values. The selection of polynomials was more difficult simulations.
curve,
introduced
equation
(5)
the
spectrofluori-
is not fitted precisely enough to the scattering
data of the second derivative (Fig. 4). The scattering can be reduced
by
using
larger windows, but then the slope of the v(n) function is falsified by undue smoothing. Obviously, the inoving-tangent approach is very sensitive to irregu[P]lnM) 4-00
/
dsas
. 3n£ = IS 10 34-
13
16
t
•Adv/dt(v)
a .5 188 183 34.
MP]
t
fe'
0
• loo I
1
I
1_
i
i
40 time(min) dv/dt
i
i
i 80
Fig. 4. Curve-fitting of experimental data using the moving-tangent. The screen copy shows the inhibition of activated cathepsin L (vo = 7.3 nM rtiin-1) by 0.2 nM low-Mr kininogen. The rate curve, v, was derived from the product concentration curve, (P), using a window of 15 samples (2.5 samples/min had been collected). Due to irregular noise, the dv/dt data (derived over a window of 10 samples from the rate curve) are scattering. The kinetic constants obtained from the coefficients of equation (5) fitted to these scattering data are shown as assay 4 T of Table 2 in comparison with the results of polynomial fitting (4 P) of the same experiment.
714
5 00 : d 205-3 \
n S
=2 - 5 1
ji'dv/dt(v)
n = S . 71 f v = F=: 1 '200 [p'j 10 85 J} " • dP
V I
M
V'
,r
100
I
1
I
!
I
I
I
I
I
200
I
40 time(min) dv/dt
Fig. 5. Polynomial curve-fitting of experimental data. Activated cathepsin L (vo = 8.4 nM min-1) was inhibited by low-Mr kininogen (0.15 nM). Data samples 1-200 (80 min) were selected for fitting a polynomial of an order of 5. (P 1 ), product concentration calculated from the polynomial, v, rate curve obtained as the first derivate of the polynomial. The scattering points, dP, represent the percent deviation of experimental concentrations from the calculated ones (+/0.5 % full scale on the ordinate). The dv/dt(v) data were obtained as the second derivative of the polynomial between samples 10 and 86 (4 and 34 min reaction time). The kinetic constants and concentrations calculated from equation (5) are shown as assay 2 in Table 2. lar long-term fluctuations of the fluorimeter output. Fluctuations of this may
result
from
insufficient
stabilisation
of
temperature gradients, possibly also from dispersion by dust bubbles
type
the light source and/or from particles
or
gas
in the cell. The polynomial fit is able to smooth short irregular fluc-
tuations so that quite reasonable results have been obtained with this method. Table
2
summarizes the kinetic constants for the inhibition of human cathepsin
L by human low-Mr kininogen obtained from some experiments nomial
curve-fitting
(Fig.
evaluated
by
poly-
5). The apparent Ki and (Et) agree reasonably well
with the corresponding values obtained from a conventional Stedman plot. As be
seen
from
the
first
experiment
in
Table
2,
(Et)
sensitively to disturbance of product curves by long-term the
determination
can
and kcat react most fluctations,
whereas
of kass yields stable results over a wide range of different
fits. Table
3
compares
the
kinetic
low-Mr kininogen determined by previously
constants for the inhibition of cathepsin H by polynomial
for cathepsin H and°(CPI
curve-fitting
with
those
reported
(which is identical with low-Mr kininogen).
715
Table 2. Kinetic constants for the inhibition of human cathepsin L (50 pM) by different concentrations of human low-Mr kininogen as obtained by polynomial curve-fitting. Ki and (Et) determined from a Stedman plot of the same experiments is given for comparison. (It)
Ki
kass
kdiss
kcat
(Et)
no.
(pM)
(pM)
(M_1s_1)
(s _ 1 )
(s _ 1 )
(pM)
1
100
18..6
CO
Assay
X
10 b
8,.9
X
IQ" 4
7..4
20,.3
6
9,.5
X
10" 4
3,.2
44,.3
2
150
18,.9
5.0
X
10
3
150
19..2
5.2
X
10 6
10,.1
X
10" 4
3..9
41,.5
6
9 .5
X
10" 4
3,.1
39,.4
8,.2
X
10" 4
3..9
31,.8
4
3,.1
55,.5
4 P
200
19,.5
4.8
X
10
4 T
200
18..0
4.6
X
10 6
5
250
Stedman plot
19,.8
4.3
X
10
6
8,.5
X
10"
40.3
18.8
Table 3. Kinetic constants for the inhibition of human cathepsin H by low-Mr kininogen determined by curve-ftting (fit) and Stedman plot (plot). Inhibitor
Reference
Low-Mr kininogen
this work
Method
Ki
kass
kdiss
(nM)
(M'V1)
is" 1 )
fit
0.7
1.8 x 10 6
-3 1.3 x 10'
plot
0.9 2.0 x 10 6
-3 1.0 x 10'
1.5 x 10 6
-3 1.5 x 10'
Pagano & Engler
o(2CPI o( CPI High-Mr kininogen
0.9
Gounaris et al.
plot
1.1
this work
fit
1.0
plot
1.1
Pagano & Engler (11), Gounaris et al. (4).
Because
of
the
higher
concentration of inhibitor needed in these experiments
the approach to enzyme-inhibitor equilibrium is much faster sin fits.
L
and
than
with
cathep-
10-20 data samples per min had to be collected to obtain reasonable
716
Conclusions
Computer simulations showed that the product concentration curve versus
time)
of
(concentration
each single assay can provide good estimates of the rate con-
stants of association and dissociation of the complex between enzyme and sible
inhibitors.
The
outlined
curve-fitting
approach
should
applicable also to the special case of irreversible inhibition (kdiss irreversible
dissociation
of
an
cover all cases of biologically
rever-
be generally =
0)
or
enzyme-inhibitor complex (kass = 0) and thus
significant
interactions
between
proteinases
a
and
and their inhibitors (7). In its present stage, the alternative
to
curve-fitting
approach
offers
fast
the existing methods for the determination of kass (6,7,8) that
seems to be at least equally accurate. In contrast to the existing precise
knowledge
of
the
methods,
no
of the enzyme concentration is needed, which, in the case of
cysteine proteinases, may be difficult to determine and to bination
simple
the
reproduce.
By
com-
curve-fitting method with one of the established equili-
brium methods for the determination of Ki (9,10), all kinetic parameters can obtained
that
be
are needed to predict the biological significance of proteinase-
proteinase-inhibitor interactions (7, J.G. Bieth, this volume).
The
Ki
deter-
mined by equilibrium methods should be used to control the curve-fitting data. Concerning the determination of Ki and (Et) precision
and
accuracy
of
results
(or
(It))
by
curve-fitting,
collected raw data from the fluorimetric assay. As can be seeen from lations,
a
random
noise
as
high
the
is highly dependent on the quality of the the
simu-
as 1 % would not significantly falsify the
results. Actually the short-term random noise of both fluorirneters used in study
is
lasting (3-5 min) in relation to the rapidly decreasing slope tion
this
below 1 %. There are, however, irregular fluctuations which are longof
most
inhibi-
curves. These fluctuations, which cannot be smoothed by linear regression,
are reponsible for the fact that the moving-tangent
method
is
hardly
working
and the product curve has to be approximated by a polynomial. Due to limitations of the applied computers our preliminary restricted
to
studies
have
been
polynomials with an order below 7, and even these have been cal-
culated with limited accuracy. Presently we are implementing the programs on IBM-compatible
larger
personal
computer
offering
the
option
of
arithmethic processor in order to increase the quality of polynomial Essential
future
improvement
fitting.
seems possible regarding the long-term stability
of fluorirneters. Instruments fulfilling the requirements deduced from puter simulations
an
a special
would open hitherto
the
com-
unexploited approaches to fluorimetric
717 enzyme-inhibitor
kinetics.
For
example,
working
with
a defined amount of a
stable inhibitor, it should be possible to determine less than picomole of
an
active
amounts
proteinase from one or few assays, or kinetic constants could be
estimated using minute amounts of enzymes
and
inhibitors
highly
purified
by
modern HPLC techniques.
Acknowledgements We would like to thank Dr. J.G. Bieth for stimulating discussion of preceding
this
us prior to publication in this volume. The the
the
poster
paper, and Dr. M. Pagano for making available his manuscript to
Sonderforschungsbereich
investigations
were
supported
by
207 of the University of Munich {grant C-2) and by
the Bundesministerium fUr Forschung und Technologie FRG (grant PTB 8651).
References 1.
Turk, V., J. Brzin, M. Longer, A. Ritonja, M. Eropkin, U. Borchart, W. Machleidt. 1983. Hoppe-Seyler1s Z. Physiol. Chem. 364, 1497.
2.
Mlil ler-Esterl, W., G. Rauth, HG. Fritz, F. Lottspeich, A. Henschen. 1983. In: Kininogenases/Kal1ikrein VI (G.L. Haberland, J.W. Rohen, H. Fritz, F. Huber, eds.). Schattauer Verlag, Stuttgart, p. 3.
3.
Mlil ler-Esterl, W., M. Vohle-Tirnmermann, Biochem. Biophys. Acta 706, 145.
4.
Gounaris, A.D., M.A. Brown, A.J. Barrett. 1984. Biochem. J. 221_, 445.
5.
Laidler, K.J., The Chemical Kinetics of Enzyme Action. 1958. Oxford University Press, London.
