Lipmann Symposium. Energy transformation in biological systems: [Symposium on Energy Transformation in Biological Systems, London, 2.–4. July, 1974] [Reprint 2019 ed.] 9783110830903, 9783110049763

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Lipmann Symposium

Lipmann Symposium Energy, Regulation and Biosynthesis in Molecular Biology Editor Dietmar Richter

W DE G

Walterde Gruyter • Berlin • New York 1974

Editor Dr. Dietmar Richter Professor, Head of Abt. Cell Biochemistry, Institute of Physiological Chemistry, University of Hamburg, Hamburg, Germany.

Photographs on pp. 699 - 701 by P. Herrlich, Z. Ku&n, and M. Miller ©Copyright 1974 by Walter de Gruyter & Co., Berlin. - All rights reserved, including those of translation into foreign languages. No 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. Cover design: R. Hiibler. - Printing: Druckerei Gerike, Berlin. - Binding: Liideritz & Bauer, Berlin. Printed in Germany ISBN 3 11 004976 7

Photograph by Z. Kucan

Dedicated to the 75th Birthday of Dr. Fritz Lipmann

Preface

D r . F r i t z L i p m a n n c e l e b r a t e d h i s 75th b i r t h d a y o n J u n e 1 2 t h , 1974. T o m a r k this event a symposium w a s a r r a n g e d and t o o k p l a c e f r o m J u l y 7th to 9th at t h e M a x - P l a n c k - I n s t i t u t f u r M o l e k u l a r e G e n e t i k in B e r l i n - D a h l e m , t h e city w h e r e , fifty y e a r s a g o , t h e c a r e e r of t h i s g r e a t s c i e n t i s t b e g a n . A b o u t 80 L i p m a n n a l u m n i , g u e s t s a n d f r i e n d s of t h e L i p m a n n f a m i l y f o r e g a t h e r e d to m e e t t h e i r t e a c h e r a n d m e n t o r , to d i s c u s s t h e p a s t , p r e s e n t a n d f u t u r e of b i o c h e m i s t r y , a n d t o e x c h a n g e m e m o r i e s of t h e i r d a y s in t h e L i p m a n n L a b o r a t o r y . T h e y c a m e f r o m all o v e r E u r o p e a n d t h e U n i t e d S t a t e s , a n d f r o m c o u n t r i e s a s f a r a w a y a s Chile, J a p a n , and A u s t r a l i a . B e c a u s e of t h e s p e c i a l n a t u r e of t h i s v e r s a t i l e b i o c h e m i s t , t h e l e c t u r e s of t h i s S y m p o s i u m c o v e r e d m a n y f i e l d s of b i o c h e m i s t r y . T h e b o o k b e g i n s w i t h vivid a n d a m u s i n g a c c o u n t s b y t h e t w o " o l d - t i m e r s " of b i o c h e m i s t r y , S i r H a n s K r e b s a n d F r i t z L i p m a n n of life a n d c o n d i t i o n s in t h e " G o l d e n T w e n t i e s " in t h e l a b o r a t o r i e s of W a r b u r g a n d M e y e r h o f at t h e f o r m e r K a i s e r - W i l h e l m - I n s t i t u t . T h e volume c l o s e s with the l e c t u r e o n " S t r u c t u r e s of Biotin E n z y m e s " b y D r . F . L y n e n . T h i s l e c t u r e m a r k s t h e b e g i n n i n g of a n e w e v e n t in t h e s c i e n t i f i c c a l e n d a r , t h e " F r i t z L i p m a n n L e c t u r e " , w h i c h will n o w b e g i v e n a n n u a l l y , a n d in t h i s c o n n e c t i o n I a m e s p e c i a l l y g r a t e f u l to t h e B o e h r i n g e r M a n n h e i m C o r p o r a t i o n f o r t h e i r f i n a n c i a l support. A s e d i t o r of t h i s v o l u m e I a m i n d e b t e d t o D r . F r i t z L i p m a n n f o r t h e t i m e t h a t I s p e n t in h i s l a b o r a t o r y at t h e R o c k e f e l l e r U n i v e r s i t y in N e w Y o r k . A s S i r H a n s K r e b s s o aptly p u t it, " T h e d e s i r e of t h e f o l l o w e r s to e x p r e s s a s e n s e of loyalty, g r a t i t u d e a n d a f f e c t i o n " w a s b y n o m e a n s t h e l e a s t of t h e r e a s o n s why the S y m p o s i u m w a s such a s u c c e s s . It i s o n e t h i n g to c o n c e i v e t h e i d e a of a r r a n g i n g s u c h a S y m p o s i u m , b u t its m a n a g e m e n t a n d o r g a n i z a t i o n i s quite a n o t h e r m a t t e r ; it h a s r e q u i r e d t h e a s s i s t a n c e a n d s u p p o r t of a n u m b e r of p e o p l e a n d i n s t i t u t i o n s w h o m I w o u l d like t o t h a n k . In p a r t i c u l a r , I w o u l d like to m e n t i o n D r . F . L y n e n a n d D r . H . U . B e r g m e y e r , b o t h of w h o m m a d e t h e initiation

VIII

of t h e a n n u a l F r i t z L i p m a n n L e c t u r e p o s s i b l e ; a l s o D r . E . H e l m r e i c h the c h a i r m a n , D r . E . A u h a g e n the t r e a s u r e r , a n d D r . H . G i b i a n t h e s e c r e t a r y of t h e G e s e l l schaft f u r Biologische C h e m i e . T h e meeting would h a v e b e e n m u c h m o r e difficult to o r g a n i z e w i t h o u t t h e a d v i c e a n d c o o p e r a t i o n of D r . H . G . W i t t m a n n . T h e f i n a n c i a l s u p p o r t g i v e n by the pharmaceutical companies S c h e r i n g A G and B o e h r i n g e r Mannheim C o r p o r a t i o n , a s well a s the V o l k s w a g e n Stiftung, Max-Planck-Gesellschaft, Gesellschaft fur Biologische Chemie and Walter de G r u y t e r P r e s s is gratefully a c k n o w l e d g e d . T h e planning and organization would have been inconceivable w i t h o u t t h e h e l p , s u p p o r t a n d c o n t i n u o u s e n c o u r a g e m e n t of my wife Heidi. F o r this I am v e r y thankful. I a m g r a t e f u l to t h e W a l t e r d e G r u y t e r P r e s s f o r m a k i n g it p o s s i b l e f o r u s to p u b l i s h t h e c o n t r i b u t i o n s in t h e i r e n t i r e t y in a s p e c i a l b i r t h d a y v o l u m e w h i c h i s d e d i c a t e d to D r . F r i t z L i p m a n n with b e s t w i s h e s . I do not w a n t to e n d this P r e f a c e without r e m a r k i n g on h o w D r . L i p m a n n a p p e a r e d t o h i s f o r m e r c o l l e a g u e s , m a n y of w h o m m a y not h a v e s e e n him f o r s o m e y e a r s - , e v e r y o n e w a s p l e a s e d to s e e t h a t h i s o w n p e r s o n a l s t o r e of h i g h - e n e r g y p h o s p h a t e b o n d s a p p e a r s to b e s u f f i c i e n t t o k e e p h i m g o i n g f o r a good while y e t . We w i s h him s i n c e r e l y m a n y m o r e creative and energetic y e a r s .

July

1974

Dietmar

Richter

Contents

L i s t of P a r t i c i p a n t s of t h e L i p m a n S y m p o s i u m L i s t of C o n t r i b u t o r s

Opening Helmreich, Wittmann,

E.J.M.: H.G.:

XIV XIX

Remarks

W e l c o m e to P r o f e s s o r

Lipmann

W e l c o m e to t h e M a x - P l a n c k - I n s t i t u t

1 5

Introduction K r e b s , H . A . and L i p m a n n , Nineteen Twenties

F.:

D a h l e m in t h e

Late 7

C ontributions A d l e r , S . P . and S t a d t m a n , E . R . : E . c o l i Glutamine Synthetase

C a s c a d e C o n t r o l of 28

Baddiley, J . , B u r n e t t , J . P . , H a n c o c k , I . C . , and H e p t i n s t a l l , J . : S t r u c t u r e a n d B i o s y n t h e s i s of a n A c i d i c G l y c o p r o t e i n in an B a c t e r i a l C e l l E n v e l o p e

40

B a u e r , K . : D e g r a d a t i o n of T h y r o t r o p i n R e l e a s i n g H o r m o n e ( T R H ) . Its Inhibition b y P y r o g l u - H i s - O C H ß a n d t h e E f f e c t of t h e I n h i b i t o r in A t t e m p t s t o S t u d y t h e B i o s y n t h e s i s of T R H 53 B e n n e t t , T . P . : A m p h i b i a n C e l l s in C u l t u r e : to M e t a m o r p h o s i s

An

Approach 63

B e s s m a n , S . P . : T h e Hexokinase-Mitochondrial B i n d i n g T h e o r y of Insulin A c t i o n

77

Bloemendal, H . and S t r o u s , A c e t y l a t i o n of P r o t e i n s ,

89

G . J . A . M . : N-Terminal a Post-Initiational Event

X B o m a n , H . G . , N i l s s o n - F a y e , I . , and R a s m u s o n , T . : Why is Insect Immunity I n t e r e s t i n g ?

103

Brodie, A . F . , Lee, S . - H . , P r a s a d , R . , Kalra, V . K . , a n d K o s m a k o s , F . C . : E n e r g y T r a n s d u c t i o n in M e m b r a n e s of M y c o b a c t e r i u m p h l e i 115 C a b r e r , B . , San-Millan, M . J . , Gordon, J . , V a z q u e z , D . , and Modolell, J . : C o n t r o l by P e p t i d y l - t R N A of E l o n g a t i o n F a c t o r G I n t e r a c t i o n with the R i b o s o m e

131

C h a n t r e n n e , H . : A Cytoplasmic Function f o r the P o l y a d e n y l i c S e q u e n c e s of M e s s e n g e r R N A

144

C h r i s t m a n , J . K . and A c s , G . : Purification and B i o l o g i c a l P r o p e r t i e s of a P l a s m i n o g e n A c t i v a t o r C h a r a c t e r i s t i c of M a l i g n a n t l y T r a n s f o r m e d C e l l s

150

E b a s h i , S . : I n t e r a c t i o n s of T r o p o n i n S u b u n i t s U n d e r lying R e g u l a t i o n of M u s c l e C o n t r a c t i o n b y C a I o n : A S t u d y on H y b r i d T r o p o n i n s

165

E d e n s , B . , T h o m p s o n , H . A . , and Moldave, K . : S t u d i e s o n t h e B i n d i n g of A c y l a m i n o a c y l O l i g o n u c l e o t i d e to R a t L i v e r 6 0 S R i b o s o m a l S u b u n i t s a n d Its P a r t i c i p a t i o n in t h e P e p t i d y l t r a n s f e r a s e Reaction

179

Engstrom, L . , Berglund, L . , Bergstrom, Hjelmquist, G . , and L j u n g s t r o m , O . : P h o s p h o r y l a t i o n of P u r i f i e d P i g L i v e r Kinase

192

G., Regulatory Pyruvate

G a n o z a , M . C . and F o x , J . L . : A Soluble Elongation F a c t o r R e q u i r e d f o r P r o t e i n S y n t h e s i s with m R N A ' s other than Poly ( U )

205

G e r l a c h , U . a n d F e g e l e r , W . : V a r i a t i o n s in t h e L a c t a t e D e h y d r o g e n a s e I s o e n z y m e P a t t e r n in Arteries

216

G e v e r s , W . , J o n e s , P . A . , C o e t z e e , G . A . , and Westhuyzen, D . R . van d e r : Biochemical D e v e l o p m e n t of t h e H e a r t in S y r i a n H a m s t e r s

225

G o l d b e r g , I . H . , K a p p e n , L . S . , and S u z u k i , H . : O n t h e M e c h a n i s m of Inhibition of G l o b i n C h a i n Initiation b y P a c t a m y c i n

237

XI G o r d o n , J . , H o w a r d , G . A . , Stöffler, G . , and H i g h l a n d , J . H . : T h e R i b o s o m a l B i n d i n g S i t e of the Antibiotic T h i o s t r e p t o n

250

Haenni, A . L . , P r o c h i a n t z , A . , and Yot, P . : t R N A S t r u c t u r e s in V i r a l R N A G e n o m e s

264

Hess,

B . : R e m a r k s o n t h e A c q u i s i t i o n of A c t i v e Quaternary Structure of E n z y m e s

277

H i e r o w s k i , M. and B r o d e r s e n , R . : Covalent Binding of B i l i r u b i n t o A g a r o s e a n d U s e of t h e P r o d u c t f o r Affinity C h r o m a t o g r a p h y of S e r u m A l b u m i n

281

Hildebrand, J . G . and Kravitz, E . A . : T r a n s m i t t e r B i o c h e m i s t r y of S i n g l e , Identified N e u r o n s

298

Huberman, A . , Rodriguez, J . M . , Franco, R . , B a r a h o n a , E . : R e g u l a t i o n of A p o f e r r i t i n B i o s y n t h e s i s in R a t L i v e r

and 308

H ü l s m a n n , W . O . and J a n s e n , H . : High Lipoprotein L i p a s e Activity and C a r d i o v a s c u l a r D i s e a s e

322

K l e i n k a u f , H . and Koischwitz, H . : Gramicidin S - S y n t h e t a s e : A c t i v e F o r m of t h e M u l t i e n z y m e Complex is Undissociable by Sodium Dodecylsulfate

336

K r i s k o , I. and G y o r k e y , F . : S t u d i e s on the B i o s y n t h e s i s a n d S t r u c t u r e of R e n a l G l o m e r u l a r Basement Membrane v K u c a n , Z . : R i b o s o m e s and the T a r g e t T h e o r y Lee, Lill,

Liu,

S . G . : Interrelation Between Tyrocidine a n d S p o r u l a t i o n in B a c i l l u s b r e v i s

Synthesis

U . I . , B e h r e n d t , E . M . , and H a r t m a n n , I n t e r g e n e r i c C o m p l e m e n t a t i o n of R N A P o l y m e r a s e Subunits

G.R.:

C . K . , Legocki, A . B . , and Weissbach, S t u d i e s on L i v e r Elongation F a c t o r 1

H.:

345 359 368

377

M a a s , W . K . : S o m e T h o u g h t s o n t h e R e g u l a t i o n of A r g i n i n e B i o s y n t h e s i s a n d Its R e l a t i o n to Biochemistry

384

399

XII Mano,

Y . , S u z u k i , N . , Murakami, K . , and K a n o , K . : M e c h a n i s m of t h e C e l l C y c l e in D N A S y n t h e s i s in S e a U r c h i n E m b r y o s

M a r c h i s - M o u r e n , G . , Marvaldi, J . , and C o z z o n e , R i b o s o m e M e t a b o l i s m in S t a r v i n g R e l a x e d E .coli C e l l s

404

A.: 410

M a t s u u r a , T . and J o n e s , M . E . : Subcellular L o c a l i z a t i o n in t h e E h r l i c h A s c i t e s C e l l of t h e E n z y m e which Oxidizes D i h y d r o o r o t a t e to O r o t a t e

422

N o m b e l a , C . , N o m b e l a , N . A . , and O c h o a , S . : C o m p a r i s o n of P o l y p e p t i d e C h a i n Initiation F a c t o r s f r o m A r t e m i a salina and Rabbit Reticulocytes

435

Nose,

Y . , Iwashima, A . , and Matsuura-Nishino, T h i a m i n e - B i n d i n g P r o t e i n of E s c h e r i c h i a coli

A.: 443

Ofengand, J . and S c h w a r t z , I . : Photoaffinity L a b e l l i n g of t R N A B i n d i n g S i t e s o n E . c o l i Ribosomes

456

O r r e g o , F . : Identification of C e n t r a l T r a n s m i t t e r s Electrically Stimulated Brain Slices Park,

with

J . H . and M e r i w e t h e r , B . P . : T h e Acetylation of C y s t e i n e - 2 8 1 in G l y c e r a l d e h y d e - 3 - P h o s p h a t e D e h y d r o g e n a s e by an S S T r a n s f e r Reaction

Parmeggiani, A . , Sander, G . , Marsh, R . C . , Voigt, J . , Nagel, K . , and Chinali, G . : R e g u l a t i o n of E l o n g a t i o n F a c t o r G - R i b o s o m a l G T P a s e Activity Penit,

C . , P a r a f , A . , and Chapeville, F.: D e o x y n u c l e o t i d e - P o l y m e r i z i n g E n z y m e s of Myelomas

471

487

499

Murine 511

R i c h t e r , D . and M i l l e r , W . : P r o p e r t i e s and Functions of E t h a n o l - P o t a s s i u m C h l o r i d e E x t r a c t a b l e P r o t e i n s f r o m 80S R i b o s o m e s and their Interchangeability with the B a c t e r i a l P r o t e i n s L 7 / L 1 2

524

R o s k o s k i , R . : Choline A c e t y l t r a n s f e r a s e : of t h e A c t i v e S i t e S u l f h y d r y l G r o u p

534

Reactions

XIII Schweiger, M., Herrlich, P . , Rahmsdorf, H . J . , Pai, S . H . , Ponta, H . , Hirsch-Kauffmann, M.: C o n t r o l of G e n e E x p r e s s i o n by E . c o l i V i r u s T 7

547

S p e c t o r , L . B . : Covalent E n z y m e - S u b s t r a t e I n t e r m e d i a t e s in C a r b o x y l Activation

564

S r e r e , P . A . and S i n g h , M . : T h e R o l e of P a n t o t h e n a t e in C i t r a t e L y a s e

575

S t r e e c k , R . E . , F i t t i e r , F . , and Z a c h a u , H . G . : C l e a v a g e of S m a l l D N A s with R e s t r i c t i o n Nucleases

589

Sy,

J . : T h e R i b o s o m a l and N o n r i b o s o m a l of G u a n o s i n e P o l y p h o s p h a t e s

Synthesis 599

S z a f r a n s k i , P . , Filipowicz, W . , Wodnar-Filipowicz, and Z a g o r s k a , L . : P h a g e f2 R N A S t r u c t u r e in Relation to S y n t h e s i s of P h a g e P r o t e i n s T a k e d a , M . and N i s h i z u k a , Y . : P r o t e i n from Eukaryotic Organisms Tao,

A., 610

Kinases 623

M . , Y u h , K . - C . , and H o s e y , M . M . : T h e Interaction of R e d Blood Cell P r o t e i n F a c t o r s with C y c l i c A M P

636

W a k a b a y a s h i , K . : On t h e L i n k a g e and R e c o m b i n a t i o n of Mitochondrial G e n e s

647

Wiegers, U . , K r a m e r , G . , Klapproth, K . , Wiegers, U . , and H i l z , H . : D e t e r m i n a t i o n of m R N A and 28 S R N A T u r n o v e r in P r o l i f e r a t i n g H e L a C e l l s

658

Annual F r i t z Lipmann Lynen,

F.:

Photographs

Lecture

S t r u c t u r e s of Biotin E n z y m e s

671

699

List of Participants of the Lipmann Symposium ACS,

G.

