Cysteine Proteinases and their Inhibitors: Proceedings of the International Symposium Portoroz, Yugoslavia, September 15–18, 1985 9783110846836, 9783110107241

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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.

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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

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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.

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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

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50

75

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I

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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

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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

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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

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Inhibition.

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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

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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.

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3.

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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