6.
Nicklin, M.J.H., A.J. Barrett. 1984. Biochem. J. 223, 245.
7.
Bieth, J.G. 1984. Biochem. Medicine 32, 387.
8.
Baici, A., M. Gyger-Marazzi. 1982. Eur. J. Biochem. 229, 33.
9.
Henderson, P. 1972. Biochem. J. 127,
B.
Boos,
B.
321.
10. Green, W.R., M.T. Hakala. 1979. J. Biol. Chem. 254, 12104. 11. Pagano, M., R. Engler. 1984. FEBS Lett. J66, 62.
Dittmann.
1982.
INHIBITION OF CATHEPSINS B, H and L BY RAT THIOSTATIN, THE CIRCULATING a, CYSTEINE PROTEINASE INHIBITOR, AND BY AN ACTIVE FRAGMENT.
F. G a u t h i e r , T. M o r e a u , N. Gutraan, A. El M o u j a h e d , F. Esnard Laboratoire de Biochimie, Faculté de Médecine, 2 bis, Bd. Tonnellé, 37032
Tours (France).
Introduction
Rat thiostatin, p r e v i o u s l y
reported as a ^ cysteine proteinase inhibitor
oijCPI) (1) or Oj major acute phase globulin (a jMAP) (2) has been shown to be
recently
identical with a rat low Mr kininogen based on the nucleotide
sequence analysis (3). This low Mr kininogen seems to correspond to the so called
T kininogen (4,5) which liberates T kinin (ILE SER bradykinin) upon
•kininogenase treatment. Thiostatin therefore possesses at least two potential biological
functions and in addition, behaves as a major acute phase
reactant in the rat (1,2). No c l e a r relationship has been so far d e m o n strated b e t w e e n these different potential properties. In this work, the proteinase inhibitory
function of rat thiostatin and of one of its tryptic
fragments has been investigated using purified rat lysosomal cathepsins B, H and L.
Experimental
Rat
thiostatin was purified
as described
previously -(1) and its activity
was determined using commercial papain first titrated with the irreversible inhibitor compound E 64 (6). Rat liver cathepsin B, H and L were purified following Kirschke (7) procedure with some modifications (8). Specific activities were determined using A b b r e v i a t i o n s used : A r g - N M e c , arginine 4-methyl-7-coumarylamide. Z-ArgArg-NMec, benzyl oxycarbonyl-arginine-arginine-4-methy 1-7-coumarylamide. ZPhe-Arg-NMec, benzyl oxycarbony1-phenylalanine-arginine-4-methy1-7-couraarylamide. E 64, L-3-carboxypropionyl-leucylamido-(4-guanidino)butane.
Cysteine Proteinases and their Inhibitors © 1 9 8 6 Walter d e Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y
720 Z-Arg-Arg-NMec, Arg-NMec and Z-Phe-Arg-NMec for cathepsin B, H and L respectively after the enzymes were titrated with E 64 (6). Reaction rates were measured at pH 6.0 for cathepsin B and L and pH 6.8 for cathepsin H with a Jobin-Yvon .JY 3 D Spectrofluorimeter.
Values of the constants are
given in table 1.
k
cat (s _ 1 )
Cathepsin B (Z-Arg-Arg-NMec)
27.5
Cathepsin H (Arg-NMec)
0.93
Cathepsin L (Z-Phe-Arg-NMec)
15.9
Table 1 : Specificity
K M (mM)
k cat/ K M (10~3 M"1 s""1)
0.22
122
0.09
10.3
0.004
of rat liver cathepsins
3900 B, H and L.
Purified thiostatin (2 mg) was partially hydrolyzed by trypsin (58 ug) for 30 min at 37'C
then fractionated
on an AcA 54 column (30.5 x 1.6 cm)
equilibrated in 0.1 M Tris HC1 , pH 7.4 , Brij 35 0,1 % after trypsin was inhibited with PMSF. Fractions were screened for their papain inhibiting activity, concentrated then equilibrated in 0.1 M Tris HC1 buffer, pH 8.0, 0.2 M NaCl, 1 mM EDTA and applied
to a Thiol-Sepharose column prepared in
the same buffer. After washing with the equilibration buffer, elution was carried out making the buffer 5 mM with dithiothreitol. Eluted fractions were equilibrated in 0.1 M Tris HC1, pH 7.4 concentrated and analysed,by SDS polyacrylamide gel electrophoresis. A single band of Mr 17,000 was obtained. The product exhibited strong inhibiting activity and was titrated by papain as before.
Results and discussion Ki determinations were made choosing experimental conditions such as non linear dose-response curves were obtained . Specifically, cathepsin B, H, L
721
were used
at
4 x
r e s p e c t i v e l y with v a r i a b l e
a final
c o n c e n t r a t tori o f
were r e p e a t e d a t 3 d i f f e r e n t mechanism o f i n h i b i t i o n .
1.75 x 1 0 " M, 6 . 2 5 x 1 0 " M and
amounts o f i n h i b i t o r .
substrate
concentrations
to determine
The l i n e a r i z a t i o n of the steady s t a t e e q u a t i o n
( e q u a t i o n 1) a l l o w s t h e r a p i d d e t e r m i n a t i o n o f Ki^ the c u r v e obtained when ( I ) Q / l - a i s p l o t t e d a g a i n s t ^ 1 -a
Experiments
"
-
a
Ki
(app)+
the (9)
j from t h e s l o p e o f 1/a
< E >o
„,
with a = vi/vo and K i ( a p p ) = Ki (1 + (S) Q /K M ) Fig I r e p r e s e n t s a t y p i c a l
a n a l y s i s of
the i n t e r a c t i o n
between r a t
t a t i n and o a t ' i e n s i n 7.,. Ki was o b t a i n e d from t h e r e p l o t o f Ki.^ substrate concentration.
thios-
j against
S i m i l a r experiments were c a r r i e d out using c a t h e p -
s i n L and the t r y p t i c d i g e s t of t h i o s t a t i n a f t e r i t was t i t r a t e d by papain.
F i g . l : Ki f o r t h e i n t e r a c t i o n b e t w e e n r a t t h i o s t a t i n and c a t h e p s i n L a c c o r d i n g to Henderson ( 9 ) . M e a s u r e m e n t s were made in 0.1 M p h o s p h a t e b u f f e r (pH 6 . 0 ) c o n t a i n i n g 1 mM EDTA, 2 mM DTT, 0.1 % B r i j 35 ( f i n a l volume : 400 u l ) . Cathepsin L was used a t a f i n a l c o n c e n t r â t ion of 4.0 x with i n c r e a s i n g amounts of t h i o s t a t i n (2.5 x 1 0 " 1 0 t o 12.5 x 1 0 " 1 0 M ) . A f t e r i n c u b a t i o n a t 3 0 ° C , 10 u l o f 0 . 2 mM Z - P h e - A r g - N M e c were added t o t h e m i x t u r e t o s t a r t t h e r e a c t i o n . Three c o n c e n t r a t i o n s of s u b s t r a t e were used i . e . Km ( - Q - ) , 3 Km ( - A - ) and 5 Km ( - » - ) . K i ( a p p ) i n c r e a s e s as a f u n c t i o n of the substrate c o n c e n t r a t i o n . The i n s e t shows a r e p l o t o f e x p e r i m e n t a l \ values v e r s u s s u b s t r a t e c o n c e n t r a t i o n . I n t e r c e p t on the o r d i n a t e g i v e s Ene t r u e Ki
722 Results for cathepsin B, H and L are reported La table 2. Alternatively the g r a p h i c a l method of Dixon (10) may be used to d e t e r m i n e Ki. Initial rates are plotted against (I) at a given enzyme concentration and lines are drawn from v Q on the vertical axis through points of the curve such as ^ 0 ~ v ^ = v 0 / n (n = 1 , 2 , 3 ...). The difference between intercepts on the h o r i z o n t a l axis gives
from which Ki is determined p l o t t i n g K i ( a p p ) against (S) as
before. Fig 2 represents a typical analysis of cathepsin B interacting with
Fig. 2 : Ki determination for the interaction between rat thiostatin and cathepsin B according to Dixon. Measurements were made in 0.1 M phosphate buffer (pH 6.0) containing 1 mM EDTA, 2 mM DTT, 0.1 Z Brij 35 (final v o l u m e 200 ul). Cathepsin B was used at a final concentration of 1.75 x 10 with increasing amounts of thiostatin (5.0 x 1 0 ~ S M to 2.5 x 10~ 7 M). After 2 min of incubation at 30°C 10 ul of 0.2 mM Z - A r g - A r g - N - M e c were added to the mixture to start the reaction. Three concentrations of substrate were used i.e. 10 uM, 40 uM and 100 uM. The insert shows a replot of experimental ^ i ( a p n ) v a l u e s versus substrate concentration. Intercept on the ordinate gives tne true Ki. Rate constants for association were determined
by monitoring
the time
dépendance of the association under second order conditions considering that no dissociation occurs during the time of experiment (cathepsin L) or
723 taking into account the reverse reaction when it was supposed to intervene significantly (cathepsin B and H). W h e n i d e n t i c a l c o n c e n t r a t i o n s of e n z y m e ( ( E ) Q ) and i n h i b i t o r used and the reverse reaction is neglected during process, the integrated form of 1 — = (E)
J_ (E)„
k 1
the
(2)
in the nanomolar
range, kj may be
deter-
from the slope of 1/(E) versus time.