T h e M t . S i n a i S c h o o l of M e d i c i n e ,

New York,

AUHAGEN, E. T r e a s u r e r of t h e G e s e l l s c h a f t f ü r B i o l o g i s c h e Wuppertal, Germany BADDILE Y, J. T h e U n i v e r s i t y of N e w c a s t l e , BAUER,

Chemie,

N e w c a s t l e upon T y n e ,

England

K.

Technische Universität Berlin, BENNETT, Florida

USA

BERGMEYER,

Tallahasse,

S.

Tutzing,

BLOEMENDAL,

USA

Nymegen,

The

Netherlands

G.

U n i v e r s i t y of U m e a , A.

Los Angeles,

H.

U n i v e r s i t y of N i j m e g e n ,

BRODIE,

Germany

P.

U n i v e r s i t y of S o u t h e r n C a l i f o r n i a ,

H.

USA

H.-U.

Mannheim C o m p a n y ,

BESSMAN,

BOMAN,

Germany

T.P.

State University,

Boehringer

Berlin,

Umea,

Sweden

F.

U n i v e r s i t y of S o u t h e r n C a l i f o r n i a , CHANTRENNE,

Los Angeles,

H.

L a b o r a t o i r e de Chimie Biologique, CHAPEVILLE,

USA

Bruxelles,

Belgium

F.

Institut d e B i o l o g i e M o l é c u l a i r e ,

Paris,

France

CRAMER, F. Max-Planck-Institut f ü r Experimentelle Medizin, G eRrO mK a nAyE R T , R . C Université L i b r e de Bruxelles, E UB n iA v eSrH s iIt ,y ofS . T o k y o ,

Tokyo,

Bruxelles, Japan

Göttingen,

Belgium

XV E H R E N S T E I N , G . von M a x - P l a n c k - I n s t i t u t für E x p e r i m e n t e l l e Medizin, Germany

Göttingen,

E I G E N , M. Max-Planck-Institut für Biophysikalische Chemie, Germany ELLIOTT,

W.

H.

U n i v e r s i t y of A d e l a i d e , ENGSTRÖM,

Adelaide,

Australia

L. Uppsala,

Institute of Medical C h e m i s t r y , FISCHER, J.

GERLACH,

Austin,

USA

Münster,

Germany

H.

Schering A G ,

Berlin,

GOLDBERG,

I.

Germany

H.

H a r v a r d Medical S c h o o l ,

Boston,

USA

J.

F r i e d r i c h M i e s c h e r Institut, B a s e l , HAENNI,

Germany

U.

Universität M ü n s t e r ,

GORDON,

Freiburg,

L.

U n i v e r s i t y of T e x a s ,

GIBIAN,

Sweden

H.

M a x - P l a n c k - I n s t i t u t für Immunbiologie, FOX,

Göttingen,

A.

L.

Institut de B i o l o g i e M o l e c u l a i r e , HARTMANN,

Switzerland

Paris,

France

G.

Universität München,

München,

Germany

HESS, B. M a x - P l a n c k - I n s t i t u t für E r n ä h r u n g s p h y s i o l o g i e , D o r t m u n d , Germany HELMREICH, E . C h a i r m a n of the G e s e l l s c h a f t für B i o l o g i s c h e C h e m i e , W bu Hü E rRzR L rIgC, H G , e rPm. a n y M a x - P l a n c k - I n s t i t u t f ü r Molekulare G e n e t i k , HnI E U i vRe O r sW i t yS KofI , A M a r.h u s ,

Aarhus,

Denmark

Berlin,

Germany

XVI HILDEBRAND, J. G. H a r v a r d Medical S c h o o l , HUBERMAN,

Boston,

USA

A.

Instituto N a c i o n a l d e l a N u t r i c i ó n , HÜLSMANN,

Mexico City,

W.C.

Erasmus

University,

JONES,

M.

Rotterdam,

The

KeLcEh nI N F ,n i v H T i sK c hAeU U e r.s i t ä t B e r l i n , KREBS, H. A. Oxford, England

Los Angeles,

Berlin,

USA

G e r m/a n y

I.

V e t e r a n s Administration Hospital, KUCAN,

Houston,

USA

Z.

" R u g j e r Bo&kovic" Institute, LIPMANN,

Zagreb,

New York,

LYNEN, F. Max-Planck-Institut für Biochemie, G eAr N mO a n, y Y . M U n i v e r s i t y of T o k y o , MARCHIS-MOUREN,

Tokyo,

M0LLER,

USA

Martinsried/München,

Japan

G.

Institut d e C h i m i e B i o l o g i q u e ,

Carlsberg

Yugoslavia

F.

The Rockefeller University,

Marseille,

France

K. Laboratorium,

NIEMEYER,

Copenhagen,

Denmark

H.

Universidad de Chile, NOSE,

Netherlands

E.

U n i v e r s i t y of S o u t h e r n C a l i f o r n i a ,

KRISKO,

Mexico

Santiago,

Chile

Y.

K y o t o P r e f e c t u r a l U n i v e r s i t y of M e d i c i n e , OFENGAND,

Japan

J.

R o c h e Institute of M o l e c u l a r B i o l o g y , ORREGO,

Kyoto,

Nutley,

USA

F.

Instituto i on.ni va el r sdiet y C M a reddi ioclaol g iSac, h oMo el ,x i cNo a sChivt yi l,l e ,M eU x iSc oA V PA a nRd eKr ,b i N l tJa.cUH

XVII P ARMEGGIANI, A . Gesellschaft für Molekularbiologische Stöckheim/Braunschweig, Germany RICHTER,

Forschung,

D.

Universität H a m b u r g , ROSKOSKI,

Hamburg,

Germany

R.

U n i v e r s i t y of I o w a , SCHUSTER,

Iowa City,

USA

H.

Max-Planck-Institut für Molekulare Genetik, SCHWEIGER,

STADTMAN,

New

Dallas,

York,

USA

USA

E.R.

N a t i o n a l I n s t i t u t e s of H e a l t h ,

Bethesda,

USA

J.

The Rockefeller University, SZAFRANSKI,

New York,

USA

Warsaw,

Poland

P.

P o l i s h A c a d e m y of S c i e n c e s , TAKEDA,

M.

Kobe University, TAO,

Germany

P.A.

T h e U n i v e r s i t y of T e x a s ,

SY,

Berlin,

L.

T h e Rockefeller University, SRERE,

Germany

M.

Max-Planck-Institut für Molekulare Genetik, SPECTOR,

Berlin,

Kobe,

Japan

M.

U n i v e r s i t y of I l l i n o i s , TRAUT,

R.

Chicago,

R.

U n i v e r s i t y of C a l i f o r n i a , TRAUTNER,

USA

T.

Davis,

USA

A.

Max-Planck-Institut f ü r Molekulare Genetik, URETA,

Berlin,

T.

Universidad de Chile,

Santiago,

V A D W U F onE Ari N vsKceN A hr suE BintS AygLY s sof At eN SlTH l oeI,k, yVoeBK ,n. n. Te os lkaynod, ,

Chile

JBaepral ni n ,

Germany

Germany

XVIII

WITT, H . T . Technische Universität Berlin, B e r l i n ,

Germany

WITTMANN, H. G . Max-Planck-Institut für Molekulare Genetik, B e r l i n ,

Germany

W I T T M A N N - U E B Ofür L D ,Molekulare B. Max-Planck-Institut Genetik, B e r l i n , ZACHAU, H. G. Universität München, München, Germany

Germany

List of Contributors Acs, G. 150 Adler, S . P . 28 Baddiley, J . 40 Barahona, E . 308 Bauer, K. 53 Behrendt, E . M . 377 Bennett, T . P . 63 Berglund, L . 192 Bergström, G. 192 Bessman, S . P . 77 Bloemendal, H . 89 Boman, H . G . 103 Brodersen, R . 281 Brodie, A . F . 115 Burnett, J . P . 40 Cabrer, B. 131 Chantrenne, H . 144 Chapeville, F . 511 C h i n a l i , G . 499 C h r i s t m a n , J . K . 150 Coetzee, G . A . 225 Cozzone, A . 410 Ebashi, S . 165 E d e n s , B . 179 Engström, L . 192 Fegeier, W. 216 Filipowicz, W. 610 Fittier, F . 589 Fox, J . L . 205 Franco, R. 308 Ganoza, M . C . 205 Gerlach, U. 216 Gevers, W. 225 Goldberg, I . H . 237 Gordon, J . 131, 250 Gyorkey, F . 345 Haenni, A . L . 264 Hancock, I . C . 40 Hartmann, G . R . 377 Helmreich, E . J . M . 1

Heptinstall, J . 40 Herrlich, P . 547 Hess, B. 277 Hierowski, M. 281 Highland, J . H . 250 Hildebrand, J . G . 298 Hilz, H . 658 Hirsch-Kauffmann, M. 547 Hjelmquist, G . 192 Hosey, M.M. 636 Howard, G . A . 250 Huberman, A . 308 Hülsmann, W . C . 322 Iwashima, A . 443 Jansen, H . 322 Jones, M . E . 422 Jones, P . A . 225 Kalra, V . K . 115 Kano, K. 404 Kappen, L . S . 237 Klapproth, K . 658 Kleinkauf, H . 336 Koischwitz, H . 336 Kosmakos, F . C . 115 Kramer, G. 658 Kravitz, E . A . 298 Krebs, H . A . 7 K r i s k o , I. 345 Kucan, £ . 359 Lee, S . - H . 115 L e e , S .G . 368 Legocki, A . B . 384 Lill, U . I . 377 Lipmann, F . 7 Liu, C . K . 384 Ljungström, O. 192 Lynen, F . 671 Maas, W . K . 399 M a n o , Y. 4 0 4 Marchis-Mouren, G . 410

XX Marsh, R . C . 499 Marvaldi, J . 410 Matsuura, T . 422 Matsuura-Nishino, A . 443 Meriwether, B . P . 487 Modolell, J . 131 Moldave, K . 179 M^fller, W . 524 Murakami, K . 404 499 Nagel, K . N i l s s o n - F a y e , I. 103 Nishizuka, Y . 623 Nombela, C . 435 Nombela, N . A . 435 N o s e , Y. 4 4 3 Ochoa, S . 435 Ofengand, J . 456 O r r e g o , F . 471 Pai, S . H . 547 Paraf, A. 511 P a r k , J . H . 487 Parmeggiani, A . 499 Penit, C . 511 Ponta, H. 547 P r a s a d , R . 115 P r o c h i a n t z , A . 264 Rahmsdorf, H . J . 547 Rasmuson, T . , J r . 103 Richter, D . 524 Rodriguez, J.M. 308 Roskoski, R . 534 Sander, G. 499 San-Millan, M . J . 131 S c h w a r t z , I. 456 S c h w e i g e r , M. 547 Singh, M. 575 Spector, L . B . 564 S r e r e , P . A . 575 Stadtman, E . R . 28 Stöffler, G . 250 Streeck, R . E . 589 S t r o u s , G . J . A . M . 89 Suzuki, H . 237 Suzuki, N . 404

599 Sy, J. Szafranski, P . 610 T a k e d a , M. 623 T a o , M. 636 Thompson, H . A . 179 Vazquez, D . 131 Voigt, J . 499 Wakabayashi, K . 647 W e s t h u y z e n , D . R . v a n d e r 225 Weissbach, H . 384 Wiegers, U . 658 Wiegers, U. 658 Wittmann, H . G . 5 W o d n a r - F i l i p o w i c z , A . 610 Yot, P . 264 Y u h , K . - C . 636 Zachau, H . G . 589 Zagorska, L . 610

Welcome to Professor Lipmann

Ernst J. Vorstand

M. Helmreich der Gesellschaft fur Biologische

Chemie

It i s i n d e e d a m e m o r a b l e e v e n t t o h o n o r P r o f e s s o r F r i t z L i p m a n n at t h e o c c a s i o n of h i s 75th b i r t h d a y h e r e in B e r l i n . In 1970 P r o f e s s o r L i p m a n n w r o t e a p e r s o n a l a c c o u n t of h i s life a s a B i o c h e m i s t w h i c h w a s p u b l i s h e d b y W i l e y I n t e r s c i e n c e 1971 w i t h t h e t i t l e : " W a n d e r i n g s of a B i o c h e m i s t " . T h i s d e l i g h t f u l little b o o k , b e c a u s e it i s s o h o n e s t a n d u n p r e t e n t i o u s , h a s i m p r e s s e d m e a s I a m c e r t a i n it h a s i m p r e s s e d e v e r y B i o c h e m i s t w h o h a s w o r k e d at l e a s t in p a r t d u r i n g t h i s p e r i o d . M o r e o v e r , it m a k e s c l e a r w h y t o h o n o r P r o f e s s o r ' L i p m a n n h e r e in B e r l i n , f i t s t h e o c c a s i o n b e c a u s e it w a s in B e r l i n at t h e K a i s e r W i l h e l m I n s t i t u t e f o r B i o l o g y a t D a h l e m , w h e n h e j o i n e d O t t o M e y e r h o f ' s L a b o r a t o r y in 1927 w h e r e h e w a s e x p o s e d to p e r h a p s t h e m o s t i m p o r t a n t intellectual i n f l u e n c e s in h i s life w h i c h h a v e m o l d e d h i s s c i e n t i f i c p h i l o s o p h y a n d i n t e r e s t s . A s P r o f e s s o r L i p m a n n w r i t e s : "In t h e F r e u d i e n s e n s e all t h a t I did l a t e r w a s s u b c o n s c i o u s l y m a p p e d o u t h e r e a n d it s t a r t e d t o m a t u r e b e t w e e n 1930 a n d 1940 a n d w a s m o r e e l a b o r a t e l y r e a l i z e d f r o m t h e n o n " . It i s n o t m y t a s k t o e n u m e r a t e the many d i s c o v e r i e s which P r o f e s s o r Lipmann and h i s a s s o c i a t e s h a v e m a d e o v e r t h e y e a r s in s o m a n y d i f f e r e n t a r e a s of B i o c h e m i s t r y . T h e y a r e b i o c h e m i c a l h i s t o r y a n d a r e t a u g h t all o v e r t h e w o r l d t o s t u d e n t s . W h e n I h a v e s e l e c t e d a f e w e x a m p l e s of P r o f e s s o r L i p m a n n ' s c o n t r i b u t i o n it i s o b v i o u s that my choice w a s a r b i t r a r y and dictated by my o w n r e s e a r c h i n t e r e s t s . B u t p e r h a p s in a t i m e w h e r e m a n y y o u n g e r c o l l e a g u e s a n d s t u d e n t s l a c k s e n s e f o r h i s t o r i c a l r e l a t i o n s h i p s , it might b e w o r t h w h i l e to s h o w on a f e w e x a m p l e s h o w i n f l u e n tial P r o f e s s o r L i p m a n n ' s w o r k w a s . T h i s m i g h t h e l p t o a r r i v e at a m o r e b a l a n c e d v i e w of t h e e v e n t s w h i c h s e t t h e p a c e a n d h e l p e d s h a p e t h e B i o l o g y of o u r a g e . Among the two examples which I have chosen one dates back t o 1*932/33. It w a s t h e c h a r a c t e r i z a t i o n of t h e p h o s p h o r y l s e r y l e s t e r b o n d in c e r t a i n p r o t e i n s . P r o f e s s o r L i p m a n n w r i t e s in h i s m e m o i r s : " W h e n 4 0 y e a r s a g o I c h o s e t o p r o b e into t h e b i n d i n g of t h e p h o s p h a t e in t h e s e p h o s p h o p r o t e i n s ,

2 unwittingly, it now t u r n s out, I s t r u c k a gold m i n e " . P h o s p h o rylation not only of e n z y m e s but of a v a r i e t y of p r o t e i n s , s o different a s the histone and n o n - h i s t o n e p r o t e i n s of c h r o m a t i n , o r m e m b r a n e and c o n t r a c t i l e p r o t e i n s a r e among the most i n t e r e s t i n g control d e v i c e s w h i c h h a v e e m e r g e d in the e v o l u t ion of living s y s t e m s . H o w e v e r , t h e s e and many o t h e r i m p o r t ant d i s c o v e r i e s a r e o v e r s h a d o w e d by p e r h a p s P r o f e s s o r L i p m a n n ' s most important c o n t r i b u t i o n . T h a t is the c o n c e p t of the e n e r g y r i c h bond and the i d e a of a p h o s p h o r y l g r o u p t r a n s f e r potential. T h e r e a r e s o m e of u s h e r e w h o h a d the h o n o r to b e invited to contribute to the F r i t z L i p m a n n d e d i c a t o r y volume " C u r r e n t A s p e c t s of B i o c h e m i c a l E n e r g e t i c s " w h i c h w a s edited by Nathan O . K a p l a n and E u g e n e P . K e n n e d y at the 25th A n n i v e r s a r y of the P u b l i c a t i o n of F r i t z L i p m a n n ' s c l a s s i c p a p e r on the " M e t a b o l i c G e n e r a t i o n and Utilization of P h o s p h a t e B o n d E n e r g y " . On the c o v e r of that book it w a s rightly stated that t h i s contribution had o p e n e d the w a y to an u n d e r s t a n d i n g of the e n e r g e t i c b a s i s of b i o synthetic r e a c t i o n s and g r e a t l y c l a r i f i e d the relation b e t w e e n e x e r g o n i c and e n d e r g o n i c p r o c e s s e s in the living c e l l . B u t a s i d e f r o m h i s scientific a c h i e v e m e n t s t h e r e i s a n o t h e r a s p e c t w h i c h should not b e f o r g o t t e n at t h i s o c c a s i o n . W e only h a v e to look at the p r o g r a m of this S y m p o s i u m to r e a lize w h a t P r o f e s s o r L i p m a n n h a s done f o r B i o c h e m i s t r y , especially also for European B i o c h e m i s t r y . T h e contributors to the S c i e n t i f i c P r o g r a m of t h i s S y m p o s i u m a r e h i s s t u d e n t s and b i o c h e m i c a l r e s e a r c h in E u r o p e h a s g r e a t l y benefitted f r o m t h e m . Many of them a r e H e a d s of D e p a r t m e n t s o r l e a d independent r e s e a r c h g r o u p s . I am p l e a s e d to a n n o u n c e t o d a y that the G e s e l l s c h a f t f u r B i o l o g i s c h e C h e m i e a s a t o k e n of o u r gratitude h a s installed an A n n u a l F r i t z L i p m a n n L e c t u r e m a d e p o s s i b l e by a g e n e r o u s financial contribution of B o e h r i n g e r Mannheim C o m p a n y . I am e s p e c i a l l y indebted to P r o f e s s o r H . U . B e r g m e y e r without its help this would not h a v e b e e n p o s s i b l e . P r o f e s s o r L y n e n will g i v e the f i r s t F r i t z L i p m a n n L e c t u r e at t h e c o n c l u s i o n of this s y m p o s i u m . A s a b i o c h e m i s t , coming f r o m W u r z b u r g I w a s thinking about something r e l a t e d with that U n i v e r s i t y and with B i o c h e m i s t r y and w h i c h might b e of i n t e r e s t to P r o f e s s o r L i p m a n n . T h e n I r e c a l l e d that my c o l l e a g u e Guido H a r t m a n n s o m e t i m e ago w h e n h e w a s still at W u r z b u r g U n i v e r s i t y had p r e s e n t e d m e with r e p r i n t s of two p a p e r s p u b l i s h e d by R o b e r t E . K o h l e r

3 f r o m t h e D e p a r t m e n t of H i s t o r y of S c i e n c e at H a r v a r d U n i v e r s i t y . T h e p a p e r s a p p e a r e d in t h e J o u r n a l of t h e H i s t o r y of B i o l o g y 1971, 1972 a n d a r e e n t i t l e d : " T h e B a c k g r o u n d to E d u a r d B u c h n e r ' s D i s c o v e r y of C e l l F r e e F e r m e n t a t i o n a n d t h e R e c e p t i o n of E d u a r d B u c h n e r ' s D i s c o v e r y of C e l l F r e e F e r m e n t a t i o n . A s P r o f e s s o r L i p m a n n w r i t e s in h i s r e c o l l e c t i o n s r " W h a t I delight in h e r e i s B u c h n e r ' s p r e o c c u p a t i o n w i t h t h e a c c i d e n t a l " . K o h l e r ' s h i s t o r i c a l s t u d y quite c o n v i n c i n g l y s h o w s t h a t H a n s B ü c h n e r , w h o w a s an I m m u n o l o g i s t a n d P r o f e s s o r of M e d i c a l M i c r o b i o l o g y a n d P u b l i c H e a l t h in M u n i c h w a s t h e i n t e l l e c t u a l d r i v i n g f o r c e a n d t h a t t h e d i s c o v e r y of z y m a s e actually e m e r g e d f r o m H a n s B u c h n e r ' s immunological s t u d i e s . E d u a r d B u c h n e r ' s e x p e r i m e n t s w e r e actually u n d e r t a k e n to e l u c i d a t e t h e r o l e of P r o t o p l a s m i c p r o t e i n s f o r i m m u n i t y . It w a s in t h e c o u r s e of t h e s e s t u d i e s t h a t E d u a r d B u c h n e r w h o l a t e r b e c a m e p r o f e s s o r of b i o c h e m i s t r y a n d o r g a n i c c h e m i s t r y in W ü r z b u r g t o g e t h e r with M a r t i n H a h n d e v e l o p e d a m e t h o d f o r getting p r e s s j u i c e w h i c h f a c i l i t a t e d t h e s t u d y of i n t e r c e l l u l a r e n z y m e s a n d p r o v e d cell f r e e f e r m e n t a t i o n in 1897. T h i s d i s c o v e r y h a s long b e e n r e c o g n i z e d a s t h e r e s o l u t i o n of o n e of t h e m o s t f a m o u s s c i e n t i f i c c o n t r o v e r s i e s of t h e 19th c e n t u r y , namely the c o n t r o v e r s y between L o u i s P a s t e u r and J u s t u s v o n L i e b i g o v e r t h e n a t u r e of a l c o h o l i c f e r m e n t a t i o n . T h e r e f o r e G u i d o H a r t m a n n a n d I, t w o B i o c h e m i s t s f r o m W ü r z b u r g t a k e t h e p l e a s u r e to p r e s e n t to P r o f e s s o r L i p m a n n t o d a y t o g e t h e r with t h e s e t w o r e p r i n t s a r e p r i n t of a p a p e r " U b e r Z e l l f r e i e G ä r u n g " in w h i c h E d u a r d B u c h n e r himself g a v e a n a c c o u n t of h i s d i s c o v e r y in a l e c t u r e g i v e n 1909 a n d p u b l i s h e d in t h e Z e i t s c h r i f t d e s " O s t e r r e i c h i s c h e n I n g e n i e u r u n d A r c h i t e k t e n V e r e i n s " . In t h i s l e c t u r e E d u a r d B u c h n e r s a i d : " - D e r m i s s l i c h s t e P u n k t ist a b e r j e d e n f a l l s d e r , d a ß w i r d u r c h a u s n i c h t in d e r L a g e s i n d , die E n z y m e z u i s o l i e r e n . B e i R e i n i g u n g s b e s t r e b u n g e n ist d e r e r s t e E r f o l g m e i s t d e r , d a ß a u c h die W i r k s a m k e i t d e s b e t r e f f e n d e n P r ä p a r a t e s a b n i m m t " . T h i s s o u n d s f a m i l i a r to e v e r y e n z y m o l o gist but it a l s o s h o w s that t h a n k s to P r o f e s s o r L i p m a n n a n d t h e b i o c h e m i s t s of h i s t i m e w e h a v e finally s o l v e d t h i s a n d many other p r o b l e m s b e c a u s e w e have followed G o e t h e ' s a d v i c e with w h i c h E d u a r d B u c h n e r c o n c l u d e d its l e c t u r e . H e s a i d : "Wenn a b e r einmal u n s e r e B e m ü h u n g e n t a t s ä c h lich j a h r e l a n g o h n e E r f o l g b l e i b e n s o l l t e n , d a n n l a s s e n S i e uns recht d e r Worte Goethes eingedenk sein: " D e r Mensch muß bei d e m G e d a n k e n v e r h a r r e n , daß d a s U n b e g r e i f l i c h e b e g r e i f l i c h s e i ; e r w ü r d e s o n s t nicht f o r s c h e n ! "

4 B e f o r e P r o f e s s o r Wittmann o u r h o s t h e r e in B e r l i n will officially o p e n t h e F r i t z L i p m a n n S y m p o s i u m w h i c h w a s o r g a nized by D r . D i e t m a r R i c h t e r , I w i s h to a c k n o w l e d g e the v e r y g e n e r o u s c o n t r i b u t i o n s of t h e VW F o u n d a t i o n , t h e S c h e r i n g A . G . and the Walter de G r u y t e r V e r l a g , B e r l i n . I a m s u r e t h a t all t h e p a r t i c i p a n t s a r e l o o k i n g f o r w a r d t o b e with f r i e n d s a n d f d r h a v i n g t h e o p p o r t u n i t y t o d i s c u s s s c i e n c e and r e m i n i s c e about the time they spent t o g e t h e r and with P r o f e s s o r L i p m a n n in t h e l a b o r a t o r y . I w i s h P r o f e s s o r L i p m a n n , his students and his f r i e n d s a v e r y good and e n j o y a b l e t i m e w h i l e h e r e in B e r l i n .

Welcome to the Max-Planck-Institut

H . G . Wittmann Max-Planck-Institut fur Molekulare Berlin-Dahlem, Germany Dr.

Lipmann,

L a d i e s and

Genetik

Gentlemen:

It g i v e s m e g r e a t p l e a s u r e t o w e l c o m e y o u all to t h e M a x P l a n c k - I n s t i t u t f u r M o l e k u l a r e G e n e t i k in B e r l i n - D a h l e m . It w a s h e r e in D a h l e m w h e r e F r i t z L i p m a n n , o u r g u e s t of h o n o u r t o d a y , b e g a n h i s s c i e n t i f i c c a r e e r a s a b i o c h e m i s t a l m o s t fifty y e a r s a g o . A t t h a t t i m e , D a h l e m w a s t h e s c i e n t i f i c c e n t e r in G e r m a n y . T h i s w a s m a i n l y d u e t o t h e w o r k d o n e in t h e v a r i o u s K a i s e r - W i l h e l m - I n s t i t u t e s l o c a t e d h e r e . Y o u will h e a r m o r e a b o u t t h i s p e r i o d of t i m e in t h e n e x t t w o l e c t u r e s . A t t h e e n d of t h e w a r , m a n y of t h e s e i n s t i t u t e s m o v e d f r o m B e r l i n to W e s t - G e r m a n y . D u r i n g t h e l a s t 25 y e a r s , t h e n u m b e r of t h e i n s t i t u t e s ( r e n a m e d t h e M a x - P l a n c k I n s t i t u t e s ) i n c r e a s e d in n u m b e r to a b o u t 50 s t a f f e d b y approximately 11.000 scientists, e n g i n e e r s and technicians. If y o u a r e i n t e r e s t e d in o b t a i n i n g m o r e d e t a i l s a b o u t t h e M a x - P l a n c k - G e s e l l s c h a f t and the institutes, t h e r e is a booklet available containing information about the o r g a n i z ation a n d t h e b u d g e t of t h e M P G , a s w e l l a s t h e s c i e n t i f i c w o r k d o n e in e a c h of t h e i n s t i t u t e s . T h e w o r k in o u r institute i s mainly c o n c e r n e d with t h e b i o c h e m i c a l a n d g e n e t i c a s p e c t s of n u c l e i c a c i d a n d p r o t e i n b i o s y n t h e s i s in b a c t e r i a a n d p h a g e s . T h e r e a r e a b o u t 2 0 0 people ( s c i e n t i s t s , technicians and g r a d u a t e s t u d e n t s ) w o r k i n g on the following s u b j e c t s : D N A replication; prophage-induction; D N A - m e m b r a n e interation; mechanism of cell division a n d c o n j u g a t i o n ; r e c o m b i n a t i o n , t r a n s f e c t i o n a n d t r a n s f o r m a t i o n ; s t r u c t u r e a n d f u n c t i o n of r i b o s o m e s , which especially includes studies on the p r i m a r y s t r u c t u r e of r i b o s o m a l p r o t e i n s , on t h e t o p o g r a p h y of t h e s u b u n i t s a n d on t h e f u n c t i o n a l r o l e of t h e r i b o s o m a l c o m ponents; p r o t e i n - R N A interaction; mechanism and r e -

6 gulation of t r a n s l a t i o n ; D N A d e p e n d e n t p r o t e i n b i o s y n t h e s i s in v i t r o . B e s i d e s t h e s e s t u d i e s o n p r o k a r y o t e s t h e r e a r e t w o g r o u p s w o r k i n g on e u k a r y o t i c s y s t e m s , namely on p h o t o t r o p i s m in P h y c o m y c e s a n d c o n j u g a t i o n in C i l i a t a . A n y b o d y w h o i s i n t e r e s t e d in d e t a i l s of t h e w o r k d o n e in o u r i n s t i t u t e , h e i s w e l c o m e to e i t h e r a p p r o a c h d i r e c t l y t h e g r o u p in w h i c h h e i s i n t e r e s t e d o r , if h e i s u n s u r e w h e r e to g o , p l e a s e c o m e a n d s e e m e s o t h a t I c a n m a k e the a p p r o p r i a t e i n t r o d u c t i o n s . P l e a s e r e g a r d o u r institute a s a n o p e n h o u s e a n d w e will t r y o u r b e s t to m a k e y o u r visit in B e r l i n a n d in o u r institute a s e n j o y a b l e a s p o s s i b l e . L e t m e e n d b y w i s h i n g all of o u r g u e s t s a p l e a s a n t t i m e in B e r l i n a n d D r . L i p m a n n a v e r y h a p p y s e v e n t y - f i f t h birthday.

Dahlem in the Late Nineteen Twenties

Hans A .

K r e b s and Fritz

Metabolic R e s e a r c h

Lipmann

Laboratory,

Nuffield D e p a r t m e n t of

Clinical Medicine, Radcliffe I n f i r m a r y , T h e Rockefeller University,

Hans A .

Oxford,

New York,

England

and

USA

Krebs

It w a s D r . R i c h t e r w h o s u g g e s t e d t h a t I might r e m i n i s c e on t h e life at D a h l e m in t h e d a y s w h e n F r i t z L i p m a n n a n d I w e r e w o r k i n g h e r e in t h e l a b o r a t o r i e s of Otto M e y e r h o f a n d Otto W a r b u r g . I w a s in D a h l e m f r o m 1926 to 1930, a n d F r i t z L i p m a n n f r o m 1927-1930. S o I will m a k e a n a t t e m p t to c o n v e y s o m e t h i n g of t h e a t m o s p h e r e , of t h e w o r k i n g a n d living c o n d i t i o n s , of o u r a t t i t u d e s t o w a r d s o u r w o r k a n d of o u r outlook in g e n e r a l . T h e K a i s e r Wilhelm G e s e l l s c h a f t D a h l e m w a s at t h a t t i m e t h e m a i n c a m p u s of t h e K a i s e r Wilhelm G e s e l l s c h a f t (in 1948 r e n a m e d M a x - P l a n c k - G e s e l l s c h a f t ) . T h i s S o c i e t y w a s initiated in 1910 with t h e intention of p r o v i d i n g o u t s t a n d i n g s c i e n t i s t s with f i r s t - r a t e r e s e a r c h f a c i l i t i e s . T h e attitude of t h e f o u n d e r s w a s c l e a r l y e x p r e s s e d b y E m i l F i s c h e r w h e n h e t r i e d to p e r s u a d e - s u c c e s s f u l l y R i c h a r d W i l l s t a t t e r to a b a n d o n h i s p r o f e s s o r s h i p at Z u r i c h a n d to join t h e S o c i e t y . F i s c h e r , a c c o r d i n g to W i l l s t a t t e r ( 1 ) , d e s c r i b e d t h e attitude in t h e s e w o r d s : " Y o u will b e c o m p l e t e ly i n d e p e n d e n t . N o - o n e will e v e r t r o u b l e y o u . N o - o n e will e v e r i n t e r f e r e . Y o u m a y w a l k in t h e w o o d s f o r a f e w y e a r s , if y o u l i k e ; y o u m a y p o n d e r o v e r s o m e t h i n g b e a u t i f u l " . O n the w h o l e this policy ( b a s e d on utmost c a r e and c o m p e t e n c e in s e l e c t i n g t h e r i g h t p e o p l e ) h a s p a i d m a g n i f i c e n t d i v i d e n d s : Otto W a r b u r g , Otto M e y e r h o f , A l b e r t E i n s t e i n , M a x v o n L a u e , F r i t z H a b e r , Otto H a h n , L i s e M e i t n e r , C a r l E r i c h C o r r e n s ,

8 R i c h a r d Goldschmidt, Michael P o l a n y i , C a r l N e u b e r g and m a n y o t h e r s m a d e t h e f u l l e s t u s e of t h e o p p o r t u n i t i e s . B y t h e l a t e 1 9 2 0 ' s , within 15 y e a r s of its f o u n d a t i o n , a n d d e s p i t e t h e u p s e t c a u s e d b y W o r l d W a r I, D a h l e m h a d b e c o m e o n e of t h e w o r l d c e n t r e s of s c i e n t i f i c r e s e a r c h . N o t only did it a t t r a c t m a n y of t h e b e s t s c i e n t i s t s in G e r m a n y but a l s o y o u n g p e o p l e f r o m all o v e r t h e w o r l d . C o l l a b o r a t o r s of W a r b u r g a n d M e v e r h o f .