Preliminary experiments carried the
the initial part of
equation giving kj is :
Using cathepsin L concentrations mined
( ( I ) Q ) are
rate constant
to be n e g l e c t e d determined opposing
using
even
at an e a r l y
the
reactions: (EI)
integrated
the initial
stage
of
equation
Ln )
enzyme
B and H indicated
too high for the reverse the
r e a c t i o n , kj
reported
was
by L a i d l e r
^ (EI
concentration,
then
(11) for ) -
(E)
that
reaction
( (E) 2 - (EI) (EI)
(El)
equ
t( (E)2 - ( E I ) 2 e q u with (E)
out with cathepsin
for dissociation was
(3)
EI
< >equ " < > > (EI) and ( E I ) e q u the complex
concentration at any time t before equilibrium and at equilibrium
respecti-
ve ly. ( E I ) e q U may be e a s i l y d e t e r m i n e d values were
read on the p l o t
of
f r o m e q u i l i b r i u m m e a s u r e m e n t s and ( E I ) v^/vQ
versus
time
as s h o w n on
fig
3.
Experiments
0
SO
100
T i m e (sec)
Fig. 3 : k j d e t e r m i n a t i o n for o p p o s i n g r e a c t i o n s . E q u i m o l a r a m o u n t s of c a t h e p s i n H and t h i o s t a t i n ((a) : 1.25 x 1 0 ~ 8 M and (b) : 2.5 x 1 0 " 8 M ) ) w e r e used in two s u c c e s s i v e e x p e r i m e n t s and r e s i d u a l a c t i v i t y ( v i / v 0 ) aliquots was measured as a function of time by adding 10 ul of 0.5 m M Arg""he ri^liitL'/ii .v.tivLty Is plotted against time.
724
were
repeated
at
2 or
3 different
enzyme and i n h i b i t o r
concentration.
R e s u l t s are r e p o r t e d in t a b l e 2.
- Dissociation
r a t e s c o n s t a n t s of t h i o s t a t i n - c a t h e p s i n L c o m p l e x e s were
determined s t a r t i n g 10
7M)
slight
and s h i f t i n g
from c o n d i t i o n s the r e a c t i o n
molar excess of
macroglobulin
rat " j i g .
family
which
for
maximal
association
a proteinase
inhibitor
forms e n z y m i c a l l y
active
c y s t e i n e p r o t e i n a s e s ( 1 3 ) . The i n c r e a s e of enzymic a c t i v i t y different
times
after
a
process t o be f i r s t o r d e r ,
il3
was
added
to
= 2.4 x
=
belonging
to
complexes
the with
was recorded
the m i x t u r e . C o n s i d e r i n g
the d i s s o c i a t i o n r a t e e q u a t i o n (EI) (EI)0
In
((EI)Q
t o w a r d s d i s s o c i a t i o n ( 1 2 ) by a d d i n g a
at the
is
- k_j t
(4)
from which k _ j may be d e t e r m i n e d .
The r a t e c o n t a n t s f o r d i s s o c i a t i o n o f c o m p l e x e s w i t h c a t h e p s i n s B and H were found t o be too high to s a t i s f y between the p r o t e i n a s e
the c o n d i t i o n
and the i n h i b i t o r
required
of
complete
to s t a r t
the
association reaction.
T h e r e f o r e k _ j v a l u e s w e r e i n f e r r e d f r o m p r e v i o u s l y d e t e r m i n e d Ki and k j v a l u e s as f o l l o w s
:
= Ki x k j
Results are given in table
2.
Ki (M) a)
k j (M
1
s
[
)
k_! (s
')
b)
CATHEPSIN B
8 x 10 - 8
7 x 10~S
4 x 106
(0.30)
CATHEPSIN H
1.3ix 10-7
8 x 10~ 8
2.5 x 106
(0.26)
CATHEPSIN L
1.8 x 1 0 " 1 0
1.6 x 1 0 - 1 0
1.1 x 1 0 " 1 0
(Mr 17,000
6 x 106
1.2 x 10~3
peptide)
T a b l e 2 : K i n e t i c constants f o r the i n t e r a c t i o n nases and r a t t h i o s t a t i n . a ) Henderson's procedure, b ) Dixon's procedure.
between c y s t e i n e
protei-
725
From t h e s e
r e s u l t s it appears ft — 1 — 1
e n o u g h (> 10
M
.s
)
to
by t h l o s t a t l n may o c c u r inhibition
of
that
rate
suppose
constants
high
t h a t b i n d i n g of c a t h e p s i n B, H a n d L
physiologically.
proteinases
for a s s o c i a t i o n are
depends
on t h e i r
However,
t h e In v i v o r a t e
concentration
at
the
site
of of
i n h i b i t i o n . T h i o s t a t i n c o n c e n t r a t i o n has been determined o n l y in plasma ( 1 , 2 ) b u t n o t h i n g i s known a b o u t i t s c o n c e n t r a t i o n lar
compartments
proteinases
The d i f f u s i o n of related During
I.e.
where
it
from lysosomes (14, thiostatin
inflammation
supposed
to
and
extravascu-
intervene
to
the blood
compartment
could
release
kinlns
thiostatin
concentration
and
possibly
low Mr i n h i b i t o r y
k i n i n o g e n s a s f o u n d i n t h i s s t u d y . The s u b s e q u e n t permeability
would
extravascular
then promote
dramatiactivated
fragments
from
vascular
activity
observed
f o r c a t h e p s i n B or H
thiostatin
i s somewhat d i s t u r b i n g when compared t o t h e r e s u l t s g e n e r a l l y tight
carrier.
rises
i n c r e a s e of
t h e e x p o r t of i n h i b i t i n g
binding plasma I n h i b i t o r s
with the expected b i o l o g i c a l
towards
(17). This could appear
complexes
obtained
p r e c i s e l y d e t e r m i n e d and p h y s i o l o g i c a l l y c o n s i s t e n t r a n g e of a v o i d i n g s u d d e n l i b e r a t i o n of c y s t e i n e p r o t e i n a s e
inconsistent
to cause
function as a t r a n s i t o r y
i n h i b i t o r which,
t i g h t b i n d i n g or i r r e v e r s i b l e i n h i b i t o r such as in v i t r o
very e a s i l y .
concentration, proteolytic
f o r human t h i o s t a t i n
(18),
could well
that
in the presence
a macroglobulins
T h i s mechanism,
previously
gets
of rid
demonstrated
occur in plasma
t h i o s t a t i n h a s c o n v e y e d c y s t e i n e p r o t e i n a s e s f r o m t i s s u e s and
is
In a
d a m a g e . The s e c o n d a n d p o s s i b l y c o m p l e m e n t a r y h y p o t h e s i s w o u l d b e
of bound p r o t e i n a s e s
for
f u n c t i o n of an i n h i b i t o r u n l e s s h y p o t h e s i s
p u t f o r w a r d t h a t t h i o s t a t i n p e r m i t s t o m a i n t a i n t h e enzyme a c t i v i t y
thiostatin
be
spaces.
The r a p i d d i s s o c i a t i o n other
well
f u n c t i o n of t h e m o l e c u l e i . e . k i n i n
response,
c a l l y i n p l a s m a ( 1 , 2) and a t t h e same t i m e k i n i n o g e n a s e s a r e (16) which
inhibit
15).
outside
to the other p o t e n t i a l the
is
In c e l l s
after
extravascular
spaces.
References 1 . E s n a r d , F. and G a u t h i e r , F. R a t a l - c y s t e i n e p r o t e i n a s e i n h i b i t o r « An a c u t e p h a s e r e a c t a n t with a 1 - a c u t e phase g l o b u l i n . J . B i o l . Chem., 1 9 8 3 , 2 5 8 , 1 2 4 4 3 - 1 2 4 4 7 .
identical
726
2 . Urban, J . , Chan, D. and S c h r e i b e r , G. A r a t serum g l y c o p r o t e i n whose s y n t h e s i s inflammation. J . B i o l . Chem., ( 1 9 7 9 ) , 254, 10565-10568. 3. C o l e , T . , Major acute and c o n t a i n s mRNA l e v e 1. FEBS L e t t . ,
rate increases g r e a t l y
during
I n g l i s , A . S . , Roxburgh, C.M., H o w l e t t , G . J . and S c h r e i b e r , G. phase o ^ - p r o t e i n of the r a t i s homologous to b o v i n e k i n i n o g e n the sequence f o r bradykinin : i t s s y n t h e s i s i s r e g u l a t e d a t the (1985),
182, 57-61.
4. Okamoto, H. and Greenbaum, L.M. I s o l a t i o n and s t r u c t u r e of T - k i n i n . Biochem. B i o p h y s . Res. Commun., ( 1 9 8 3 ) , 112 , 7 0 1 - 7 0 8 . 5. B e d i , G . S . , B a l b i e r c z a k , J . and Back, N. A new v a s o p e p t i d e formed by the a c t i o n of a Murphy-Sturm lymphosarcoma p r o t e a s e on rat plasma k i n i n o g e n . Biochem. B i o p h y s . Res. Commun., ( 1 9 8 3 ) , 112, 6 2 1 - 6 2 8 .
acid
6. B a r r e t t , A . J . , K e m b h a v i , A.A., Brown, M.A., K i r s c h k e , H., K n i g h t , C.G., Tamai, M. and Hanada, K. L - t r a n s e p o x y s u c c L n y 1 - 1 e u c y 1 ami n o - ( 4 - g u a n l d i n o ) b u t a n e (E 6 4 ) and its analogues as i n h i b i t o r s of c y s t e i n e p r o t e i n a s e s i n c l u d i n g c a t h e p s i n B, H and L . Biochem. J . , 1982, 201, 189-198. 7. B a r r e t t , A . J . and K i r s c h k e , H. Cathepsin B, c a t h e p s i n H, and c a t h e p s i n L. Methods i n Enzymology, (1981), 80, 535-560. 8 . Moreau, T . , Esnard, F . , Gutman, N . , El Moujahed, A. and G a u t h i e r , in p r e p a r a t i o n .