1926-1930

D u r i n g m y s t a y at D a h l e m t h e p e o p l e w o r k i n g in t h e l a b o r a t o r y of W a r b u r g i n c l u d e d E r w i n N e g e l e i n , H a n s G a f f r o n , Robert E m e r s o n , Fritz Kubowitz, W e r n e r C r e m e r , E r w i n H a a s , W a l t e r C h r i s t i a n , W a l t e r K e m p n e r , Akiji Fiy'ita a n d several other J a p a n e s e . Meyerhof's laboratory, accommodate d in t h e s a m e b u i l d i n g , a f e w s t e p s a w a y , i n c l u d e d K a r l Lohmann, Karl Meyer, Fritz Lipmann, Hermann Blaschko, S e v e r o Ochoa, F r a n k Schmitt, Ralph G e r a r d , Dean B u r k , David N a c h m a n s o h n , Louis G e n e v o i s , Ken Iwasaki. Other y o u n g b i o l o g i s t s w o r k i n g in t h e s a m e building w e r e V i c t o r H a m b u r g e r , C u r t S t e r n a n d J o a c h i m H a m m e r l i n g . M a n y of t h e s e left t h e i r m a r k o n l a t e r s c i e n t i f i c d e v e l o p m e n t s . A c h i e v e m e n t s of t h e L a b o r a t o r i e s of W a r b u r g a n d in t h e N i n e t e e n T w e n t i e s

Meverhof

T h e d i s c o v e r i e s m a d e in t h e m i d d l e a n d l a t e r p a r t of t h e 1920's in W a r b u r g ' s l a b o r a t o r y i n c l u d e d t h e d i s c o v e r y of t h e a e r o b i c g l y c o l y s i s of t u m o u r s , t h e g e n e r a l o c c u r r e n c e of t h e P a s t e u r e f f e c t , t h e a c c u r a t e q u a n t i t a t i v e m e a s u r e m e n t s of cell r e s p i r a t i o n a n d cell g l y c o l y s i s , t h e c a r b o n m o n o x i d e inhibition of cell r e s p i r a t i o n a n d t h e light s e n s i t i v i t y of t h i s inhibition w h i c h m a d e it p o s s i b l e to m e a s u r e t h e a c t i o n s p e c t r u m of t h e o x y g e n t r a n s f e r r i n g e n z y m e in r e s p i r a t i o n ( n o w r e f e r r e d to a s c y t o c h r o m e 3 3 ) a n d to identify t h e c a t a l y s t a s an i r o n p o r p h y r i n , t h e d e v e l o p m e n t of s p e c t r o p h o t o m e t r i c m e t h o d s of a n a l y s i s ( t w e n t y y e a r s l a t e r c o m m e r cially i n c o r p o r a t e d b y B e c k m a n into h i s b l a c k b o x ) , t h e d i s c o v e r y of c o p p e r in b l o o d s e r u m a n d t h e fall of its c o n c e n t r a t i o n in a n a e m i a s . M e y e r h o f ' s l a b o r a t o r y m a d e d e c i s i v e c o n t r i b u t i o n s to w h a t i s n o w c a l l e d t h e E m b d e n - M e y e r h o f p a t h w a y of g y c o l y s i s . It laid t h e g r o u n d w o r k l e a d i n g t o t h e d i s c o v e r y of h e x o k i n a s e ,

9 aldolase and other e n z y m e s . Monumental d i s c o v e r i e s by L o h m a n n w e r e t h o s e of A T P , f i r s t identified a s a c o f a c t o r of g l y c o l y s i s a n d of t h e " L o h m a n n r e a c t i o n " - t h e i n t e r a c t i o n between A T P and c r e a t i n e . O n e of t h e s e c r e t s of t h e s e o u t s t a n d i n g a c h i e v e m e n t s in both l a b o r a t o r i e s w a s t h e c r e a t i o n of n e w m e t h o d s , s u c h a s t h e t i s s u e slice t e c h n i q u e , m a n o m e t r y and s p e c t r o p h o t o m e t r y by W a r b u r g , a n d L o h m a n n ' s m e t h o d of d i s t i n g u i s h i n g b e t w e e n the many different phosphate e s t e r s by m e a s u r i n g their r a t e of h y d r o l y s i s at 100° in 2 N H C 1 . N o w , s o m e 45 y e a r s later, w e can a s s e s s the achievements of W a r b u r g a n d M e y e r h o f in p r o p e r p e r s p e c t i v e . M a n y s c i e n tific p a p e r s m a y s e e m to b e v e r y i m p o r t a n t at t h e t i m e of t h e i r a p p e a r a n c e b u t a s t h e field d e v e l o p s it i s a p p r e c i a t e d t h a t t h e y w e r e l e s s s i g n i f i c a n t t h a n w a s at f i r s t t h o u g h t ; a s time g o e s on the r e a l l y significant contributions stand out a s lasting l a n d m a r k s . A n a m a z i n g f e a t u r e of t h e t e a m s of W a r b u r g a n d M e y e r h o f w a s their smallness. Altogether there w e r e hardly m o r e t h a n t w o o r t h r e e d o z e n p e o p l e w h o p a r t i c i p a t e d in t h e s e g r e a t developments I h a v e listed. Meyerhof had f o u r o r five s m a l l r o o m s a n d t h e total n u m b e r of h i s c o l l a b o r a t o r s at a n y o n e t i m e w a s not m o r e t h a n f i v e . T h e r e w a s o n l y o n e t r a i n ed technician, Walter S c h u l z , and a p a r t - t i m e typist. T h e technician w a s Meyerhof's p e r s o n a l assistant and together with a " D i e n e r " h e l o o k e d a f t e r t h e g e n e r a l l a b o r a t o r y a f f a i r s s u c h a s m a i n t e n a n c e of a p p a r a t u s a n d o r d e r i n g of m a t e r i a l s . When I joined W a r b u r g t h e r e w a s o n e l a r g e r o o m f o r s i x p e o p l e in a l l , p l u s s e v e r a l i n s t r u m e n t r o o m s , p l u s one D i e n e r . W a r b u r g h a d n o s e c r e t a r i a l h e l p - w e all t y p e d o u r s e l v e s . T h e r e w a s no t e c h n i c i a n in t h e o r d i n a r y s e n s e of h e l p e r s . It i s t r u e W a r b u r g ' s l o n g - s t a n d i n g c o l l a b o r a t o r s - Negelein, Kubowitz, Christian, H a a s - w e r e o r i ginally t e c h n i c i a n s but not in t h e o r d i n a r y s e n s e . T h e y w e r e r e s e a r c h assistants who had been primarily trained a s i n s t r u m e n t m e c h a n i c s in w o r k s h o p s in t h e f a c t o r i e s of t h e S i e m e n s C o m p a n y in B e r l i n . T h e y k n e w h o w to h a n d l e i n s t r u m e n t s a n d h o w to m a k e a c c u r a t e m e a s u r e m e n t s , a n d W a r b u r g t a u g h t t h e m all t h e c h e m i s t r y t h e y n e e d e d . In 1928 W a r b u r g obtained one m o r e r o o m f o r f o u r e x t r a people and

10 when he moved into his new Institute in 1931 t h e r e w e r e a few m o r e p l a c e s but the total number r e m a i n e d deliberately s m a l l . Meyerhof of c o u r s e a l s o had m o r e s p a c e and m o r e staff when he moved to H e i d e l b e r g in 1930. T o t h o s e who participated what w e did s e e m e d to u s quite normal and natural, a matter of c o u r s e . W a r b u r g , h o w e v e r , w a s a l w a y s fully c o n s c i o u s of the monumental nature of his contributions. H e h a s stated this in writing m o r e than o n c e , f o r intellectual modesty w a s not his s t r o n g e s t point. H e c o n s i d e r e d himself to b e in direct line with the giants of biology and in p a r t i c u l a r the chemically orientated biologists - a direct s u c c e s s o r of P a s t e u r , with n o - o n e of c o m p a r a b l e c a l i b r e between him and P a s t e u r (1822-1895). Meyerhof a p p e a r e d much m o r e humble, and not c o n c e r n e d with his own a s s e s s ment of his position in the history of s c i e n c e . H e w a s content to l e a v e this to his p e e r s and to p o s t e r i t y . And not only did the genius of l e a d e r s h i p by W a r b u r g and Meyerhof make outstanding contribution to the subject and inspire, the small band of deeply motivated and committed young c o l l a b o r a t o r s . In the p r o c e s s W a r b u r g and Meyerhof also educated (I do not s a y " t r a i n " b e c a u s e " e d u c a t e " m e a n s m o r e than t r a i n ; educating includes the t r a n s m i s s i o n of an outlook, not m e r e l y of technicalities) a future generation of leading s c i e n t i s t s . T h i s happened without actually aiming at doing it, without any policy of p o s t g r a d u a t e training p r o g r a m m e s . It happened naturally, and I believe something of this s o r t will a l w a y s happen naturally. B o r n l e a d e r s attract b o r n f o l l o w e r s who develop into l e a d e r s - a s long a s b u r e a u c r a c y and the e r r o n e o u s c o n c e p t s of equality do not i n t e r f e r e . D a v - t o - D a v L i f e in the L a b o r a t o r i e s We all w o r k e d v e r y h a r d and intensively, though the atmos p h e r e w a s r e l a x e d . In W a r b u r g ' s l a b o r a t o r y the working h o u r s w e r e f r o m 8 a . m . to 6 p . m . f o r six d a y s a w e e k . Most of the r e a d i n g and most of the writing had to b e done at home in the e v e n i n g s , at w e e k e n d s and during the s u m m e r v a c a t i o n . W a r b u r g and Meyerhof w e r e in attendance m o r e o r l e s s all the time and a l w a y s a c c e s s i b l e to their c o l l a b o r a t o r s . T h e r e w e r e no committee meetings and hardly any a c a d e m i c t o u r i s m . T h e r e w a s a brief luncheon interval w h e r e the y o u n g e r people f r o m different departments ( e s p e c i a l l y f r o m

11 t h e l a b o r a t o r i e s of W a r b u r g a n d M e y e r h o f ) met in a c o m m o n r o o m f o r a s i m p l e s n a c k c o n s i s t i n g u s u a l l y of e g g s , s a n d w i c h e s and milk. Coffee and t e a b r e a k s w e r e u n k n o w n . T h e main v a c a t i o n w a s r a t h e r l o n g . W a r b u r g c l o s e d h i s l a b o r a t o r y f o r 8 w e e k s d u r i n g A u g u s t a n d S e p t e m b e r but d u r i n g t h i s time h e w r o t e m o s t of h i s p a p e r s w h i l e o n h i s e s t a t e in t h e I s l a n d of R i i g e n . W a r b u r g liked to point out that t h e w o r k i n g h o u r s w e r e m u c h l e s s t h a n t h e y h a d b e e n in h i s y o u n g e r d a y s . W h e n h e w o r k e d in H e i d e l b e r g in K r e h l ' s D e p a r t m e n t of M e d i c i n e , K r e h l often m a d e a r o u n d of t h e l a b o r a t o r i e s on S u n d a y e v e n i n g s a n d e x p e c t e d m o s t of t h e w o r k e r s to b e in a t t e n d a n c e . In M e y e r h o f ' s l a b o r a t o r y w o r k i n g h o u r s w e r e l e s s r i g i d but h a r d l y s h o r t e r . W a r b u r g ' s c o n t r o l of t h e L a b o r a t o r y w a s v e r y a u t o c r a t i c but w e n e v e r q u e s t i o n e d t h e justification of h i s a u t h o r i t a r i a n r u l e b e c a u s e w e t h o u g h t h e w a s entitled to t h i s on a c c o u n t of h i s outstanding intellect, his a c h i e v e m e n t s and his integrity, q u a lities w h i c h w e a d m i r e d e n o r m o u s l y . O n t h e w h o l e h i s r u l e w a s b e n e v o l e n t but it could a l s o b e f i e r c e . O n o n e o c c a s i o n he d i s m i s s e d a r e s e a r c h w o r k e r instantaneously after an inc i d e n t in w h i c h W a r b u r g t h o u g h t h e h a d not s h o w n p r o p e r r e s p e c t and c o u r t e s y . F o r W a r b u r g autocratic control w a s e s s e n t i a l in t h e i n t e r e s t of high s t a n d a r d s of t h e w o r k a s w e l l a s of p e r s o n a l c o n d u c t . H i s w a s a u t o c r a t i c r u l e at its b e s t . H e n e v e r exploited the j u n i o r , a s d o e s a u t o c r a t i c r u l e at its w o r s t . D e m o c r a t i c r u l e m a y at b e s t m a k e full u s e of t h e p o o l e d r e s o u r c e s but at w o r s t it m a y c r e a t e a situation w h e r e i g n o r a n c e and obstruction p r e v a i l s o v e r c o m p e t e n c e a n d e f f i c i e n c y . W a r b u r g w a s m o s t g e n e r o u s in giving c r e d i t to h i s c o l l a b o r a t o r s . M a n y p i e c e s of r e s e a r c h to w h i c h h e had m a d e the main contribution and which he h a d written w e r e p u b l i s h e d w i t h o u t h i s n a m e , e x c e p t p e r h a p s in an a c k n o w l e d g e m e n t b y t h e a u t h o r . A r e v i e w of h i s w o r k ( 2 ) w h i c h e s t a b l i s h e d t h e o x y g e n t r a n s f e r r i n g c a t a l y s t of cell r e s p i r a t i o n a s a n i r o n p o r p h y r i n e n d e d with t h e p a s s a g e "In c o n c l u d i n g I w i s h to e m p h a s i z e t h a t t h e r e s u l t s w h i c h I h a v e p r e s e n t e d a r e l a r g e l y d u e to t h e w o r k of my c o l l a b o r a t o r s , D r s . Negelein and K r e b s " . Yet the whole w o r k w a s c o n c e i v e d b y W a r b u r g h i m s e l f a n d t h e g r e a t e r p a r t of t h e c r i t i c a l e x p e r i m e n t s w e r e c a r r i e d out b y him w i t h h i s o w n hands. I k n o w of at l e a s t o n e s p e c i f i c i n c i d e n t w h e r e M e y e r h o f

was

12 also v e r y f a i r . In 1929 Lipmann had d i s c o v e r e d ( a f t e r E i n a r L u n d s g a a r d had reported muscular contractions without lactate production in the p r e s e n c e of iodoacetate) that on anaerobic contraction muscle b e c o m e s initially alkaline even in the absence of iodoacetate, the r i s e of p H being due to the hydrolysis of creatine phosphate. Lipmann measured the pH change mano metric ally by the uptake of C O ^ which r e a c t s with the OH ions f o r m e d . T h i s w a s an important finding because it helped to establish the now generally accepted concept that c r e a t i n e - P , through the Lohmann reaction, can e n e r g i s e contraction. A s it w a s v e r y important to him to find a job he w a s anxious that he should get p r o p e r recognition and he said in a somewhat r e s i g n e d spirit to one of his colleagues, Hermann Blaschko . " T h i s will be just another p a p e r by Meyerhof and L i p m a n n " . Blaschko then encouraged Lipmann to ask Meyerhof whether he may not be the first author. M e y e r h o f ' s immediate reply w a s " B u t of c o u r s e " ( 3 ) . A n d F r i t z w a s the sole author of a second paper printed directly after the joint o n e , in which he s h o w e d that fluoride can act similarily to iodoacetate ( 4 ) . Financial Position of Young R e s e a r c h

Workers

Our financial position w a s v e r y restricted and quite a f e w people in the laboratory r e c e i v e d no s a l a r y o r grant at all. Hermann Blaschko tells me that when he asked Meyerhof to be accepted in his laboratory Meyerhof eventually a g r e e d to have him but he told him "I cannot give you any payment" whereupon Blaschko replied "1 did not expect o n e " . F r i t z Lipmann w a s not paid during the first 2 y e a r s of his stay with M e y e r h o f , nor w a s S e v e r o Ochoa paid. I w a s lucky and r e c e i v e d a starting s a l a r y of 300 marks which r o s e to 400 after one y e a r . It is difficult to equate this with present p r i c e s but it meant that w e had to live frugally and count e v e r y p e n n y . If w e w e r e careful w e could afford one modest holiday a y e a r ; w e could afford c o n c e r t s and theatres in the cheapest s e a t s . T h e r e w e r e no travel grants f o r attending meetings. T h i s did not mean that w e w e r e isolated b e c a u s e there w e r e plenty of opportunities f o r learning something about new scientific developments within Berlin itself, through the colloquia at Dahlem and through the B e r l i n Chemical and Medical S o c i e t i e s . Who then, financed our maintenance? A s f a r as I know our p a r e n t s , even though they could ill afford

13 it, f o r inflation h a d d e v a l u e d t h e p r e - w a r M a r k b y a f a c t o r of 10 12 ( a million million) b y t h e e n d of 1923. P a r e n t s w e r e willing to m a k e s a c r i f i c e s f o r a g o o d t r a i n i n g of t h e i r s o n s . Of c o u r s e , w e felt v e r y u n c o m f o r t a b l e to b e a b u r d e n t o o u r p a r e n t s w h e n w e w e r e b e t w e e n 25 a n d 3 0 y e a r s of a g e . F r a n z K n o o p i m p r e s s e d m e in 1920 b y s a y i n g d u r i n g h i s l e c t u r e s to m e d i c a l s t u d e n t s t h a t h e h a d e a r n e d nothing until h e w a s 37 y e a r s of a g e , although h e h a d m a d e a n o u t s t a n d i n g d i s c o v e r y - that of ^ - o x i d a t i o n - w h e n h e w a s 3 0 . It w a s u n d e r s t o o d t h a t an a c a d e m i c c a r e e r m e a n t w i l l i n g n e s s to p u t u p with v e r y m o d e s t m a t e r i a l s t a n d a r d s of l i v i n g . W e w e r e m o t i v a t e d b y a k e e n d e d i c a t i o n to o u r w o r k a n d w e w e r e maintained by the h o p e that w h e n w e had r e c e i v e d a t h o r o u g h t r a i n i n g - w h i c h w e e x p e c t e d w o u l d l a s t until w e w e r e a b o u t 30 - w e w o u l d e v e n t u a l l y g e t a w o r t h w h i l e j o b satisfying professionally a s well a s financially. T h e s a c r i f i c e s n e e d e d f o r a long p e r i o d of t r a i n i n g m e a n t a l s o t h a t only t h e k e e n e s t did not g i v e u p . M o s t of u s w e r e m e d i c a l g r a d u a t e s a n d c o u l d , if w e w a n t e d t o , at a n y t i m e b r a n c h off into a r e l a t i v e l y l u c r a t i v e m e d i c a l c a r e e r - but w e p r e f e r r e d research. T h e f i n a n c i a l a n d s o m e o t h e r a s p e c t s of t h e s c e n e at D a h l e m w e r e of c o u r s e not u n i q u e . E r w i n C h a r g a f f - m y c o n t e m p o r a r y w o r k i n g at that t i m e in V i e n n a - r e c e n t l y w r o t e ( 5 ) " N o - o n e w h o e n t e r e d s c i e n c e within t h e p a s t 30 y e a r s o r s o c a n imagine how small the scientific establishment then w a s . T h e selection p r o c e s s o p e r a t e d mainly t h r o u g h a f o r m of a n initial v o w of p o v e r t y . A p a r t f r o m i n d u s t r i a l e m p l o y m e n t in a f e w s c i e n t i f i c d i s c i p l i n e s , s u c h a s c h e m i s t r y , t h e r e w e r e few university p o s t s , and they w e r e mostly ill-paid". N o w a d a y s t h e r e a r e many undergraduate students who i n s i s t o n t h e i r " r i g h t s " t o b e fully s u p p o r t e d b y t h e s t a t e . W e e x p e c t e d n o r i g h t s e v e n at t h e p o s t - g r a d u a t e a n d p o s t d o c t o r a l l e v e l . W e w e r e s a t i s f i e d if w e c o u l d w o r k h a r d u n d e r r e a s o n a b l e c o n d i t i o n s a n d l e a r n . W e did not f e e l e n titled t o e x p e c t m u c h , let a l o n e m a k e d e m a n d s , b e f o r e w e had l e a r n e d a lot. In s p i t e of o u r r e s t r i c t e d e c o n o m i c c i r c u m s t a n c e s w e w e r e o n t h e w h o l e v e r y h a p p y b e c a u s e w e felt t h a t w e w e r e r e c e i v i n g a f i r s t r a t e t r a i n i n g a n d w e r e doing s o m e t h i n g w o r t h while . We had no u n d u e w o r r i e s about o u r l o n g - t e r m f u t u r e

14 although this looked v e r y uncertain in view of the economic and political difficulties in G e r m a n y . I am not a w a r e that any of u s anticipated a particularly s u c c e s s f u l c a r e e r . B e i n g c l o s e to giants of s c i e n c e w e felt v e r y small and W a r b u r g himself did not do much to e n c o u r a g e o u r s e l f - c o n f i d e n c e . In fact when I had to l e a v e his l a b o r a t o r y he told me that he c o n s i d e r e d my c h a n c e s in biochemistry a s slight and a d v i s e d me to return to clinical medicine (which I did) . Relations between Dahlem and G e r m a n

Universities

Of a p e c u l i a r s o r t w e r e the relations of W a r b u r g and M e y e r hof to the official r e p r e s e n t a t i v e s of G e r m a n physiology and b i o c h e m i s t r y , i . e . the G e r m a n university d e p a r t m e n t s . W a r b u r g r e g a r d e d himself a s an o u t s i d e r , and Meyerhof too, but p e r h a p s l e s s s o . It i s r e m a r k a b l e that G e r m a n u n i v e r s i ties had not appreciated officially Meyerhof's qualities. B y the time he got the Nobel P r i z e in 1923, at the a g e of 3 9 , he held the post of an a s s i s t a n t in the Physiological Institute of Kiel U n i v e r s i t y ; he had been p a s s e d o v e r f o r a junior p r o f e s s o r i a l appointment ( P r o f e s s o r E x t r a o r d i n a r y ) in f a v o u r of a man called P ü t t e r , w h o s e claim to distinction r e m a i n e d slight. A f t e r the a w a r d of the Nobel P r i z e to Meyerhof, W a r b u r g s u c c e e d e d in p e r s u a d i n g the K a i s e r Wilhelm G e s e l l schaft to offer Meyerhof a p o s t . T h i s s e n s e of being o u t s i d e r s had to do with the C i n d e r e l l a treatment of biochemistry by the G e r m a n u n i v e r s i t i e s . T h e number of c h a i r s and departments of biochemistry o r p h y s i o logical chemistry w a s v e r y small in G e r m a n y . Independent departments existed in four universities only, in F r a n k f u r t with E m b d e n at its h e a d , at F r e i b u r g , Tiibingen and L e i p z i g . In other universities biochemistry w a s a s u b - s e c t i o n of p h y siology and the h e a d s of t h e s e s u b - s e c t i o n s did not h a v e the rank of full p r o f e s s o r . In s o m e universities the p r o f e s s o r of physiology w a s essentially a biochemist. T h i s applied, f o r i n s t a n c e , to Heidelberg w h e r e K o s s e l w a s the P r o f e s s o r of P h y s i o l o g y and w h e r e f i r s t - r a t e w o r k w a s done on protein chemistry. T h u s it c a m e about that L e o n o r Michaelis, one of the e s t biochemists of his time could not b e a b s o r b e d into G e r m a n university s y s t e m and t h e r e f o r e left G e r m a n y f o r J a p a n , to move later to J o h n s Hopkins University

brightthe in 1921 and the

15 R o c k e f e l l e r I n s t i t u t e . While in G e r m a n y h e h a d to e a r n h i s living a s a clinical b i o c h e m i s t in o n e of t h e m u n i c i p a l h o s p i t a l s in B e r l i n . H o p k i n s , in a g e n e r a l a d d r e s s at t h e I n t e r n a t i o n a l C o n g r e s s h e l d in S t o c k h o l m in 1926 c o m m e n t e d o n t h e n e g l e c t of b i o c h e m i s t r y in G e r m a n u n i v e r s i t i e s a n d s p o k e a b o u t t h e i m p o r t a n c e of " s p e c i a l i s e d i n s t i t u t e s of g e n e r a l b i o c h e m i s t r y " . H e r e f e r r e d to t h e a p p e a l b y H o p p e - S e y l e r in V o l u m e 1 of h i s Z e i t s c h r i f t f ü r P h y s i o l o g i s c h e C h e m i e in 1877 that i n s t i t u t e s of b i o c h e m i s t r y s h o u l d b e g e n e r a l l y s e t up in t h e u n i v e r s i t i e s . In 1877 t h e o n l y institute of t h i s kind w a s t h a t in S t r a s b o u r g with H o p p e - S e y l e r at its h e a d . H o p p e - S e y l e r ' s a p p e a l w a s at o n c e o p p o s e d b y t h e p h y s i o l o g i s t E . P f l ü g e r in h i s J o u r n a l , a n d 4 9 y e a r s l a t e r H o p k i n s r e m a r k e d t h a t in f a c t H o p p e S e y l e r ' s a p p e a l f o r t h e r e c o g n i t i o n of b i o c h e m i s t r y a s a n i n d e p e n d e n t s u b j e c t h a d still not y e t f o u n d p r o p e r r e s p o n s e in h i s o w n c o u n t r y a n d h e a d d e d "It i s difficult to s e e h o w G e r m a n y c a n c o n t i n u e t h e l e a d along t h e p a t h w h i c h f o r a long t i m e s h e h a s a l m o s t t r o d a l o n e " . H e e m p h a s i s e d that a c a d e m i c c e n t r e s in g e n e r a l in E u r o p e a r e in t h i s r e s p e c t b e h i n d t h o s e in A m e r i c a . T h e s e r e m a r k s , i n c i d e n t a l l y , were m a d e at t h e s u g g e s t i o n of F . K n o o p f o r t h e b e n e f i t of t h e G e r m a n r e a d e r s h i p a n d t h e y w e r e r e p r i n t e d in t r a n s l a t i o n in the Münchener Medizinische Wochenschrift ( 6 ) . Now and

Then

T o d a y t h e s c e n e of s c i e n t i f i c r e s e a r c h s e e m s v e r y d i f f e r e n t . In t h e 1920's p u r e r e s e a r c h w a s still w i d e l y l o o k e d u p o n a s a l u x u r y o r e x t r a v a g a n c e t h a t did not d e s e r v e m a j o r s u p p o r t f r o m t h e S t a t e . T h e K a i s e r Wilhelm G e s e l l s c h a f t , a f t e r a l l , w a s f o u n d e d in 1910 a s a p r i v a t e o r g a n i s a t i o n ( w h i l e t h e G e r m a n U n i v e r s i t i e s w e r e all s t a t e - c o n t r o l l e d ) . N o w , 50 y e a r s l a t e r , s c i e n t i f i c r e s e a r c h i s r e g a r d e d a n e c e s s i t y f o r t h e s u r v i v a l of a n a t i o n , a n d l a r g e s u m s a r e provided by G o v e r n m e n t s f o r training people and f o r p u r s u ing r e s e a r c h . But although the s c e n e h a s c h a n g e d s o m e fundamental p r i n c i p l e s g o v e r n i n g s u c c e s s f u l r e s e a r c h a r e still t h e s a m e , a n d will a l w a y s r e m a i n t h e s a m e - t h e r e c o g n i t i o n of l e a d e r s h i p in r e s e a r c h , t h e v a l u e of long t r a i n i n g , t h e n e e d f o r h a r d

16 w o r k and f o r dedication,

a n attitude of h u m i l i t y .

What h a s g o n e , a m o n g o t h e r t h i n g s , a r e t h e biting p o l e m i c s in s c i e n c e in w h i c h W a r b u r g liked to i n d u l g e , h u r t i n g a n d r i d i c u l i n g t h e o p p o n e n t s - t h e r e a r e m a n y e x a m p l e s of t h e s e in W a r b u r g ' s b o o k s a n d in t h e B i o c h e m i s c h e Z e i t s c h r i f t ( 7 ) . Gone, also, h a s the autocratic rule which W a r b u r g , his t e a c h e r s a n d h i s c o n t e m p o r a r i e s p r a c t i c e d . T h i s kind of b e n e v o l e n t ( o r m o s t l y b e n e v o l e n t ) d i c t a t o r s h i p at its b e s t a s I a l r e a d y s t a t e d , h e l p e d to m a i n t a i n high s t a n d a r d s - but it could e a s i l y d e g e n e r a t e into a r b i t r a r y i n j u s t i c e s , e x p l o i t a t i o n and mismanagement. T o d a y a d i f f e r e n t b a s i s of t h e r e l a t i o n s b e t w e e n s e n i o r s a n d j u n i o r s h a s e v o l v e d . T h e m a s t e r m a y still r u l e , a n d r u l e f i r m l y , b u t t h e b a s i s of h i s a u t h o r i t y i s n o w a n a t u r a l r e s p e c t , a n a t u r a l m i x t u r e of a d m i r a t i o n a n d a f f e c t i o n w h i c h h e h a s e a r n e d b y h i s w o r k a n d c o n d u c t ; in a g o o d l a b o r a t o r y a u t h o r i t y i s n o l o n g e r b a s e d o n t h e p o w e r i n v e s t e d in a h e a d of a l a b o r a t o r y . I think t h i s v e r y o c c a s i o n h e r e is a n i l l u s t r a t ion of t h e k i n d of h u m a n r e l a t i o n s b e t w e e n t h e m a s t e r a n d h i s f o l l o w e r s w h i c h n o w a d a y s e x i s t in a r e a l l y i d e a l t e a m . It w a s t h e d e s i r e of t h e f o l l o w e r s t o e x p r e s s a s e n s e of l o y a l ity, g r a t i t u d e a n d affection w h i c h h a s b r o u g h t t h i s s y m p o s i u m into b e i n g . T h i s a s s e m b l y of s o m a n y p e o p l e f r o m s o f a r a w a y , m o t i v a t e d b y mutual goodwill, I find d e e p l y g r a t i f y i n g a n d m o v i n g , e s p e c i a l l y h e r e t o d a y in D a h l e m w h e r e , w i t h i n a s t o n e ' s t h r o w in t h e F r e e U n i v e r s i t y , I a m t o l d , s e n s e l e s s and unpleasant confrontations b e t w e e n t e a c h e r s and taught r e p r e s e n t a m e n a c e to a c a d e m i c l i f e , a m e n a c e l i a b l e t o d e s t r o y t h e old a c a d e m i c i d e a l s , t h e s e a r c h , in a s p i r i t of t o l e r a n c e , f o r knowledge and t r u t h . T h e m o t i v e s w h i c h b r o u g h t u s t o g e t h e r r e m i n d m e of a r e m a r k w h i c h W a r b u r g m a d e to m e d u r i n g a c a s u a l c h a t in t h e l a b o r a t o r y . F o r two y e a r s I s h a r e d an island b e n c h with him, w o r k i n g o p p o s i t e to e a c h o t h e r in c l o s e p r o x i m i t y . A l t h o u g h both of u s w e r e not e x a c t l y t a l k a t i v e w h i l e p r e o c c u p i e d w i t h our experiments there w e r e occasional conversations touching o n m a n y a s p e c t s of l i f e . O n e d a y h e r e m a r k e d " T h e w o r s t d e f e a t a s c i e n t i s t c a n s u f f e r i s t o die e a r l y b e c a u s e t h e f r u i t s of h i s l a b o u r m a t u r e v e r y s l o w l y " . A l t h o u g h t h e s e w o r d s - like m a n y o t h e r of h i s c a s u a l s a y i n g s a r e still d e e p ly i n g r a i n e d in m y m e m o r y - I did not p r o p e r l y a p p r e c i a t e

17 their full meaning at the t i m e . But today their meaning is c l e a r to m e . T h e f r u i t s of a scientist's labour a r e of s e v e r a l k i n d s . P r o m o t i o n to a g o o d post and a N o b e l a w a r d a r e s o m e ; a v e r y important o n e is the long t e r m r e s p o n s e f r o m students and c o l l a b o r a t o r s . Much of a r e s e a r c h scientist's time is spent in helping to shape the outlook, . c a r e e r and life of his j u n i o r s , and the s e e d s w h i c h he plants take a long time to g r o w . T o d a y w e s e e the r i c h h a r v e s t that has c o m e to F r i t z f r o m helping and guiding his y o u n g e r a s s o c i a t e s - a h a r v e s t not only in the f o r m of gratitude and affection but also in the f o r m of the intensely p l e a s i n g k n o w l e d g e that t h e r e is a n e w g e n e r a t i o n willing to c a r r y on the w o r k and to uphold the s t a n d a r d s and i d e a l s w h i c h motivated the m a s t e r . S u c h kinds of thoughts, I s u s p e c t , must h a v e b e e n in W a r b u r g ' s mind w h e n he suddenly s p o k e about the i m p o r t a n c e of living to old a g e . P e r h a p s s o m e e x p e r i e n c e s of his f a t h e r ( 8 ) ( a f o u n d e r of a l a r g e and d e v o t e d s c h o o l of p h y s i c i s t s ) , had i m p r e s s e d him*

F u r t h e r material on D a h l e m in the 1920's and the p e r s o n alities of W a r b u r g and M e y e r h o f will b e found in r e f e r e n c e s ( 6 ) , ( 9 ) , ( 1 0 ) , (11), (12) and ( 1 3 ) .

References (1) (2)

Willstätter, R . : A u s m e i n e m L e b e n . Z w e i t e A u f l a g e . Weinheim V e r l a g C h e m i e , p . 2 0 0 ( 1 9 5 8 ) . W a r b u r g , O . : U b e r die c h e m i s c h e Konstitution d e s A t m u n g s f e r m e n t e s . N a t u r w i s s e n s c h a f t e n ]6_, 3 4 5 - 3 5 0

(1928). (3)

(4)

(5) (6)

L i p m a n n , F . , M e y e r h o f , O . : U b e r die R e a k t i o n s ä n d e r u n g d e s tätigen M u s k e l s . B i o c h e m . Z . 2 2 7 . 84-109 (1930) . L i p m a n n , F . : U b e r den T ä t i g k e i t s s t o f f w e c h s e l d e s f l u o r i d - v e r g i f t e t e n M u s k e l s . B i o c h e m . Z . 227 110-115 ( 1 9 3 0 ) . C h a r g a f f , E . : Building the T o w e r of B a b b l e . N a t u r e 248. 776-779 ( 1 9 7 4 ) . H o p k i n s , F . G . : U b e r die N o t w e n d i g k e i t v o n Instituten f ü r p h y s i o l o g i s c h e C h e m i e . Münch . M e d . W o c h . 73, 1586-1587 ( 1 9 2 6 ) .

18 (7)

(8) (9)

(10)

(11) (12) (13)

Fritz

K r e b s , H . A . : Otto H einrich W a r b u r g . Biographical Memoirs of F e l l o w s of the R o y a l S o c i e t y . 18., 629-699 (1972). F r a n c k , J . : Emil W a r b u r g zum Gedächtnis. N a t u r wissenschaften 993-997 (1931). W e b e r , H . H . : Otto Meyerhof - W e r k und P e r s ö n l i c h keit, in Molecular B i o e n e r g e t i c s and Macromolecular Biochemistry p p . 2-13, S p r i n g e r V e r l a g , B e r l i n , H e i d e l b e r g , N e w Y o r k , (1972). P e t e r s , R . A . (with a contribution by H . B l a s c h k o ) : Otto M e y e r h o f . Obituary Notices of F e l l o w s of the R o y a l S o c i e t y . 2 , 175-200 (1954). Muralt, A . v o n : Otto M e y e r h o f . E r g e b n . P h y s i o l . 47, I - X X (1952). Nachmansohn, D . , O c h o a , S . , Lipmann, F . : Otto Meyerhof 1884-1951. S c i e n c e ]15, 363-369 (1952). Nachmansohn, D . : Biochemistry as part of my life. A n n . R e v . B i o c h e m . 41_, 1-28 (1972).

Lipmann

I am going now to d e s c r i b e my relationship to Dahlem when I w a s in M e y e r h o f ' s l a b o r a t o r y , and h e r e I would also like to include B e r l i n . F r o m my e a r l y youth B e r l i n , the g r e a t city, had been f o r me a magnet. I w a s born in a small town, K o e n i g s b e r g , then in East P r u s s i a , and my first contact with B e r l i n w a s after absolvation of my abiturium, as w e called the final examination ending gymnasium time and giving the right to enter into U n i v e r s i t y . T h e n , my parents g a v e me as a gift, a w e e k all on my own in B e r l i n to e x p e r i e n c e the theater and to e x p e r i e n c e the g r e a t city. I spent m o r e time in B e r l i n later on studying medicine, and again I w a s i m p r e s s ed by the e x p e r i e n c e of what happens in that city. A n d then I came back to B e r l i n f o r quite a lopg p e r i o d after I had finished my medical studies. Still under th$ influence of medical f r i e n d s , I spent the latter half of my practical y e a r t h e r e , and the first t h r e e months with L u d w i g P i c k , an excellent pathologist, b e c a u s e it w a s thought that to b e c o m e a physician one had to do some pathology. T h e n I h e a r d about

19 a b i o c h e m i s t r y c o u r s e w h i c h w a s g i v e n at t h e C h a r i t é , t h e M e d i c a l S c h o o l of B e r l i n U n i v e r s i t y , w h e r e a l a r g e n u m b e r of p h y s i c i a n s w h o b e c a m e v e r y g o o d b i o c h e m i s t s , i n c l u d i n g H a n s K r e b s and David N a c h m a n s o h n , w e r e t r a i n e d . T h a t w a s R o n a ' s l a b o r a t o r y . W h e n I w e n t t o L u d w i g P i c k t o tell h i m t h a t I w o u l d s p e n d t h e l a s t t h r e e m o n t h s of m y p r a c t i c a l y e a r t a k i n g t h e b i o c h e m i s t r y c o u r s e of P e t e r R o n a , h e t h r e w h i s h a n d s u p in a m a z e m e n t . B i o c h e m i s t r y t h e n w a s still a n u n k n o w n e n t i t y in G e r m a n y . R o n a ' s n a m e i s p r o b a b l y k n o w n t o v e r y f e w of y o u ; f o r q u i t e a w h i l e h e w a s a c o l l a b o r a t o r of L e o n o r M i c h a e l i s . O n e of t h e r e a s o n s I w a n t e d to mention him w a s that I f o u n d an a m u s i n g p i c t u r e of t h e m e m b e r s of t h e c o u r s e w h e n I t o o k it a n d I w o u l d like to s h o w y o u t h i s ( F i g u r e 1 ) .