F.
9 . Henderson, P . J . F . A l i n e a r equation that d e s c r i b e the s t e a d y - s t a t e k i n e t i c s of enzymes and s u b c e l l u l a r p a r t i c l e s i n t e r a c t i n g with t i g h t l y bound i n h i b i t o r s . Biochem. J . , ( 1 9 7 2 ) , 127, 32 1 - 3 3 3 . 10. Dixon, M. The g r a p h i c a l d e t e r m i n a t i o n of K^ and K^. Biochem. J . , 1972 , 129 , 1 9 7 - 2 0 2 . 11. L a i d l e r , K . J . The chemical k i n e t i c s of enzyme a c t i o n » Oxford U n i v e r s i t y P r e s s , 1953. 12. B i e t h , J.G. P a t h o p h y s i o l o g i c a l i n t e r p r e t a t i o n of k i n e t i c constants of p r o t e a s e tors. B u l l . Eur. P h y s i p a t h . R e s p . , ( 1 9 8 0 ) , 16 ( s u p p l . ) , 1 8 3 - 1 9 5 .
inhibi-
727 13. Esnard, F . , Gutman, N . , El Moujahed, A. and G a u t h i e r , F. Rat plasma a l - i n h i b i t o r 3 : a member of the a - r a a c r o g l o b u l i n f a m i l y . FEBS L e t t . , 1 9 8 5 , 182, 1 2 5 - 1 2 9 . 14. B a r r e t t , A . J . i n Enzyme R e g u l a t i o n and Mechanism of A c t i o n ( M i l d n e r , P. and R i e s , P . , e d s . ) , Pergamon P r e s s , Oxford, 1980, 305-315. 15. M e l l o n i , E . , P o n t r e m o l i , S . , S a l a m i n o , F . , S p a r a t o r e , E., M i c h e t t i , M. and Horecker, B.L. C h a r a c t e r i z a t i o n of three r a b b i t l i v e r lysosomal p r o t e i n a s e s with f r u c t o s e 1, 6 b i p h o s p h a t a s e converting enzyme a c t i v i t y . Arch. Biochem. B i o p h y s . , 1981, 208, 175-183. 16. Kaplan, A . P . , S i l v e r b e r g , M., Dunn, J . T . and Ghebrehivet, B. I n t e r a c t i o n of the c l o t t i n g , k i n i n - f o r m i n g , complement and f i b r i n o l y t i c pathways in inflammation. Ann. N. Y. Acad. S c i . , ( 1982) , 2 5 - 3 8 . 17. B i e t h , J . G . In v i v o s i g n i f i c a n c e of k i n e t i c c o n s t a n t s of m a c r o m o l e c u 1 a r inhibitors. Adv. Exp. Med. B i o l . , ( 1 9 8 4 ) , 1 6 7 , 9 7 - 1 0 9 . 18. Pagano, M., Esnard, F . , E n g l e r , R. e t G a u t h i e r , F . I n h i b i t i o n of human l i v e r c a t h e p s l n L by a 2 - c y s t e i n e - p r o t e I n a s e and the low-Mr c y s t e i n e p r o t e i n a s e I n h i b i t o r from human serum. Biochem. J . , 1 9 8 4 , 2 2 0 , 1 4 7 - 1 55.
proteinase
inhibitor
TUMOR CYSTEINE PROTEINASES AND THEIR
B.F. S l o a n e 1 , 2 , 3, T . T . L a h B a n d o 2 a n d K . V . H o n n 2, 3
INHIBITORS
N . A . D a y 1 , J. R o z h i n 2 , Y.
D e p a r t m e n t s of P h a r m a c o l o g y 1 , R a d i a t i o n O n c o l o g y 2 a n d Biological Sciences3, Wayne State University Detroit, Michigan 48201, USA
I.
PROTEINASES AND METASTASIS
P r o t e o l y t i c e n z y m e s h a v e b e e n h y p o t h e s i z e d to p l a y r o l e s in v a r i o u s a s p e c t s of t u m o r g r o w t h a n d m e t a s t a s i s . In o r d e r for tumor c e l l s to m o v e f r o m the p r i m a r y site m e t a s t a t i c s i t e s the t u m o r c e l l s h a v e to c r o s s
to
through
s e v e r a l c o n n e c t i v e t i s s u e b a r r i e r s , p e r h a p s the m o s t i m p o r t a n t of t h e s e is the b a s e m e n t m e m b r a n e t h a t the e n d o t h e l i a l c e l l s of the v a s c u l a t u r e .
The
underlies
basement
m e m b r a n e , in c o n t r a s t to other c o n n e c t i v e t i s s u e s , t y p e IV c o l l a g e n .
In a d d i t i o n the b a s e m e n t
contains glycoproteins proteoglycans.
membrane
(laminin, fibronectin)
A n u m b e r of i n v e s t i g a t o r s h a v e
and hypothesized
that tumor c e l l s m u s t b e a b l e to d e g r a d e type IV in o r d e r to invade t h r o u g h the b a s e m e n t
contains
collagen
membrane.
M e t a s t a t i c v a r i a n t s of the B16 m e l a n o m a h a v e b e e n s h o w n t o s e c r e t e a type IV c o l l a g e n a s e
(1).
H o w e v e r , type IV
c o l l a g e n a s e is s e c r e t e d by t u m o r c e l l s i n a l a t e n t
form
w h i c h requires activation
IV
(2) s u g g e s t i n g t h a t t y p e
c o l l a g e n a s e is n o t b y itself s u f f i c i e n t for
basement
Cysteine Proteinases and their Inhibitors © 1 9 8 6 Walter de Gruyter & Co., Berlin • N e w York - Printed in G e r m a n y
730 membrane invasion by tumor cells and that additional proteinases or a proteolytic cascade may participate in basement membrane invasion.
Proteinases of three classes
(serine, metallo and cysteine) have been implicated in the ability of tumor cells to degrade connective tissue matrices (for review see 3-6).
This chapter focuses on the
role of one cysteine proteinase, cathepsin B, and its endogenous inhibitors in tumor cell metastasis. The requirement or lack thereof for proteolytic degradation during basement membrane invasion remains an open question.
Tumor cells may be able to penetrate
through the type IV collagen lattice found in the basement membrane in the absence of tumor cell or host cell collagenolytic activity.
The type IV collagen lattice has
holes whose sides are ~ 800nm (7), i.e., a latticework that a tumor cell could traverse.
Alternatively, penetration of
the basement membrane could require limited digestion of the nonhelical regions of type IV collagen or degradation of the proteoglycans which form the ground substance of the basement membrane.
This limited degradation of type IV
collagen could be accomplished by proteinases such as plasmin formed by the action of plasminogen activator (3,8,9) or cathepsin B.
In addition, heparanase (10) or
cathepsin B could singly or in concert induce degradation of basement membrane proteoglycans. Since we (11) and others (3) have observed local dissolution of endothelial basement membrane in regions of contact between tumor cells and basement membrane, proteolytic degradation does appear to play a role in
731
basement membrane invasion.
We recently studied (at the
light and electron microscopic levels) the arrest and extravasation of B16 amelanotic melanoma (B16a) cells injected intravenously (11).
Figure 1 is a diagrammatic
representation of the process(es) by which a B16a tumor cell extravasates into the perivascular space at metastatic sites.
The upper figure illustrates a tumor cell arrested
in a pulmonary capillary.
The middle figure illustrates
the retraction of the underlying endothelial cell resulting in exposure of the basement membrane.
The lower figure
illustrates the dissolution of the basement membrane at the site of contact with the tumor cell plasma membrane.
Such
focal sites of dissolution would suggest that a proteolytic enzyme (such as cathepsin B) or a glycolytic enzyme localized in the tumor cell plasma membrane or released locally could be an effective mediator of B16a extravasation. With the B16a line there was not any evidence of diapedesis or active invasion by the tumor cells.
II.
DEGRADATION OF BASEMENT MEMBRANE
Cathepsin B was purified from normal human liver and human ovarian and colon carcinoma using a modification of the method described by Willenbrock and Brocklehurst (12).
On
SDS-PAGE under reducing conditions one band of 24 kDa was seen for liver. The ovarian and colon carcinoma exhibited two bands of 35 and 24 kDa (Figure 2).
We studied the abilities
732
Figure 1. Arrest and extravasation of B16 amelanotic melanoma cells in pulmonary microvasculature. Top, arrest and attachment to endothelial cell. Middle, retraction of endothelial cell-cell junctions exposing basement membrane Bottom, focal dissolution of basement membrane at site of contact with tumor cell plasma membrane. CFrom Sloane et al.(56) with permission.]
66 45 36 29 24 20 14 Liver
Ov. Care.