Figure

1

In t h e c e n t e r i s P e t e r R o n a ; in t h e m i d d l e a young man - h e died a f e w w e e k s a g o ; right c o r n e r a m I, with a b o w tie - s o m e a p p o i n t e d t h a t I d o n ' t w e a r it a n y m o r e a s early y e a r s .

is H . H . W e b e r a s a n d in t h e t o p people a r e disI u s e d t o d o in

I t h i n k it w a s a n e x t r a o r d i n a r y c o u r s e . I l e a r n e d t h e r e t h e l a t e s t a d v a n c e s in t h e b i o c h e m i s t r y of t h a t t i m e : m a n o m e t r y , pH m e a s u r e m e n t , e l e c t r o p h o r e s i s , and so o n . Actually, I s t a y e d on with R o n a a n d did a m e d i c a l d o c t o r a t e t h e s i s w h i c h

20 w a s o b l i g a t o r y in G e r m a n y . It did not n e e d to b e v e r y i m p o r t a n t . Mine w a s on the e l e c t r o p h o r e t i c b e h a v i o r of iron o x i d e c o l l o i d s , mainly c o n c e r n e d with the r e v e r s a l of the p o s i t i v e c h a r g e to n e g a t i v e in the p r e s e n c e of c i t r a t e . C o l l o i d c h e m i s t r y w a s v e r y m o d e r n in t h o s e d a y s - many p e o p l e u s e d it to d e s c r i b e the p r o t o p l a s m . It s e e m e d enough then to call it a colloid to imply o n e u n d e r s t o o d s o m e t h i n g about it. We h a v e learned better. T h a t w a s in 1921-1922. T h e n I d e c i d e d to g o b a c k to my h o m e town to l e a r n c h e m i s t r y s i n c e I w a s lucky enough to b e a b l e to do this with H a n s M e e r w e i n w h o w a s the p r o f e s s o r of c h e m i s t r y at the U n i v e r s i t y . I s h o u l d g u e s s that his n a m e i s known to t h o s e of y o u c o n v e r s a n t with o r g a n i c c h e m i s t s . H e w a s a s u p e r i o r c h e m i s t , and l a t e r m o v e d f r o m K o e n i g s b e r g to M a r b u r g . In t h r e e y e a r s I l e a r n e d a g r e a t d e a l , p a r t i c u l a r ly f r o m l e c t u r e s ; all d u r i n g t h o s e y e a r s all s t u d e n t s h a d to attend h i s l e c t u r e s and it w a s a t r e m e n d o u s p l e a s u r e . H e g a v e them all h i m s e l f ; t h e r e w a s no substitution by a s s i s t a n t s , and that g a v e u s s t u d e n t s a contact with h i s p e r s o n a l i t y . T h e n I got my V e r b a n d s - e x a m e n , which e n a b l e d me to s t a r t on a t h e s i s . A f t e r that f i r s t s t e p w a s finished I b e c a m e s o m e what r e s t l e s s . I felt it w a s now time f o r m e to find a p l a c e w h e r e I could do b i o c h e m i s t r y , f o r which I had b e e n p r e p a r ing m y s e l f all this t i m e . It w a s not without o t h e r r e a s o n s that I c h o s e B e r l i n ; but I w a s mostly motivated by the e x i s t e n c e in B e r l i n - D a h l e m of the two institutions which at that time s e e m e d to me to do w o r k in the field I had b e g u n to b e c o m e i n t e r e s t e d in, i n t e r m e d i a r y m e t a b o l i s m . T h e s e w e r e the l a b o r a t o r i e s of C a r l N e u b e r g and Otto M e y e r h o f , and I d e b a t e d f o r a time w h e t h e r I s h o u l d join Meyerhof o r N e u b e r g . I eventually d e c i d e d f o r Meyerhof b e c a u s e of his much m o r e p h y s i o l o g i c a l l e a n i n g , and j u s t a s H a n s K r e b s h a s told y o u , I l i k e w i s e didn't e x p e c t any s a l a r y and f o r the f i r s t t w o y e a r s I didn't get anything. I j u s t a s k e d him if I could w o r k t h e r e and a s I had studied c h e m i s t r y and had s o m e e x p e r i e n c e I w a s lucky enough to b e a c c e p t e d ; he a s k e d m e , i n t e r e s t i n g l y e n o u g h , if I had iany p r o b l e m to w o r k on and I w a s a s h a m e d to s a y that I hadri'i. I had to get a p r o b l e m f r o m h i m . T h e p r o b l e m s I w o r k e d on in the e a r l y d a y s t h e r e w e r e not v e r y i m p o r t a n t . I did s o m e w o r k on f l u o r i d e inhibition

21 of g l y c o l y s i s and f e r m e n t a t i o n , which w a s published in B i o c h e m i s c h e Z e i t s c h r i f t . T h e s e p a p e r s I could put t o g e t h e r and u s e as my c h e m i c a l d o c t o r ' s t h e s i s . It r e f l e c t s interestingly on the status of the h e a d s of l a b o r a t o r i e s at the K a i s e r W i l helm Institutes that M e y e r h o f w a s unable to b e my d o c t o r " f a t h e r " b e c a u s e he couldn't accept g r a d u a t e students although he w a s a titular p r o f e s s o r . H o w e v e r , N e u b e r g , w h o s e institute w a s next d o o r , could; he w a s a p r o f e s s o r at the T e c h n i s c h e H o c h s c h u l e and I actually had to f a r m out my t h e s i s , s o to s a y , to N e u b e r g , w h o b e c a m e my doctoral " s t e p f a t h e r " in a w a y . H e a l w a y s t r e a t e d me v e r y k i n d l y . I will n o w s a y a little about the M e y e r h o f l a b o r a t o r y . While I w a s sitting h e r e just now and thinking about my c h o i c e of l a b o r a t o r y , I am almost s u r p r i s e d to d i s c o v e r that although most i m p r e s s e d by W a r b u r g , I n e v e r e v e n d a r e d to think of going to w o r k with h i m . O n e of the important a s p e c t s of M e y e r h o f w a s that he w a s not a s s t e r n and w a s much l o o s e r than W a r b u r g . But he w a s W a r b u r g ' s pupil and t h e r e is a p a p e r by W a r b u r g and M e y e r h o f w h i c h c a m e f r o m the N a p l e s L a b o r a t o r y . T h e M a r i n e L a b o r a t o r y in N a p l e s w a s o n e of the meeting g r o u n d s f o r b i o c h e m i s t s in the s a m e s e n s e that W o o d s H o l e is o r w a s in the U . S . A . In his e a r l i e r y e a r s M e y e r h o f tended to b e v e r y i n t e r e s t e d in p h i l o s o p h y , had j o i n e d a s c h o o l of p h i l o s o p h e r s , and w r o t e s e v e r a l p a p e r s of a philosophical c h a r a c t e r . It w a s the influence of W a r b u r g , I u n d e r s t a n d , that d e c i d e d him to b e c o m e a b i o c h e m i s t ; and he took all the traits of W a r b u r g , the feeling that to do g o o d w o r k o n e n e e d s the most exact methodology and has to h a v e full c o n f i d e n c e in o n e ' s r e s u l t s . T h e w o r k in his l a b o r a t o r y , as y o u h a v e h e a r d a l r e a d y , c e n t e r e d about the m u s c l e and I w o r k e d during that time l a r g e ly with m u s c l e o r m u s c l e e x t r a c t s , mostly r e l a t e d to g l y c o l y s i s . It is only in the l a t e r p e r i o d after the l a b o r a t o r y had m o v e d to H e i d e l b e r g that I did a f a i r l y n i c e p i e c e of w o r k on a determination of c r e a t i n e phosphate b r e a k d o w n in living m u s c l e w h i c h I m e a s u r e d m a n o m e t r i c a l l y . T h e s e m a n o m e t e r s that I u s e d w e r e s o m e w h a t difficult to construct b e c a u s e I w a n t e d to stimulate the m u s c l e in the m a n o m e t e r and o n e had to seal in platinum e l e c t r o d e s , w h i c h w a s v e r y h a r d to do without a l e a k . T h i s w o r k started w h e n M e y e r h o f s u g g e s t e d that I should t r y to s e e w h a t happens in muscle contraction at r e l a tively high acidity, that i s , in a b i c a r b o n a t e s o l u t i o n with C O 2

22 in t h e g a s p h a s e . It w a s t h e n t h a t I f o u n d , d u r i n g t h e e a r l y p h a s e of a s e r i e s of c o n t r a c t i o n s , a n a l k a l i n i z a t i o n , i . e . , m a n o m e t r i c a l l y , C O 2 a b s o r p t i o n i n s t e a d of t h e e x p e c t e d C O 2 l i b e r a t i o n f r o m l a c t i c a c i d f o r m a t i o n . C h e m i c a l a n a l y s i s of t h e m u s c l e s h o w e d that t h e alkali that f o r m e d e a r l y c o r r e s p o n d e d v e r y nicely to a c r e a t i n e p h o s p h a t e b r e a k d o w n ; t h i s h a d b e e n f o u n d t o y i e l d a l k a l i b e c a u s e of t h e s t r o n g a l k a l i n i t y of t h e guanidinium b a s e l i b e r a t e d . T h i s w a s an e a r l y proof that w i t h out inhibitor u n d e r t h e s e acid- c o n d i t i o n s c r e a t i n e p h o s p h a t e b r e a k d o w n could c a u s e c o n t r a c t i o n .

Figure

2

It i s n o w t i m e to s h o w y o u M e y e r h o f . T h i s p i c t u r e ( P i g u r e 2 ) i s r a t h e r t y p i c a l b e c a u s e it s h o w s t h a t it w a s n o t e a s y t o a p p r o a c h M e y e r h o f . W e a c t u a l l y t a l k e d v e r y little a n d w h a t I l e a r n e d f r o m him w a s l a r g e l y by diffusion. But this w a s to i n f l u e n c e m e all t h r o u g h m y l i f e . T h i s p i c t u r e of u s t o g e t h e r w a s t a k e n in 1941 at a c o n f e r e n c e in M a d i s o n , a n d y o u m i g h t s a y w e l o o k a little u n e a s y , w h i c h w a s b e c a u s e I h a d g i v e n a l e c t u r e t h e r e on the P a s t e u r effect and h a d s h o w n d i s a g r e e ment with his interpretation, and h e w a s n ' t too h a p p y about it. O u r w o r k i n g h o u r s w e r e m u c h m o r e r e l a x e d t h a n t h o s e at W a r b u r g . I r e m e m b e r that w e took p r e t t y long lunch i n t e r m i s s i o n s a n d w e n t r a t h e r o f t e n t o a little r e s t a u r a n t , w h i c h

w a s next to a f u r t h e r - u p s u b w a y station, and sat t h e r e and happily talked in the g a r d e n . When I s a y w e , in the next p i c t u r e ( F i g u r e 3 ) you c a n s e e s o m e of the p e o p l e w h o were "we" .

Figure

3

Y o u c a n s e e , f r o m left to r i g h t , K e n I w a s a k i , K a r l L o h m a n n , W a l t e r S c h u l t z , and S c h r o e d e r , who w a s something in b e t w e e n a scientific a s s i s t a n t and a d i e n e r j t h e n David N a c h m a n sohn and P a u l R o t h s c h i l d ; and again m e , this time with a long t i e . K e n I w a s a k i , N a c h m a n s o h n , P a u l R o t h s c h i l d , and I w e r e the o n e s w h o often w e n t to that little r e s t a u r a n t , and not only that, w e e v e n went t o g e t h e r to m a s q u e r a d e b a l l s w h i c h w e r e v e r y f a s h i o n a b l e and much attented at that t i m e } but t h e y w e r e v e r y good entertainment and a s f r e e in spirit a s in p r e s e n t day t e r m s . At o n e of t h e m , the s o c i a l i s t b a l l , which had nothing to do with s o c i a l i s t s , I met F r e d a Hall w h o w a s to b e c o m e my w i f e . S o that w a s an important event during my B e r l i n d a y s . A c t u a l l y , at this p a r t i c u l a r b a l l , D a v i d N a c h m a n s o h n d a n c e d m o r e with F r e d a than I did, but h e w a s already married. Now to r e t u r n to the l a b o r a t o r y . I had s o m e contact with R a l p h G e r a r d - I think w e s h a r e d a l a b o r a t o r y w h e n I e n t e r ed the M e y e r h o f L a b o r a t o r y ; w e met again in l a t e r life and I w a s r a t h e r fond o f h i m . T h e n I moved into a n o t h e r l a b o -

24 r a t o r y and w o r k e d v e r y c l o s e to K e n I w a s a k i w h o s p e n t a g o o d d e a l of t i m e in B e r l i n a n d I a m s u r e h a d a v e r y g o o d t i m e t h e r e ; h e w a s n o t m a r r i e d t h e n . I a m told t h a t M r . T a k e d a , M a s a o ' s f a t h e r , b e c a m e a v e r y g o o d f r i e n d of K e n I w a s a k i ; t h e y s p e n t m u c h t i m e t o g e t h e r in B e r l i n a n d a r e still v e r y g o o d f r i e n d s . K e n I w a s a k i i s n o w r e t i r e d f r o m h i s b i o c h e m i s t r y p r o f e s s o r s h i p a n d h a s a l a b o r a t o r y in t h e T a k e d a C o m p a n y , w h i c h i s o n e of t h e l a r g e s t p h a r m a c e u t i c a l c o m p a n i e s in J a p a n . T h e t o p i c s o n w h i c h t h e w o r k w a s d o n e in M e y e r h o f ' s l a b o r a t o r y w e r e n o t t o o v a r i e d ; it w a s m a i n l y c o n c e n t r a t e d o n t h e m u s c l e , b u t it a l s o i n c l u d e d n i t r o g e n f i x a t i o n o n w h i c h D e a n B u r k a n d K e n I w a s a k i w o r k e d . T h e s t a t u s of o u r u n d e r s t a n d i n g at t h a t t i m e m a y b e s h o w n b y w h a t K a r l M e y e r d i d . H e w a s t r y i n g , in p a r a l l e l t o w h a t h a d b e e n c a l l e d z y m a s e b y H a r d e n , t o i s o l a t e t h e " g l y c o l y t i c e n z y m e " ; it w a s n ' t q u i t e r e a l i z e d t h e n t h a t t h e g l y c o l y t i c e n z y m e w a s of c o u r s e c o m p o s e d , a s w e n o w k n o w , of n u m e r o u s e n z y m e s and that t h e s e e n z y m e s could eventually b e s e p a r a t e d . T h a t c a m e n o t m u c h l a t e r , b u t in 1928 it w a s r e a l l y s u r p r i s i n g t h a t o n e c o u l d e v e n a i m at t h i n k i n g of i s o l a t i n g a s a u n i t s o m e t h i n g like a glycolytic e n z y m e . T h e n , shortly before w e moved away from Berlin, I w o r k e d f o r a little w h i l e w i t h L o h m a n n , a n d I l e a r n e d m u c h f r o m h i m . A s y o u h e a r d a l r e a d y , h e r e a l l y w a s a n a r t i s t in d e t e r m i n i n g by acid h y d r o l y s i s the different p h o s p h o r y l a t e d c o m p o u n d s that w e r e in a m i x t u r e . F o r e x a m p l e , in t h i s w a y h e d i s c o v e r e d t h e e q u i l i b r a t i o n of g l u c o s e 6 - p h o s p h a t e w i t h f r u c t o s e 6 - p h o s p h a t e . T h e latter h a s a much f a s t e r h y d r o l y s i s time than g l u c o s e 6 - p h o s p h a t e , w h i c h i s o n e of t h e m o s t difficult t o hydrolyze phosphate e s t e r s . On the other hand, fructose d i p h o s p h a t e is completely h y d r o l y z e d within t h r e e h o u r s . L o h m a n n u s e d t h e a c i d h y d r o l y s i s of p h o s p h a t e e s t e r s v e r y e f f e c t ively a n d , a s I s a i d , h e d i s c o v e r e d m a n y n e w c o m p o u n d s . I a m v e r y e a g e r t o s h o w o n c e in a w h i l e a n e x p e r i m e n t f r o m which the w r o n g cbnclusions w e r e d r a w n . T a b l e I s h o w s s u c h a n e x p e r i m e n t t h a t I did w i t h L o h m a n n a n d w h i c h e v e n t u ally b e c a m e of g r e a t c o n s e q u e n c e . In t h i s e x p e r i m e n t f r u c t o s e d i p h o s p h a t e w a s i n c u b a t e d in m u s c l e e x t r a c t , a n d I c a m e in t o t r y a n d s e e if t h i s c o n v e r s i o n of f r u c t o s e d i p h o s p h a t e w o u l d a l s o g o w i t h o u t f l u o r i d e t h a t w a s a d d e d in L o h m a n n ' s

25 Table

1 : C o n v e r s i o n of F D P into a c i d - s t a b l e p h o s p h a t e e s t e r in m u s c l e e x t r a c t of w i n t e r f r o g s a f t e r i n c u b a t i o n at 2 0

Incubation (min)

Phosphate Bound (mg P j )

0 20 60 120 Biochem.Z.,

0.48 0.50 0.50 0.49

Phosphate AcidConverted H y d r o l y z e d in 3 h r P h o s p h a t e (mg P j ) (mgPj) (%) 0.48 0.39 0.25 0.19

0.12 0.27 0.33

25 53 68

222.389.1930

e a r l i e r e x p e r i m e n t s . T h e t a b l e s h o w s t h e c h a n g e of f r u c t o s e d i p h o s p h a t e w i t h o u t f l u o r i d e t o w h a t w a s c a l l e d a difficult t o h y d r o l y z e h e x o s e d i p h o s p h a t e . O n e can s e e that by t h e i n c u b a t i o n in m u s c l e e x t r a c t p h o s p h a t e i s n o t r e l e a s e d . H o w e v e r , t h e h y d r o l y s i s t i m e of t h e a d d e d f r u c t o s e d i p h o s p h a t e g o e s u p if o n e u s e s t h e t h r e e h o u r s m e n t i o n e d a b o v e a s a s t a n d a r d . O n e c a n s e e t h a t w i t h t i m e it b e c o m e s c o n v e r t e d , a n d e v e n t u a l l y 70% of it i s p r e s e n t a s w h a t w a s t h o u g h t t o b e a d i f f e r e n t h e x o s e d i p h o s p h a t e w h i c h w a s m u c h m o r e difficult t o a c i d hydrolyze. T o t u r n a little m o r e t o t h e h i s t o r y of t h i s p a r t of b i o c h e m i s t r y a s I did in t h e m e e t i n g at t h e C i b a c o n f e r e n c e l a s t w e e k w h e r e I also mentioned these experiments, Nilsson had shown a little e a r l i e r in E u l e r ' s l a b o r a t o r y t h a t in t h e p r e s e n c e of f l u o r i d e , the s a m e f r u c t o s e d i p h o s p h a t e , with a y e a s t p r e p a ration, w h e n p a i r e d with acetaldehyde, g a v e p h o s p h o g l y c e r i c a c i d a s t h e o x i d a t i o n p r o d u c t p a r a l l e l w i t h r e d u c t i o n of a c e t a l d e h y d e t o e t h a n o l . T h i s w a s t h e f i r s t a p p e a r a n c e of p h o s p h o g l y c e r i c a c i d in t h e p i c t u r e of f e r m e n t a t i o n a n d g l y c o l v s i s a n d n o b o d y at t h a t t i m e c o u l d a p p r e c i a t e w h y t h i s c o m p o u n d w a s f o r m e d a s a n o x i d a t i o n p r o d u c t of f r u c t o s e d i p h o s p h a t e . N i l s s o n c a m e to t h e M e y e r h o f l a b o r a t o r y a n d w e d i s c u s s e d this s t r a n g e c o m p o u n d , p h o s p h o g l y c e r i c acid, without seeing the light. T h e right idea w a s E m b d e n ' s w h o essentially r e peated o u r e x p e r i m e n t . We had just m a d e barium precipitates

26 of w h a t w e c o n s i d e r e d a h e x o s e d i p h o s p h a t e . H e f o u n d t h a t it w a s s u r e l y difficult t o h y d r o l y z e but t h a t it w a s n o t h e x o s e d i p h o s p h a t e b u t r a t h e r a m i x t u r e of p h o s p h o g l y c e r o l a n d p h o s p h o g l y c e r i c acid w h i c h h e a s s u m e d to b e f o r m e d b y a dismutation r e a c t i o n . T h u s , w e had misinterpreted the c o n v e r s i o n of f r u c t o s e d i p h o s p h a t e . H o w e v e r , in t h e h a n d s of E m b d e n it b e c a m e t h e r e a s o n w h y w e n o w t a l k a b o u t t h e E m b d e n - M e y e r h o f cycle; f r o m this reaction he then concluded t h e d i s r u p t i o n of f r u c t o s e d i p h o s p h a t e into a n e q u i l i b r i u m m i x t u r e of t w o t r i o s e p h o s p h a t e s a n d m a p p e d o u t t h e f o u n d a t i o n of o u r p r e s e n t s c h e m e , r e c o g n i z i n g p h o s p h o g l y c e r i c a c i d t o b e t h e o x i d a t i o n p r o d u c t of p h o s p h o g l y c e r a l d e h y d e , t h e b i o c h e m i c a l l y d o m i n a n t of t h e t w o t r i o s e p h o s p h a t e s f o r m e d . T h a t ' s j u s t a sidelight on L o h m a n n ' s a r t i s t r y with h y d r o l y s i s . A s I s a i d , I l e a r n e d m u c h f r o m h i m t o h a n d l e w h a t I did s o o n a f t e r w a r d s . W h e n I w e n t to N e w Y o r k to w o r k with L e v e n e , I c h o s e t h e p h o s p h o p r o t e i n s a s a n o b j e c t of i n v e s t i g a t i o n . Y o u h e a r d in D r . H e l m r e i c h ' s t a l k t h a t I t h e r e i s o l a t ed s e r i n e p h o s p h a t e f r o m the egg yolk p h o s p h o p r o t e i n . L o h mann w a s the o n e w h o s u g g e s t e d that I w o r k with L e v e n e . H e h a d in m i n d , I t h i n k , t h a t I s h o u l d w o r k o n n u c l e o t i d e s , but I c h o s e the mentioned topic b e c a u s e L e v e n e h a d isolated a v e r y p h o s p h a t e - r i c h protein f r o m egg yolk w h i c h a t t r a c t e d my i n t e r e s t . T h e m e t h o d s I u s e d w e r e actually b o r r o w e d f r o m w h a t I h a d l e a r n e d f r o m L o h m a n n . It a p p e a r e d t h a t t h e p h o s p h a t e in t h e y o l k p r o t e i n i s a l k a l i - l a b i l e b u t v e r y a c i d s t a b l e , a n d c h o o s i n g a c i d h y d r o l y s i s a s a m e a n s of d e g r a d a t i o n of t h e p r o t e i n , I t h u s i s o l a t e d t h e s e r i n e p h o s p h a t e . M u c h l a t e r , I t u r n e d t o w h a t w a s t h e p r o m i n e n t i n t e r e s t in M e y e r h o f ' s l a b o r a t o r y , n a m e l y , b i o e n e r g e t i c s . T h i s c a m e to b e an u n d e r g r o u n d w e l l , s o to s a y , that eventually o p e n e d up a f t e r I d i s c o v e r e d acetyl p h o s p h a t e a n d led to my w r i t i n g t h e p a p e r o n g e n e r a t i o n a n d u t i l i z a t i o n of p h o s p h a t e b o n d energy. I w o u l d l i k e t o c l o s e b y s a y i n g a little m o r e a b o u t B e r l i n in t h o s e d a y s . B e f o r e going to N e w Y o r k , F r e d a Hall a n d I w e r e m a r r i e d , a n d w e w e r e a m a z e d to s e e that t h e r e w a s s u c h a n e n o r m o u s d i f f e r e n c e in t h e w a y of life b e t w e e n B e r lin a n d N e w Y o r k , p a r t i c u l a r l y a m o n g y o u n g w o m e n a n d y o u n g m e n . W e r e a d in t h e " S a t u r d a y E v e n i n g P o s t " t h r e e articles by H e r g e s h e i m e r , w h o w a s then a r a t h e r f a s h i o n -

27 a b l e n o v e l i s t , in w h i c h h e d e s c r i b e d B e r l i n a s t h e c e n t e r of E u r o p e t p e o p l e didn't go to P a r i s , t h e y w e n t , r a t h e r , to B e r l i n . T h e r e w a s the t h e a t e r , t h e r e w a s the m u s i c , t h e r e w a s the d a n c e ; you could h a v e e v e r y t h i n g . T h e r e w a s also a g r e a t f r e e d o m in B e r l i n in t h e l a t e t w e n t i e s , a s i m i l a r f r e e d o m t o t h a t w h i c h h a s d e v e l o p e d in A m e r i c a in r e c e n t y e a r s . It w a s d u e t o t h e b r e a k d o w n of t h e f a m i l y t i e s b y w h i c h y o u n g p e o p l e w e r e held b e c a u s e t h e y h a d to d e p e n d o n t h e i r f a m i l i e s , a n d it h a d t h e e f f e c t t h a t t h e y o u n g m e n and w o m e n interacted much m o r e f r e e l y with e a c h o t h e r . A f t e r 1 h a d m o v e d a w a y f r o m B e r l i n a n d f r o m G e r m a n y , it t o o k a l o n g t i m e t o f o r g e t t h e w a y of life w e h a d e x p e r i e n c e d there. I h a v e b e e n v e r y p l e a s e d and moved by this meeting's coming a b o u t , a n d don't w a n t to e n d without t h a n k i n g the m a n y p e o p l e w h o h a v e made possible this happy g e t - t o g e t h e r . I first want to mention D i e t m a r R i c h t e r w h o really h a d the lion's s h a r e in t h i n k i n g of it, w r i t i n g all t h e l e t t e r s , a n d a r r a n g i n g t h e w h o l e p r o c e e d i n g s . I w i s h to t h a n k D r . Wittmann f o r p r o v i d ing t h i s Institute a s a p l a c e f o r u s to m e e t , and D r . H e l m reich f o r his nice w o r d s and also for the e n c o u r a g e m e n t and support f r o m Gesellschaft f u r Biologische C h e m i e . And of c o u r s e I a m m o s t g r a t e f u l f o r t h e s u p p o r t of t h e p h a r m a c e u t i c a l c o m p a n i e s of B o e h r i n g e r a n d S c h e r i n g , a n d of t h e V o l k s w a g e n F o u n d a t i o n , without w h i c h w e could not h a v e had it. T h a n k y o u all v e r y

much.

Cascade Control of E. coli Glutamine Synthetase

Stuart P. Adler and Earl R. Stadtman National Institutes of Health, Bethesda, Md. 20014, USA One important mechanism for the regulation of glutamine synthetase activity in 15. coli is the covalent attachment and removal of AMP from a specific tyrosyl residue in each of the enzymes 12 identical subunits (1-4).

Adenylylation of a subunit converts it

to a less active form dependent upon Mn++ (1)•

The enzyme's

activity is thus controlled by the average number of adenylylated subunits per molecule which can vary from zero to 12.

Both

adenylylation and deadenylylation of glutamine synthetase are catalyzed by single enzyme, adenylyltransferase (ATase) (5). Adenylylation involves the transfer of AMP from ATP into an AMP-O-tyrosyl linkage (4)f whereas deadenylylation involves a phosphorolysis of this bond to yield ADP (.6). Although ATase catalyzes both reactions, its ability to adenylylate or deadenylylate glutamine synthetase (GS) is modulated by the regulatory protein Pji and metabolic effectors including a-ketoglutarate (ct-KG) , ATP, glutamine and Pi (4,7,8). ATP + GS

ATaS P

> AMP-GS + PPi IIA

It AMP-GS + Pi_Eli5».GS + ADP ATase SCHEME I The regulatory protein Pjj exists in two interconvertible forms (7).

One form, PuA> stimulates the ATase catalyzed adenylyla-

tion of glutamine synthetase, whereas the other form, P u d is required for the ATase catalyzed deadenylylation (Scheme I). When Pija i s incubated in the presence of UTP, ATP, a-KG, Mn*4" or Mg^"1", .and another enzyme, uridylyltransferase (UTase), it is converted to P u d ; this involves the covalent attachment of UMP

29 to the protein (Reaction 1) (9). UTP + PJ-J

UTase

UMP-Pn + PPi

ot-KG, ATP, Mg*4" REACTION 1

^IIA

can

regenerated from

PJJd

by a uridylyl removing enzyme

activity (UR enzyme) (Reaction 2). Plr-UMP

TOenZyMe>| Mn++

p

+

^

REACTION 2

Figure 1. Reciprocal effects of UR-enzyme catalyzed deuridylylation of PJID- H^-UMP-P was incubated with partially purified preparation of UR-UTase. The P H A and P ^ m activities as well as the release of [3H]-UMP was followed. (Exp. details, see ref. 10).

Figure 1 illustrates the relationship between uridylylation and the capacity of PJJ to stimulate adenylylation or deadenylylation of glutamine synthetase.

When P U D labelled with ^n-UMP

was

30 incubated with Mil"1"-*" and a partially purified extract containing UR enzyme activity, there was a rapid release of covalently bound 3 H -UMP from PJJ.

This release was accompanied by a parallel

increase in the ability of PJJ to stimulate the ATase catalyzed adenylylation of glutamine synthetase, and a concomitant loss in its ability to stimulate the ATase catalyzed deadenylylation of glutamine synthetase (10).

The reciprocal changes in activity

associated with deuridylylation of P U D

clearly demonstrate the

role of PJJ uridylylation in determining the adenylylation and deadenylylation capacity of ATase. TABLE 1 Properties of the Regulatory Protein PJJ

Property

Value

Molecular weight, native protein Subunit molecular weight Number of identical subunits Number of tyrosines per subunit Number of iodinatable tyrosines per subunit of Piia Number of iodinatable tyrosines per subunit of PjID Moles of UMP per mole of PJIJD subunit

44,000 11,000 4 2 2 1 1

The PJJ protein has been purified to homogeneity from IS. coli (11) and from Pseudomonas putida (12). Table 1.

Properties are shown in

The native protein has a mol. wt. of 44,000.

Based on

its homogeneous behavior during disc gel electrophoresis in the presence of SDS or 8 M urea (11) and during sedimentation in 6 M guanidine-HCl (13), and also the minimum mol. wt. calculated from amino acid composition, the native protein is a tetramer of identical subunits (11).

This is supported also by the facts:

(1) there are two tyrosyl residues per 11,000 mol. wt.; (2) following iodination of the tyrosyl residues of PiiA with tryptic digestion yields only two radioactive peptides in

31 equal molar amounts.

O n l y o n e of t h e s e two p e p t i d e s is

obtained

a f t e r t r y p t i c d i g e s t i o n of i o d i n a t e d , f u l l y u r i d y l y l a t e d S i n c e s u b s t i t u t i o n of a t y r o s y l h y d r o x y l g r o u p

prevents

i o d i n a t i o n of the a r o m a t i c r i n g , t h i s r e s u l t i n d i c a t e s P

IID

t h e

U M P

is

covalently bound in phosphodiester

h y d r o x y l g r o u p of o n e of t h e two t r y o s y l r e s i d u e s T h i s is s u p p o r t e d a l s o phosphodiesterase

in each

in the

subunit.

b y the f a c t t h a t t r e a t m e n t of P J I D w i t h

a n d t h e s t o i c h i o m e t r i c a p p e a r a n c e of p h e n o l a t e i o n

of P u

that

l i n k a g e to

r e s u l t s i n r e l e a s e of t h e c o v a l e n t l y b o u n d

measured by ultraviolet absorption spectroscopy activity

PJID*

UMP

(pH > 1 1 . 0 )

(14).

is m o d u l a t e d b y c o v a l e n t a t t a c h m e n t of U M P to

specific tyrosyl residue in each

that catalyze the u r i d y l y l a t i o n of P J I A

and the d e u r i d y l y l a t i o n of P u d

(UR -

the (UTase)

enzyme).

TABLE 2 E f f e c t of D i v a l e n t C a t i o n s a n d O t h e r E f f e c t o r s on UR and UTase Activities

Effector added

M g , A T P , ci-KG 1 Mn, ATP, a-KG Mn Mg Mg + Mn M g , A T P , ct-KG, G L N

Relative Activity UR activity UTase activity

55% 85 100 0 100 —

a

subunit.

T a b l e 2 s h o w s t h e e f f e c t of c a t i o n s a n d o t h e r e f f e c t o r s o n enzyme activities

as

Thus,

100% 100 0 0 0 7

T h e c o n c e n t r a t i o n of e f f e c t o r s u s e d w a s : 1 m M MnCl2, 10 m M M g C l 2 , 0 . 1 m M A T P , 5 m M a - k e t o g l u t a r a t e (ct-KG) , 18 m M g l u t a m i n e , a n d 2 m M K 2 M g E D T A . These reaction m i x t u r e s a l s o c o n t a i n 2 m M K 2 M g E D T A to c h e l a t e t r a c e s of Mn" 1 ^ t h a t w e r e p r e s e n t i n t h e e n z y m e p r e p a r a t i o n . (For e x p . d e t a i l s , s e e r e f . 1 0 ) . Whereas Mn++ alone supports maximal U R activity, Mg++ s u p p o r t U R a c t i v i t y e x c e p t in the p r e s e n c e of A T P

and

cannot

32 i |

a-ketoglutarate; however, activity with M g ^ is less than with Mn"*-*" alone.

In contrast, Mn-*-1" and Mg"1-*" support equal UTase

activity in the presence of ATP and a-ketoglutarate, but neither cation supports UTase activity in the absence of these effectors. Table 2 also shows that UTase activity is inhibited by glutamine whereas other data (not shown) demonstrate no effect of glutamine on UR activity. All attempts to separate the UR and UTase activities have failed. An example of their copurification is illustrated in Figure 2.

Figure 2. Chromatography of UR-UTase activities on sepharose-C5~ NH2. (A) An extract containing UR-UTase activities was applied on a sepharose-C5-NH2 column (8.5 x 1.5 cm). After the unabsorbed protein was eluted, a KC1 gradient up to 0.4 M was applied. (B) Fractions from experiment A containing UR-UTase activities were pooled, and reapplied to the same column as in A, only eluted with a shallower KC1 gradient. See ref. (15).

A partially purified extract containing UR-UTase activities was chromatographed on a hydrophobic column (Sepharose-Cg-N^). when eluted with a very shallow KC1 gradient both activities

Even

33 cochromatograph (Figure 2B).

The

facts that both activities

copurify through a variety of procedures, are stabilized by high ionic strength buffers (14) , and in the presence of Mg"1""1" require ATP and a-ketoglutarate for activity (Table 2) indicate that both activities might be catalyzed by a single enzyme or enzyme complex. Discussion Figure 3 summarizes current knowledge of the complex system that regulates glutamine synthetase activity in li. coli.

The system

consists of two opposing sets of reactions (cascades) that lead on the one hand to inactivation (adenylylation) of glutamine synthetase and on the other hand to its activation (deadenylylation).

The inactivation cascade (Fig. 3A) is initiated by the

action of UR enzyme which catalyzes the deuridylylation of PJJ-UMP (i.e., PJXD)•

Deuridylylation leads to an unmodified form of

Pjl, which together with ATase promotes the adenylylation of glutamine synthetase, thus converting it from a Mg^-dependent form of high catalytic potential and a pH optimum of 8.0, to a • | Mn

-dependent form of low catalytic potential and a pH optimum

of 6.9.

The activation cascade (Fig. 3B) is initiated by the

action of UTase, which catalyzes uridylylation of PJI.

The

PJJ'UMP, thus formed, together with ATase promotes deadenylylation of glutamine synthetase, converting it back to the Mg^"1"dependent form of high catalytic potential.