F i g u r e 2. SDS - P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (under r e d u c i n g c o n d i t i o n s ) of c a t h e p s i n B p u r i f i e d f r o m n o r m a l h u m a n liver a n d f r o m h u m a n o v a r i a n c a r c i n o m a .
734
400 200 1
125 85
60 CB 1
2
3
4
5
Figure 3. Degradation of laminin by cathepsin B purified from human colon carcinoma. Lane 1, laminin alone for 24 hr at 25° C and pH 6.5. Lane 2, laminin + tumor cathepsin B for 24 hr at 25 ° C and pH 6.5. Lane 3, same as lane 2 for 4 hr. Lane 4, laminin + human liver cathepsin B for 24 hr at 25° C and pH 6.5. Lane 5, same as lane 4 for 4 hr.
735 of t h e s e t w o e n z y m e s to d e g r a d e n a t i v e l a m i n i n a t two (6.5 a n d 7.4).
T h e s e in v i t r o a s s a y s w e r e r u n a t
t e m p e r a t u r e a t t i m e i n t e r v a l s f r o m 4 to 24 h r .
room
Cathepsin B
f r o m b o t h liver a n d c o l o n c a r c i n o m a w a s i n e f f e c t i v e d e g r a d i n g l a m i n i n a t p H 7.4.
pH's
in
S i n c e l a m i n i n is d e g r a d e d by
s e r i n e p r o t e i n a s e s a t p H 7.4, w e p r e s u m e t h a t the l a c k of d e g r a d a t i o n b y liver a n d t u m o r c a t h e p s i n B is d u e to i n a c t i v a t i o n of the e n z y m e .
A t p H 6 . 5 b o t h liver a n d
cathepsin B degraded laminin
(Figure 3; 13).
However,
l i m i t e d d e g r a d a t i o n of l a m i n i n b y t u m o r c a t h e p s i n B a t 1 0 - f o l d lower e n z y m e to s u b s t r a t e r a t i o s t h a n of l a m i n i n b y liver c a t h e p s i n B.
tumor
occurred
degradation
Tumor cathepsin B
d e g r a d a t i o n of l a m i n i n r e s u l t e d in d e g r a d a t i o n p r o d u c t s 1 2 5 , 85 a n d 61 kDa.
These products differ from
of
those
r e p o r t e d for d e g r a d a t i o n of l a m i n i n b y p l a s m i n or c a t h e p s i n G (14).
D e g r a d a t i o n of l a m i n i n b y c a t h e p s i n B (liver
tumor) w a s t o t a l l y b l o c k e d b y 10 y M E - 6 4 a n d
partially
i n h i b i t e d b y c y s t a t i n s - B p u r i f i e d f r o m h u m a n liver below).
and
(see
S t u d i e s a r e o n g o i n g to d e t e r m i n e w h i c h c h a i n of
l a m i n i n is d e g r a d e d b y c a t h e p s i n B. F i b r o n e c t i n , in c o n t r a s t to l a m i n i n , w a s n o t d e g r a d e d b y e i t h e r liver or t u m o r c a t h e p s i n B over a 24 h r p e r i o d a t t e m p e r a t u r e a n d p H 6.5.
Recklies and Poole
(15) h a v e
room
cited
u n p u b l i s h e d d a t a i n d i c a t i n g t h a t liver c a t h e p s i n B d e g r a d e s fibronectin. and Poole
W e c a n n o t c o m p a r e the two s t u d i e s as R e c k l i e s
(15) d i d n o t d e s c r i b e their
assay.
736 III.
TUMOR-ASSOCIATED CATHEPSIN B
A.
Release
The synthesis, processing and packaging of tumor cathepsin B has not been studied.
However, during the
synthesis, processing and packaging of other lysosomal enzymes a proportion of the enzyme is released extracellularly (16, 17).
Under certain pathological
conditions like lysosomal storage diseases, e.g., I-cell disease, the proportion released is enhanced.
In cells such
as macrophages or neutrophils lysosomes seem to function like secretory granules whose contents are released in response to given stimuli (18).
Thus the proportion of lysosomal enzymes
released from macrophages or neutrophils can be quite high. Cathepsin B activity has been measured extracellularly in culture medium of tumor cells and explants (19-28), ascites fluid of women with ovarian carcinomas (25, 29) and serum and urine of patients with a diverse variety of malignancies (30-35).
Arachidonic acid metabolites may play
a role in release of cathepsin B since release of lysosomal glycosidases from neutrophils has been shown to be stimulated by lipoxygenase products (principally leukotriene B ) of arachidonic acid metabolism (36, 37) and inhibited by inhibitors of the lipoxygenase pathway (38, 39).
Preliminary
evidence indicates that cathepsin B release from B16a cells can be stimulated by lipoxygenase products and inhibited by inhibitors of the lipoxygenase pathway (24, 40).
In a solid
737
tumor the c e l l u l a r s o u r c e of l i p o x y g e n a s e p r o d u c t s that s t i m u l a t e c a t h e p s i n B r e l e a s e a n d a c c o u n t for the
could
enhanced
l e v e l s of c a t h e p s i n B m e a s u r e d e x t r a c e l l u l a r l y h a s n o t b e e n identified.
T u m o r cell r e l e a s e of c a t h e p s i n B in r e s p o n s e
to
the s t i m u l u s of l i p o x y g e n a s e p r o d u c t s is n o t a s s o c i a t e d w i t h r e l e a s e of l y s o s o m a l g l y c o s i d a s e s
(24, 4 0 ) , t h u s
t h a t tumor c e l l s d o n o t r e l e a s e t h e i r e n t i r e c o n t e n t s as d o n e u t r o p h i l s in r e s p o n s e to products
(36, 37).
suggesting
lysosomal
lipoxygenase
Interactions among tumor cells, host
c e l l s , d i f f u s i b l e f a c t o r s , l i p o x y g e n a s e p r o d u c t s , etc.
which
m i g h t r e s u l t in e n h a n c e d r e l e a s e of c a t h e p s i n B in v i v o clearly require further
study.
T h e m o s t s u g g e s t i v e e v i d e n c e t h a t c a t h e p s i n B is r e l e a s e d f r o m tumor c e l l s in v i v o is that p r o v i d e d b y M o r t et al.
(25):
c a t h e p s i n B a c t i v i t y is p r e s e n t in a s c i t e s
from women w i t h ovarian carcinoma.
fluid
The ascites cells are
b e l i e v e d to b e the s o u r c e of the c a t h e p s i n B a c t i v i t y
since
the a s c i t e s c e l l s g r o w n in c u l t u r e r e l e a s e c a t h e p s i n B w h e r e a s s e r a f r o m the same p a t i e n t s h a v e n o c a t h e p s i n B activity.
H o s t c e l l s m i g h t a c c o u n t in p a r t for c a t h e p s i n B
r e l e a s e in v i v o or f r o m tumor e x p l a n t s in v i t r o .
However,
m a c r o p h a g e s a r e n o t b e l i e v e d to b e the s o u r c e of c a t h e p s i n B a c t i v i t y s i n c e R e c k l i e s et a l .
the
(28) d i d n o t
find
c a t h e p s i n B a c t i v i t y in c u l t u r e m e d i a of r e s i d e n t or stimulated murine macrophages.
B a i c i et al.
(41)
recently
r e p o r t e d that c a t h e p s i n B is r e l e a s e d b o t h f r o m e x p l a n t s of r a b b i t V2 c a r c i n o m a a n d f r o m e x p l a n t s of n o r m a l tissue from tumor-bearing rabbits.
subcutaneous
T h i s r e l e a s e , like
that
738 from human and murine mammary carcinoma explants, requires protein synthesis.
Cathepsin B release from the V2
carcinoma, normal tissue and co-cultures of the two can be stimulated by a diffusible factor present in tumor explant-conditioned culture medium.
B.
Membrane-associated
We have been exploring the possibility that cathepsin B activity in tumor cells may be associated with the plasma membrane.
Tumor cells grown in vitro have been shown to shed
plasma membrane-derived vesicles (42-44). Poste and Nicolson (44) found that by fusing shed vesicles from B16-F10 melanoma cells (high potential) with B16-F1 cells (low potential) the lung colonization potential of the B16-F1 melanoma can be increased.
We had previously established that cathepsin B
activity is higher in B16-F10 cells than B16-F1 (19, 45) and that both variants release cathepsin B in vitro (19). Therefore, we assayed membrane vesicles, from a cell line (15091A) which has been shown to shed vesicles in quantity (42, 43), for cathepsin B activity and established that the vesicles do possess cathepsin B activity (22). Others have also established that cathepsin B activity can be found in association with the plasma membrane.
When
normal and neoplastic human cervical cells are subjected to sucrose density gradient centrifugation, cathepsin B activity is found in a plasma membrane fraction of the neoplastic cells, but not of the normal cells (46).
Zucker et al. (47)
have recently reported elevated levels of cathepsin B in
739 plasma membrane fractions isolatd from human pancreatic adenocarcinoma cells.
Although Koppel et al. (48) found
cathepsin B activity in rat sarcoma plasma membranes, the cathepsin B activity in the plasma membrane of the metastatic variant is less than that in the nonmetastatic variant. Bohmer et ai. (49), on the other hand, found that the activity of an unidentified acid cysteine proteinase is similar in plasma membranes purified from normal bovine lymphoid cells and from bovine lymphosarcoma cells. Kozlowski et al. (50) have provided morphological evidence, using a fluorescent transition-state analog, that a cysteine proteinase is associated with the cell surface of HSDM C fibrosarcoma cells. We have examined the subcellular localization of cathepsin B activity by fractionating murine liver and three murine melanomas of increasing metastatic potential (Cloudman < B16-F1 < B16a) by differential and Percoll density gradient centrifugation.
We have established that the cathepsin B
activity in the three melanomas shifts in correspondence with the metastatic potential to the plasma membrane of the tumors (51, 52).
Differential centrifugation indicates that the
localization of cathepsin B activity shifts to the light mitochondrial fraction in correspondence with metastatic potential (Table 1).
Cathepsin H activity does not exhibit
this same shift (Table 1).
Percoll density gradient
centrifugation of the light mitochondrial fraction indicates that the shift in localization of cathepsin B activity is to the plasma membrane fraction of the metastatic B16 melanomas (Table 2).
The L-l fraction (density = 1.045 g/ml) of all
740 T a b l e 1. R a t i o of S p e c i f i c A c t i v i t y in L i g h t M i t o c h o n d r i a l F r a c t i o n (L) to that in H e a v y M i t o c h o n d r i a l F r a c t i o n (H) for C y s t e i n e P r o t e i n a s e s in M u r i n e L i v e r a n d Melanomas. L/H Enzyme
Liver
Cloudman
B16-F1
B16a
Cathepsin B
0.44
1.04
1.50
1.82
Cathepsin H
0.77
1.14
1.33
0.96
T a b l e 2. P e r c e n t a g e of C y s t e i n e P r o t e i n a s e A c t i v i t i e s L - l Fractions Separated by Percoll Density Gradient Centrifugation.
in
Liver
Cloudman
B16-F1
B16a
Cathepsin B
4
12
33
37
Cathepsin H
2
8
5
12
Enzyme
A light mitochondrial fraction obtained by differential centrifugation was purified by density gradient c e n t r i f u g a t i o n in 30% i s o - o s m o t i c P e r c o l l p r e p a r e d in h o m o g e n i z a t i o n b u f f e r (250 m M s u c r o s e , 25 m M H E P E S , lmM EDTA). T h e s e l f - f o r m i n g g r a d i e n t w a s g e n e r a t e d by c e n t r i f u g a t i o n for 16 m i n at 6 0 , 0 0 0 x g . T w o v i s i b l e b a n d s (L-l, u p p e r , a n d L - 2 , lower) w e r e a s p i r a t e d w i t h a P a s t e u r p i p e t t e , s e p a r a t e d f r o m the P e r c o l l m e d i u m a f t e r d i l u t i n g in the h o m o g e n i z a t i o n b u f f e r a n d r e c e n t r i f u g i n g at 120,000 x g for 55 m i n . F r a c t i o n s w e r e r e s u s p e n d e d in the h o m o g e n i z a t i o n b u f f e r a n d q u i c k - f r o z e n a t -70° C.
741 four t i s s u e s c o n t a i n e d N a + , K + - A T P a s e a c t i v i t y a n d the L - 2 fraction
(density = 1.07 g/ml) of liver c o n t a i n e d
lysosomal hydrolase activities 3 -N-acetyl-glucosaminidase and
four
(cathepsins B and H, 6 -glucuronidase).
I n the
B 1 6 m e l a n o m a s c a t h e p s i n B a c t i v i t y w a s f o u n d in b o t h L - l a n d L - 2 fractions.
S p e c i f i c a c t i v i t y of c a t h e p s i n B in the
plasma membrane
(L-l) f r a c t i o n s i n c r e a s e d in
w i t h metastatic potential
correspondence
Cathepsin H activity
(Table 2).
d i d n o t s h i f t to the p l a s m a m e m b r a n e f r a c t i o n s of the B16 melanomas
(Table 2).
T h e K i for i n h i b i t i o n b y l e u p e p t i n of c a t h e p s i n B a c t i v i t y in the L - l ( p l a s m a m e m b r a n e ) f r a c t i o n w a s 0.86 n M w h e r e a s the K i for i n h i b i t i o n of c a t h e p s i n B a c t i v i t y L-2
(lysosomal)
f r a c t i o n w a s 0.044 nM.
in the
The cathepsin B
a c t i v i t y in the L - l f r a c t i o n a l s o h a s e n h a n c e d s t a b i l i t y a t pH's g r e a t e r t h a n 7.0.
These differences
in i n h i b i t i o n
p H s t a b i l i t y s u g g e s t that the c a t h e p s i n B ' s i n the fractions may also
IV.
and
two
differ.
TUMOR CYSTEINE PROTEINASE
INHIBITORS
S i n c e the a c t i v i t y of c a t h e p s i n B in t u m o r s m a y b e m e d i a t e d in p a r t by e n d o g e n o u s c y s t e i n e p r o t e i n a s e w e h a v e r e c e n t l y p u r i f i e d the low M^ inhibitors
cysteine
inhibitors
proteinase
(~ 13,000 k D a a n d i s o e l e c t r i c p o i n t s f r o m 4.7 -
8.0) f r o m n o r m a l h u m a n liver a n d h u m a n s a r c o m a fibrous histiocytoma)
(malignant
u s i n g a m o d i f i c a t i o n of two m e t h o d s
a n d V . Turk., p e r s o n a l c o m m u n i c a t i o n ) .
T w o c l a s s e s of
(53
742
>h> I-
o
90% inhibition at the same concentration (55 and Figure 4).
Cystatins-A and
-B were isolated from the alkaline extract by fast protein liquid chromatography or chromatofocusing.
Sarcoma
cystatins-B, i.e., inhibitors with isoelectric points greater than 6.0, exhibited similar abilities compared to normal liver cystatins-B to inhibit papain and cathepsin B. However, sarcoma cystatins-A, i.e., inhibitors with acidic isoelectric points, were ~ 10-fold less inhibitory toward cathepsin B than papain when compared to liver cystatins-A. The amino acid composition and fluorescence spectra (Figure 5) of these two classes of inhibitors were also examined. Both analyses indicated that these inhibitors were proteins with distinct structure and conformation.
V.
DOES CATHEPSIN B PLAY A ROLE IN TUMOR METASTASIS? No definitive proof exists to indicate that cathepsin B
745
plays a role in tumor invasion and metastasis.
There
h o w e v e r , a n i n c r e a s i n g b o d y of c o r r e l a t i v e e v i d e n c e
relating
c a t h e p s i n B a c t i v i t y to the m a l i g n a n c y of h u m a n t u m o r s the m e t a s t a t i c c a p a b i l i t i e s of a n i m a l t u m o r s 6 , 56).
is,
and
(for r e v i e w
C o l l e c t i v e l y the s t u d i e s in our l a b o r a t o r i e s
cathepsin B and cysteine proteinase inhibitors suggest
see
on that
t u m o r c a t h e p s i n B c o u l d p l a y a role in f o c a l d i s s o l u t i o n of the e x t r a c e l l u l a r m a t r i x b y t u m o r c e l l s d u r i n g the m e t a s t a t i c cascade.
A role for c a t h e p s i n B in this p r o c e s s is
b y the c o r r e l a t i o n b e t w e e n c a t h e p s i n B a c t i v i t y
supported
in
subcutaneous tumors, isolated tumor cells and plasma membrane f r a c t i o n s i s o l a t e d f r o m s u b c u t a n e o u s t u m o r s a n d the m e t a s t a t i c p o t e n t i a l of the t u m o r s .
T h e s h i f t of c a t h e p s i n B
a c t i v i t y to p l a s m a m e m b r a n e f r a c t i o n s in m e t a s t a t i c
tumors
(52), the e n h a n c e d s t a b i l i t y a t n e u t r a l p H of c a t h e p s i n B in t u m o r cell m e m b r a n e v e s i c l e s
(22) a n d in the p l a s m a m e m b r a n e
f r a c t i o n a n d a r e d u c e d a f f i n i t y of c y s t a t i n - A t y p e s of
tumor
c y s t e i n e p r o t e i n a s e i n h i b i t o r s for c a t h e p s i n B (55) c o u l d a l l r e s u l t in a n e n h a n c e m e n t of c a t h e p s i n B a c t i v i t y in t u m o r s . Most importantly this enhanced activity may be localized at the p l a s m a m e m b r a n e a n d t h e r e b y to the sites of focal d e g r a d a t i o n w h e r e the t u m o r cell m e m b r a n e a n d e x t r a c e l l u l a r m a t r i x a r e in d i r e c t
the
contact.
Ac know1edgment s
T h e a u t h o r s t h a n k D r . J.D. C r i s s m a n for s u p p l y i n g t i s s u e a n d M r s . A . A l s t o n for t y p i n g the m a n u s c r i p t .
human This
746 w o r k w a s s u p p o r t e d in p a r t b y C A 3 6 4 8 1 a w a r d e d b y the N a t i o n a l Cancer Institute Hospitals
(BFS) a n d a g r a n t f r o m H a r p e r - G r a c e
(KVH a n d B F S ) .
Career Devlopment Award
BFS is the r e c i p i e n t of a R e s e a r c h (CA00921) f r o m the N a t i o n a l
Cancer
Institute.
REFERENCES
1.
L i o t t a , L . A . , K . T r y g g v a s o n , S. G a r b i s a , K . H a r t , C . M . F o l t z , S. S h a f i e . 1980. N a t u r e (London) 284, 61.
2.
S a l o , T. , L.A. L i o t t a , K. T r y g g v a s o n . Chem. 257, 3058.
3.
Jones, P.A., Y.A. DeClerck. Rev. 1, 289.
4.
L i o t t a , L . A . , U . P . T h o r g e i r s s o n , S. G a r b i s a . C a n c e r M e t a s t a s i s R e v . 1, 277.
5.
M u l l i n s , D . E . , S.T. R o h r l i c h . A c t a 6 9 5 , 177.
6.
Sloane, B.F., K.V. Honn. 3, 249.
7.
Kuhn, K. In: P r o t e i n a s e s in I n f l a m m a t i o n a n d T u m o r I n v a s i o n . (H. T s c h e s c h e , ed.) W a l t e r de G r u y t e r & C o . , B e r l i n , (in p r e s s ) .
8.
Q u i g l e y , J . , E. C r a m e r , S. F a i r b a i r n , R . G i l b e r t , J. L a c o v a r a , G. O j a k i a n , R . S c h w i m m e r . 1985. In: M e c h a n i s m s of M e t a s t a s i s : Potential Therapeutic I m p l i c a t i o n s (K.V. H o n n , W . E . P o w e r s , B.F. S l o a n e , eds.) M a r t i n u s N i j h o f f , B o s t o n , p. 309.
9.
G o l d f a r b , R. H . 1985. In: M e c h a n i s m s of M e t a s t a s i s : P o t e n t i a l T h e r a p e u t i c I m p l i c a t i o n s (K.V. H o n n , W . E . P o w e r s , B.F. S l o a n e , eds.) M a r t i n u s N i j h o f f , B o s t o n , p. 341.
10.
N i c o l s o n , G . L . , M . N a k a j i m a , T. I r i m u r a . 1985. In: M e c h a n i s m s of M e t a s t a s i s : Potential Therapeutic I m p l i c a t i o n s (K.V. H o n n , W . E . P o w e r s , B . F . S l o a n e , eds.) M a r t i n u s N i j h o f f , B o s t o n , p. 275.
11.
C r i s s m a n J . D . , J. H a t f i e l d , M . S c h a l d e n b r a n d , B . F . Sloane, K.V. Honn: Lab. I n v e s t . (in p r e s s ) .
1982.
1983.
1984.
1983.
Cancer
J.
Biol.
Metastasis
Biochim.
1982. Biophys.
Cancer Metastasis
Rev.
747
12.
Willenbrock, F., K. Brocklehurst. 2 2 7 , 511.
13.
L a h , T . T . , N . A . D a y , C.N. R a o , K . V . H o n n , L . A . B.F. S l o a n e . In p r e p a r a t i o n .
14.
R a o , C . N . , I . M . K . M a r g u l i e s , R . H . G o l d f a r b , J.A. M a d r i , D . T . W o o d l e y , L . A . L i o t t a . 1982. Arch. Biochem. B i o p h y s . 2 1 9 , 65.
15.
Recklies, A.D., A.R. Poole. 1982. In: L i v e r M e t a s t a s i s . (L. W e i s s , H . A . G i l b e r t , eds.) G . K . H a l l , B o s t o n , p. 77.
16.
Rosenfeld, M.G., G. Kreibich, D. Popov, K. Kato, D.D. S a b a t i n i . 1982. J. C e l l B i o l . 9 3 , 135.
17.
Sly, W.S., H.D. Fischer. 67.
18.
S k u d l a r e k , M . D . , R . T . S w a n k . 1981. 10137.
19.
S l o a n e , B . F . , K . V . H o n n , J.G. S a d l e r , W . A . T u r n e r , J.J. Kimpson, J.D. T a y l o r . 1982. C a n c e r R e s . 42., 980.
20.
P i e t r a s , R . J . , C . M . S z e g o , J.A. R o b e r t s , B.J. 1981. J. H i s t o c h e m . C y t o c h e m . 29, 440.
21.
H o n n , K . V . , P. C a v a n a u g h , C. E v e n s , J.D. T a y l o r , Sloane. 1982. S c i e n c e 217, 540.
22.
P . G . C a v a n a u g h , B.F. S l o a n e , A. B a j k o w s k i , G . J . G a s i c , T.B. Gasic, K.V. Honn. 1983. C l i n . E x p . M e t a s t a s i s 1, 297.
23.
S l o a n e , B . F . , J . G . S a d l e r , C. E v e n s , R. R y a n , A . S . B a j k o w s k i , J.D. C r i s s m a n , K.V. Honn. 1984. C a n c e r Bull. 3 6 , 196.
24.
S l o a n e , B . F . , S. M a k i m , J.R. D u n n , R . L a c o s t e , M . T h e o d o r o u , J. B a t t i s t a , R. A l e x , K . V . H o n n . 1982. In: Prostaglandins and Cancer. (R.S. B o c k m a n , T. P o w l e s , K . V . H o n n , P. R a m w e l l , eds.) A l a n L i s s , N e w Y o r k , p. 789.
25.
M o r t , J . S . , M. L e d u c , A . D . R e c k l i e s . B i o p h y s . A c t a 6 6 2 , 173.
26.
Poole, A.R., K.J. Tiltman, A.D. Recklies, T.A.M. 1980. N a t u r e (London) 2 7 3 , 545.
27.
Recklies, A.D., K.J. Tiltman, T.A.M. Stoker, A.R. 1980. C a n c e r Res. 4 0 , 550.
1982.
1985.
Biochem.
J.
Liotta,
J. C e l l . B i o c h e m .
18,
J. B i o l . C h e m .
1981.
256,
Seeler. B.F.
Biochim. Stoker. Poole.
748
28.
Recklies, A.D., J.S. Mort, Res. 4 2 , 1026.
A.R. Poole.
1982.
29.
Mort, J.S., M. Leduc, A.D. Recklies. B l o p h y s . A c t a 755, 369.
30.
P i e t r a s , R . J . , C . M . S z e g o , C.E. M a n g a n , B.J. S e e l e r , M.M. Burtnett, M. O r e v i . 1978. O b s t e t . G y n e c o l . 5 2 , 321.
31.
P i e t r a s , R . J . , C . M . S z e g o , C . E . M a n g a n , B.J. M.M. Burtnett. 1979. G y n e c o l O n c o l . 7, 1.
32.
P e r r a s , R . J . , J. C r a m e r , R. B i s h o p , H. A v e r e t t e , B.U. Sevin. 1983. P r o c . Am. A s s o c . C a n c e r R e s . 2 4 , 130.
33.
P e r r a s , J . P . , B.U. S e v i n . R e s . 2 5 , 250.
34.
D u f e k , V . , B. M a t o u s , V . K r a l .
35.
Vasishta, A., P.R. Baker, P.E. Preece, R.A.B. Wood, A. Cuschieri. 1984. E u r . J. C l i n . O n c o l . 10, 203.
36.
Bokoch, G.M., P.W. Reed. 5317.
37.
H a f s t r o m , I., J. P a m b l a d , C . L . M a l s t e n , 0. R a d m a r k , Samuelsson. 1981. F E B S L e t t . 130, 146.
38.
Walenga, R.W., H.J. Showeil, M.B. Feinstein, E.L. Becker. 1980. L i f e Sei. 27, 1047.
39.
S m i t h , R . J . , F . F . S u n , B . J . B o w m a n , S.S. I d e n , H . W . Smith, J.C. M c G u i r e . 1982. B i o c h e m . B i o p h y s . R e s . Commun. 109, 943.
40.
Sloane, B.F., K.V. Honn. 1985. In: B i o c h e m i s t r y of Arachidonic Acid Metabolism. (W.E.M. L a n d s , ed.) M a r t i n u s N i j h o f f , B o s t o n , p. 311.
41.
B a i c i , A . , M. G y g e r - M a r a z z i , P. S t r a u l i . M e t a s t a s i s 4, 13.
42.
G a s i c , G . J . , J.L. C a t a l f a m o , T . B . G a s i c , N . A v d a l o v i c . 1981. In: M a l i g n a n c y a n d the H e m o s t a t i c S y s t e m . (M.B. D o n a t i , J.F. D a v i d s o n , S. G a r a t t i n i , eds.) R a v e n P r e s s , N e w Y o r k , p. 27.
43.
G a s i c , G . J . , D. B o e t t i g e r , J.L. C a t a l f a m o , T . B . G.J. S t e w a r t . 1978. C a n c e r Res. 38, 2950.
44.
P o s t e , G . , G.L. N i c o l s o n . USA 77, 399.
1983.
Cancer
Biochim.
Seeler,
1984. P r o c . A m . A s s o c .
Cancer
1984. N e o p l a s m a 31, 99.
1981. J. B i o l . C h e m .
1984.
256, B.
Invasion
Gasic,
1980. P r o c . N a t l . A c a d .
Sei
749 45.
Sloane, B.F., J.R. Dunn, K.V. Honn. 1151.
1981. S c i e n c e
212,
46.
P i e t r a s , R . J . , J.A. R o b e r t s . 8536.
47.
Z u c k e r , S . , R . M . L y s i k , J. W i e m a n , D . W i l k i e , B. L a n e . 1985. P r o c . A m . A s s o c . C a n c e r R e s . 2 6 , 56.
48.
K o p p e l , P . , A . B a i c i , R . K e i s t , S. M a t z k u , R. K e l l e r . 1984. E x p . Cell B i o l . 52, 293.
49.
B ö h m e r , F . D . , H . E . S c h m i d t , R. S c h o n . Med. G e r m , 41, 883.
50.
Kozlowski, K.A., F.H. Wezeman, R.M. Schultz. P r o c . N a t l . A c a d . Sei. U S A 8 1 , 1135.
51.
S l o a n e , B . F . , J. R o z h i n , J.D. C r i s s m a n , A . S . B a j k o w s k i , K.V. Honn. 1985. In: T r e a t m e n t of M e t a s t a s i s : Problems and Prospects. (K. H e l l m a n n a n d S.A. E c c l e s , eds.) T a y l o r a n d F r a n c i s , L o n d o n , p. 377.
52.
S l o a n e , B . F . , J. R o z h i n , K . J o h n s o n , H. T a y l o r , J.D. Crissman, K.V. Honn. Submitted.
53.
Green, G.D.J., A.A. Kembhavi, M.E. Davies, A.J. 1984. B i o c h e m . J. 218, 939.
Barrett.
54.
J a r v i n e n , M. , A . R i n n e . 1982. B i o c h i m . B i o p h y s . 708, 210.
Acta
55.
L a h , T . T . , J. R o z h i n , K . V . H o n n , J . D . C r i s s m a n , Sloane. 1985. J. C e l l B i o l , (in p r e s s ) .
B.F.
56.
S l o a n e , B . F . , J. R o z h i n , R . E . R y a n , T . T . L a h , N . A . D a y , J.D. C r i s s m a n a n d K . V . H o n n . 1985. In: M e c h a n i s m s of Metastasis: Potential Therapeutic Implications. (K.V. H o n n , W . E . P o w e r s , B.F. S l o a n e , eds.) Martinus Nijhoff, B o s t o n , p. 377.
1981. J. B i o l . C h e m .
256,
1982. A c t a B i o l . 1984.
THIOL PROTEASE INHIBITOR RELEASED FROM HUMAN MALIGNANT MELANOMA
Y. Nishida, H. Tsushima, N. Toki, H. Sumi, H. Mihara Department of Physiology, Miyazaki Medical College, 5200 Kihara, Kiyotake-cho, Miyazaki-gun, Miyazaki-ken 889-16, Japan
Introduction Many kinds of human thiol protease inhibitor
(TPI) have baen re-
ported and the number of studies on their physiological or pathophysiological significance have been increasing. Recently several investigators have suggested that malignant cells release cathepsin B and its activity has been correlated to tumor invasion and metastasis
(1, 2, 3, 4).
In the present study, using an established cell line of human malignant melanoma and human melanoma tissue, we observed that malignant melanoma cells released TPl's, which was far greater than a thiol protease, cathepsin B. We therefore evaluated TPI's possible pathophysiological significance in cancer.
Methods and Results Cell culture. A human malignant melanoma cell line (Bowes) and a fetal lung fibroblast line were cultured in Eagle's minimum essential medium containing 10% fetal calf serum using Rijkin and Collen's method
(5). To collect large pools of conditioned media,
confluent flasks were rinsed three times and replenished with 7.5 ml of the serum-free medium per 7.5 sq cm of culture flask. Tissue extraction. Human melanoma tissue samples were obtained from several patients with primary and metastatic lesions. These tissues were minced and homogenized in 10 volumes of 2 M KSCN according to the method of Astrup and Albrechtsen (6) and incubated over night at 4°C. After being centrifuged at 15,000 x g for 30 min at 4°C, the supernatant was dialyzed against 0.1 M phosphate buffer, pH 7.4, and then concentrated to 1/10 of the original volume by ultra-
Cysteine Proteinases and their Inhibitors © 1986 Walter de Gruyter & Co., Berlin • N e w York - Printed in Germany
752
6
12
21
Incubation Time
Î8 Chrs)
Fig. 1. Accumulation of TPI in culture media of human malignant melanoma cells and human fetal lung fibroblasts. filtration. Thus we obtained the crude extract. Assay method. TPI activity was assayed by the caseinolytic technique (7) and the amidolytic technique (a synthetic substrate, H-Dvalylleucyllysyl-p-nitroanilide) (8) using ficin as a representative thiol protease. One unit of TPI was defined as the amount required to inhibit 1 yg of ficin and was calculated from the amount causing 50% inhibition under the assay conditions. The values obtained from these two techniques coincided with each other. TPI activity in the culture media of malignant melanoma cells TPI activity was found in the 24-hr incubated culture media of human malignant melanoma cells (approximately 0.2-0.3 units/ml). We then compared the accumulation of TPI in the melanoma cell culture media to that of nonmalignant cells (human fetal lung fibroblasts). As shown in Fig. 1, the TPI activity in the culture supernatant of malignant melanoma cells increased progressively with increasing incubation time. However, in the case of the fibroblasts TPI activity was barely detectable and scarcely increased. We clearly recognized that the extracellular TPI activity accumulated progressively in the culture supernatant of malignant melanoma cells but not in that of fetal lung fibroblasts. The mean specific activities of TPI in the 6-, 12-, 24- and 48-hr
753 incubated media of melanoma cells were 1.60, 1.90, 2.79, 1.64 units/mg protein, respectively. As reported previously (8), total TPI activity in 24-hr incubated media of one flask was about 1.5fold higher than total intracellular TPI activity of cells. The specific activity in the medium was about 10-fold higher than the intracellular specific activity. These facts indicate that cultured malignant melanoma cells release TPI. The molecular distribution of cultured melanoma TPI was investigated by Sephadex G-150 gel filtration of crude conditioned media.
1
2
3
4
5
6
4. 4- 4- + 4- +
60
JO Neutral Thiol Protease Activity
JO
(2hTS-Incubation)
o——O^OOOOOOQOoiXjoOOOOOOOOOOOOOOOOO—o—oo
Cathepsin B Activity
10
20
30
40
(2hrs-Incubation)
50
60
70
80
90
Fraction Number
Fig. 2. Gel filtration of crude culture media of melanoma cells on Sephadex G-150. Pooled 24-hr incubated media of 250 ml were concentrated to 2.2 ml by a solid polyethylene glycol 20,000 and lyophylization technique, and were centrifuged (1,500 x g for 10 min). The supernatant (2.0 ml) was applied to a column (1.6 x 70 cm), equilibrated with 10 mM phosphate buffer-0.2 M NaCl (pH 7.4), at 4°C. Arrows, elution positions of the following standard proteins: 1, Blue Dextran; 2, aldolase (Mr 158,000); 3, bovine serum albumin (Mr 67,000); 4, egg albumin (Mr 45,000); 5, chymotrypsinogen A (Mr 25,000); 6, cytochrome C (Mr 12,500). U, units. Neutral thiol protease activity was assayed by the modified method of Otto and Bhakdi (9) using 4.6 mM N-a-benzoyl-L-arginine-pnitroanilide and the incubation buffer, pH7.4. Cathepsin B activity was assayed by the method of Barrett (10) using 5 mM N-a-. benzoyl-DL-arginine-B-naphthylamide and a total assay volume of 1.0 ml.
754 As shown
in F i g .
corresponding w h i c h the
2, t h e T P I a c t i v i t y w a s e l u t e d a s d o u b l e
to m o l e c u l a r weights of about
latter was main. We searched
and neutral
thiol protease
through a Sephadex activities
were not
G-150
activity
column.
56,000 and
in all
fractions
passage
protease
detected.
in the h u m a n m a l i g n a n t m e l a n o m a
TPI activity
was
tissue
found in the e x t r a c t of m e l a n o m a
in the c u l t u r e m e d i a .
imately
From
30 g o f t h e m e l a n o m a
593.5 u n i t s of TPI w a s o b t a i n e d .
tissue as tissue,
The molecular
distribu-
tissue
3). Two m a i n p e a k s w e r e d e t e c t e d a n d their
respectively.
We searched
extract and all
investigated by Sephadex
weights were approximately
for c a t h e p s i n B a c t i v i t y
fractions by Sephadex
er, cathepsin B activity
40,000
was not
G-100
gel
and
well approx-
filtration
lated molecular
TPI was
extract
t i o n of m e l a n o m a (Fig.
of
activity
by
these thiol
TPI activity
as
10,000,
for cathepsin B
However,
peak,
G-100
gel
calcu-
10,000,
in the
crude
filtration.
Howev-
detected.
F i g . 3. G e l f i l t r a t i o n o f t h e c r u d e t i s s u e e x t r a c t o n S e p h a d e x G - 1 0 0 . T h e c r u d e e x t r a c t (10 m l ) w a s a p p l i e d t o a c o l u m n (2.5 x 60 c m ) , e q u i l i b r a t e d w i t h 0.1 M p h o s p h a t e b u f f e r , p H 7 . 4 . A r r o w s , e l u t i o n p o s i t i o n s of t h e f o l l o w i n g s t a n d a r d p r o t e i n s : 1, b o v i n e s e r u m a l b u m i n (Mr 6 7 , 0 0 0 ) ; 2, e g g a l b u m i n (Mr 4 5 , 0 0 0 ) ; 3, c h y m o t r y p s i n o g e n A (Mr 2 5 , 0 0 0 ) ; 4, c y t o c h r o m e C (Mr 1 2 , 5 0 0 ) .
755 Table 1. Comparative Properties of TPI's from Cultured Melanoma and Melanoma Tissue
Molecular Weight
Culture
Tissue
56,000 and 10,000
40,000 and 10,000
Inhibition Spectra^
ficin (1(19)
bromelain g)
0.1 0.2 0.3