The activities of

these two opposing cascade systems are finely modulated by the concentrations of various metabolites, including UTP, ATP, a-KG, Pi, glutamine and probably other compounds as yet unidentified. The catalytic potential of glutamine synthetase is determined by its state of adenylylation (i.e., the average number of adenylylated subunits per mole of enzyme).

When glutamine

synthetase is incubated with ATase, PJJ^, P U D and the effectors shown in Figure 3, it assumes a dynamic steady state of adenylylation in which the rates of adenylylation and deadenylylation are equal (16); moreover, the actual state of

34 H H io °V l H h

, /'Me
P u d and the various effectors.

Since

for any given steady state the rates of adenylylation and deadenylylation of glutamine synthetase (GS) are equal, it follows from theoretical considerations that the final state of adenylylation, n, is a function of the magnitude of the specific rate constants for adenylylation (k^) and deadenylylation (k 2 ) and the mole fraction of P h a . ( p H A ) f > according to the equation: " _

12 (PllA)f k 2 + k X (PiiA)f - k 2 (PilA)f

35 Figure 4 shows how the value of n varies as a function of the ( p IIA)f

an

d the ratio of ki/k.2-

Pg* PnA • P«0 Figure 4. Dependence of the steady state level of adenylylation on the mole fraction of PJIA ant^ the relative specific rate constants for adenylylation and deadenylylation. Data are calculated values derived from equation in the text (where (PlIA^f = PlIA/PlIA + ^IID)• It i® assumed that ATase is present in excess compared to Pxi and that its activity is determined by the specific rate constants for adenylylation (ki) and deadenylylation (K2) , and the proportions of P n A and P U D present. Numbers on curyes indicate the ratio, ki/k2-

Note that when k^ = k2, n is a linear function of the mole fraction of PIXA, but that nonlinear functions are obtained if the ratio of k^/k2 is varied as occurs, in the response to the differential effects of metabolites on the ATase catalyzed adenylylation and deadenylylation reactions.

Additional flexi-

bility in control derives from the fact that the mole fraction of PlIA can vary independently in response to changing concentrations of metabolites that affect the rates of the uridylylation and deuridylylation reactions (Fig. 3).

36 Cascade systems offer other advantages in the regulation of certain cellular functions.

Because they consist of a series of

reactions in which one catalyst acts upon another, they can amplify the response of the target enzyme to primary effectors acting on the initial enzyme in the series.

This could be

important if the changes in effector concentrations are small compared to the concentration of the target enzyme and in situations as described here in which the multiple protein catalysts are not present in comparable concentrations as they are in organized multienzyme complexes. In addition, cascade systems increase the number and types of allosteric effectors that can affect the activity of the ultimate target enzyme, since each enzyme in the cascade can be independently regulated.

This may be important in the regulation of

enzymes such as glutamine synthetase that occupy a central position in metabolism and therefore need to receive a massive input of regulatory information from diverse cellular functions. In such cases physical and steric limitations may preclude the existence on a single enzyme of a sufficient number of allosteric sites to accommodate the required number of regulatory effectors. This may account for the fact that the direct regulation of glutamine synthetase by eight different end products of glutamine metabolism is supplemented by a cascade system in which regulation of the UR-UTase and ATase activities is mediated by six additional effectors, including divalent cations (Fig. 3).

In

addition 3-phosphoglycerate, fructose-6-P, P-enolpyruvate, CoA, and fructose-P2 have been shown to inhibit the adenylylation of glutamine synthetase (17). Finally, another advantage of cascade systems is obtained when more than one step in the cascade is subject to control by the same effector.

Increased sensitivity of the system to negative

control is obtained when two steps are inhibited by the same ligand.

Thus, when a given concentration of a single metabolite

37 inhibits each of two steps by 50%, the overall inhibition will be 75%.

Moreover, if a metabolite stimulates two separate steps in

a cascade, the net effect is to increase the apparent reaction order with respect to that effector; therefore, under appropriate conditions activation of the last enzyme in the cascade will be a sigmoidal function 6f the metabolite concentration.

This

advantage of cascade systems appears to be realized in the glutamine synthetase cascade, since as is shown in Fig. 3, two steps, (the uridylylation of Pjl and the adenylylation of glutamine synthetase) are inhibited by glutamine and-are stimulated by both a-ketoglutarate and ATP. In the last analysis, the elaborate cascade system illustrated in Figure 3 serves as a physiological computer which is programed to sense fluctuations in the concentrations of numerous metabolites and to integrate their effects so as to modulate the activity of glutamine synthetase to meet changing metabolic demands. References 1.

Kingdon, H. S., Shapiro, B. M., Stadtman, E. R.:

Regulation

of glutamine synthetase, VIII. ATP: glutamine synthetase adenylyltransferase, an enzyme that catalyzes alterations in the regulatory properties of glutamine synthetase.

Proc.

Nat. Acad. Sci. US 58, 1703-1710 (1967). 2.

Shapiro, B. M., Kingdon, H. S., Stadtman, E. R.:

Regulation

of glutamine synthetase, VII. Adenylylglutamine synthetase: a new form of the enzyme with altered regulatory and kinetic properties. 3.

Proc. Natl. Acad. Sci. US 58, 642-649 (1967).

Mecke, D., Wulff, K., Liess, K., Holzer, H.:

Characterization

of a glutamine synthetase inactivating enzyme from Escherichia coli.

Biochem. Biophys. Res. Commun. 158,

514-525 (1966). 4.

Shapiro, B. M., Stadtnan, E. R.:

5'-Adenylyl-O-tyrosine:

The novel phosphodiester residue of adenylylated glutamine synthetase from Escherichia coli. 3771 (1968).

J. Biol. Chem. 243, 3769-

5.

Anderson, W. B., Hennig, S. B., Ginsburg, A., Stadtman, E. R.: Association of ATP: Glutamine synthetase adenylyltransferase activity with the Pi component of the glutamine synthetase deadenylylation system.

Proc. Natl. Acad. Sci. US 67_, 1417-

1424 (1970). 6.

Anderson, Wayne B., Stadtman, E. R.:

Glutamine synthetase

deadenylylation: A phosphorolytic reaction yielding ADP as nucleotide product.

Biochem. Biophys. Res. Commun. 41,

704-709 (1970). 7.

Brown, M. S., Segal, A., Stadtman, E. R.:

Modulation of

glutamine synthetase adenylylation and deadenylylation is mediated by metabolic transformation of the Pij-regulatory protein. 8.

Proc. Natl. Acad. Sci. US 68, 2949-2953 (1971).

Shapiro, B. M.:

The glutamine synthetase deadenylylating

enzyme system from Escherichia coli. Resolution into two components, specific nucleotide stimulation and cofactor requirements. 9.

Biochemistry 8, 659-670 (1969).

Mangum, John H., Magni, G., Stadtman, E. R.:

Regulation of

glutamine synthetase adenylylation and deadenylylation by enzymatic uridylylation and deuridylylation of the PJJ regulatory protein.

Arch. Biochem. Biophys. 158, 514-525

(1973). 10.

Adler, Stuart P., Mangum, J. H., Magni, G., Stadtman, E. R.: Uridylylation of the PJI regulatory protein in cascade control of Escherichia coli glutamine synthetase.

Third

International Symposium on Metabolic Interconversion of Enzymes, Springer Verlag, New York, 221-233 (1974). 11.

Adler, Stuart P., Purich, D., Stadtman, E. R.:

Cascade

control of j£. coli glutamine synthetase: Properties of the P

II regulatory protein and the uridylyltransferase—uridylyl

removing enzyme. Fed. Proc. 33, 142.7 (1974). 12.

Huang, C., Adler, S. P.:

Unpublished Data.

13.

Adler, S. P., Ginsburg, A.:

14.

Adler, S. P.:

Unpublished Data.

Unpublished Observation.

15.

Shaltiel, S.:

Hydrophobic chromatography in the study of

regulatory enzymes.

Third International Symposium on

Metabolic Interconversion of Enzymes, Springer Verlag, New York, 379-392 (1974). 16.

Segal, A., Brown, M. S., Stadtman, E. R.:

Metabolite

regulation of the state of adenylylation of glutaraine synthetase. 17.

Arch. Biochem. Biophys. 161, 319-327 (1974).

Ebner, E., Wolf, D., Gancedo, C., Elsasser, S., Holzer, H.: ATP: Glutamine synthetase adenylyltransferase from Escherichia coli B, purification and properties. Biochem. 14, 535-544 (1970).

Eur. J.

Structure and Biosynthesis of an Acidic Glycoprotein in a Bacterial Cell Envelope

J.Baddiley, J. P. Burnett, I. C. Hancock and J. Heptinstall Microbiological Chemistry Research Laboratory, The University, Newcastle upon Tyne, NE1 7RU, England INTRODUCTION Although glycoproteins a r e universally distributed in animals and plants, their occurrence in microorganisms is l e s s well-documented. Some eucaryotic microorganisms a r e known to produce glycoproteins and in particular several yeast glycoproteins have been described, (1 2)

some that a r e enzymes ' and others that appear to be envelope or (3 4) extracellular proteins

'

these yeast glycoproteins.

. Mannose is the major sugar in many of In contrast, there a r e fewer than ten

reports of glycoproteins in bacteria.

Everse and K a p l a n ^ have

described glycoprotein enzymes from Bacillus subtilis and there a r e reports of poorly-characterised glycoproteins in the envelopes of Escherichia c o l i ^ and Pseudomonas a e r u g i n o s a ^ .

The bacterial

glycoprotein that has been studied in most detail is that from the envelope of the marine pseudomonad BAL 31

, but this may be a

viral-specific component that results from infection with bacteriophage PM 2. N-Acetylglucosamine is the major sugar in all these bacterial glycoproteins; the only exception to this is the fucosecontaining glycoprotein of a corynebacterium whose biosynthesis has been studied by Strobel and his co-workers (9) . There have been three recent reports of glycoproteins that contain phosphodiester linkages in their glycan moieties.

These 'phospho-

41 glycoproteins' have been found in Hansenula h o l s t l i ^ ^ , Cladosporium werneckii^^ and in Penicillium c h a r l e s i i ^ \

"they represent an

interesting new class of macromolecules whose function is not yet known. We report here studies on the biosynthesis and preliminary characterisation of a glycoprotein from B. licheniformis that is rich in Nacetylglucosamine and phosphate and therefore appears to be a phosphogly copr otein. METHODS Growth of Cells and Preparation of Membrane B. licheniformis ATCC 9945 was grown to exponential phase and membranes were prepared by treatment of the cells -with lysozyme in (13) the absence of an osmotic stabiliser as previously described

.

Membranes were suspended at about 50 mg dry wt/ml in 0.05M T r i s HC1, pH 8.0 containing 5mM ethanethiol, and were stored frozen until required. Preparation of radioactive Glycoprotein Large scale enzyme reactions for preparation of the phosphoglycoprotein were carried out as follows: 1.0 ml membrane suspension was incubated with 0.1 ml MgCl (0. 8 M), 0.1 ml UDP-N-acetylglucosamine 14 6 (U- C; 2.98 x 10 cpm/fimole; lOmM) and tris buffer in a total o volume of 1.5 ml at 30 for 1 h. The reaction was stopped by addition of 0 . 5 ml butan-l-ol and the mixture applied as a band on the origin of a preparative paper chromatogram (Whatmann 3MM). The chromatogram was developed for 18 h in solvent system A, and the o material that remained at the origin was extracted into water at 60 for 3 h. More than 95% of the radioactivity from the origin was recovered in this way.

42 Chromatography and Electrophoresis Paper chromatography was carried out on Whatman No. 3MM paper Or No. 1 paper in the following solvents;- A propan-l-ol-ammonia (0. 88 sp. gr. )-water (6 : 3 : 1 by volume). B ethylacetate-pyridineacetic acid-water (5 : 5 : 1 : 3 by volume). Paper electrophoresis was carried out on Whatman No. 1 paper in 0.1M pyridinium acetate pH 6.5 at 40 volts/cm for

h.

Polyacrylamide gel electrophoresis in 0.1% sodium dodecylsulphate (14) (SDS) was accomplished as described by Weber and Osborn

. Gels

were stained for protein with Coomassie blue, and for carbohydrate material by the periodate-Schiff reagent of Segrest and Jackson^^. Phosphate-containing compounds were detected on paper chromatograms by the method of Hanes and I s h e r w o o d ^ \ (17) were stained with silver nitrate

Reducing sugars

. Quantitative Estimations and

radioactivity measurements were carried out as previously described^^. Preparation of polyisoprenol monophosphate (19) Polyisoprenols were phosphorylated as described by Popjak et al and purified by column chromatography on DEAE cellulose acetate in methanolic ammonium a c e t a t e ^ ^ Radioactive substrates 14 UDP-N-(acetyl described^^.

C)-acetylglucosamine was prepared as previously UDP-N-acetyl (U^C)glucosamine was purchased from

43 the Radiochemical Centre, Amersham, Bucks. U.K.

RESULTS

During a study of peptidoglycan biosynthesis in a membrane preparation from B. licheniformis the incorporation of radioactivity from 14 [

C]UDP-N-acetylglucosamine into polymeric material was

measured in the presence and absence of UDP-N-acetylmuramyl pentapeptide (Park nucleotide), the other substrate required for peptidoglycan synthesis.

Table 1 shows the results of such an

experiment.

14

Table 1 - Incorporation of

C-UDP-N-acetylglucosamine into macro-

molecular material.

cpm incorporated into polymer cpm incorporated into lipid

With Park nucleotide

no addition

10693

7664

1105

10837

Reaction mixtures contained 0.1 ml membrane suspension, UDP-N[acetyl- 1 4 C] acetylglucosamine (0.1 /¿mol, 2 . 9 8 x 10 5 cpm), MgCl 2 (8 fimol) in a total volume of 0 . 1 3 ml. The mixture was incubated for 1 h at 30° and then the reaction mixture was applied in a 2. 5 cm band to the origin of a paper chromatogram (Whatman no. 3MM paper). The paper was developed in Solvent System A for 18 h. Polymeric material remained at the origin, while lipids migrated with an Rf of 0.85. It was found that even in the absence of Park nucleotide, when peptidoglycan synthesis is impossible, a large amount of radioactivity was incorporated into high molecular weight material, and also into lipid.

44 In order to ascertain whether the radioactivity remained entirely in glucosamine residues in the product, the above experiment was 14 repeated using uniformly labelled C-N-acetylglucosamine and the baseline area from the chromatogram, and the lipid region, were o cut out and hydrolysed on the paper in 2M-HC1 at 100 for 3 h. The hydrolysate was chromatographed on Whatman no. 1 paper in Solvent system B for 18 h. All the radioactivity co-chromatographed with standard samples of glucosamine and N-acetylglucosamine, for both polymer and lipid. Properties of the polymer It was found that the material containing N-acetylglucosamine that remained at the origin of the chromatogram of reaction mixtures in Solvent A could be completely eluted from the paper into water at 60° in 3 h. Material was isolated in this way from large-scale incubation mixtures as described in 'Methods'. Column chromatography of the material was carried out on Sephadex G75 in both 10 mM-tris-HCl, pH 8.0 containing 1 mM-mercaptoethanol and in the same buffer containing 0.1% sodium dodecyl sulphate (SDS). In both cases the bulk of the radioactivity was excluded from the gel and the small amount of included material was UDP-N-acetylglucosamine.

The excluded

material contained protein as indicated by the Lowry method and absorption at 280 nm. Analytical polyacrylamide gel electrophoresis in 0.1% SDS revealed two diffuse protein bands, one at the top of the gel and the other with Rf of about 0. 75. The Rf 0. 75 band contained all the radioactivity and stained positively with periodate-Schiff.

Radioactive polymer that had been extracted from large scale incubation mixtures as described was further purified by preparative SDS polyacrylamide gel electrophoresis, under the same conditions as described for analytical electrophoresis, on a 2. 5 cm x 6. 5 cm

45 cylindrical gel. The fast running protein fraction (R^ 0. 65 - 0. 85), which contained all the radioactivity, was eluted from the gel into water, dialysed, and passed through a column of Sephadex G75, from which the excluded peak containing all the radioactivity was retained. Double-diffusion against DEAE-dextran in 1.0% agarose gel at pH 7. 3 revealed two sharp precipitin lines, indicating that the purified material contained two anionic components. This partially purified labelled material was used for further chemical studies. o Acid hydrolysis in 2N HC1, 3 h at 100 , followed by paper chromatography in solvent system B and staining with silver nitrate reagent, revealed the presence of glucose and glucosamine (with a trace of N-acetylglucosamine).

Paper electrophoresis of the same hydrolysate

revealed a non-reducing component that stained positively for phosphate and amino groups but was unidentified. This component was also detected by paper chromatography in solvent A, in which it displayed an R G l c N H ^ . 6 . p = 2.24. Table 2 shows the amino acid composition of the glycoprotein.

It

contained only small amounts of the aromatic amino acids and negligible amounts of amino acids containing sulphur. Table 2 - Amino acid composition of glycoprotein Lysine 1.35 Proline 0.30 iLeucine

0.35

Histidine

0.17

Glycine

0.91

Leucine

0.44

Arginine

1.70

Alanine

1.61

Tyrosine

0.09

Aspartic acid 1.0

half-cystine

0

Phenylalanine

0.13

Threonine

0.35

Valine

0.44

Tryptophan

0

Serine

0.52

D.a.p.

trace

Glucosamine

0.44

Methionine

0.13

Glutamic acid 1.22

The analysis was carried out on an 18 h hydrolysate. Aspartic acid was set arbitrarily at 1.0.

46 Determination of N-terminal amino acids by dansylation gave two major AAs, one of which was the dansyl derivative of glycine.

The

other has not been identified. The preliminary evidence suggested that the material containing Nacetylglucosamine was an acidic glycoprotein.

In order to examine

this hypothesis the polymer was subjected to digestion with pronase (1 mg/ml) for 24 h at pH 7. 0 in Tris-HCl containing lmM-CaCl 2 , 37° After 24 h a further 1 mg/ml pronase was added and incubation was continued for a further 24 h. The digestion mixture was subjected to paper electrophoresis in pyridinium acetate at pH 6. 5. This yielded a small, sharply defined fraction that had a slight positive charge, was radioactive and stained with ninhydrin, together with a more diffuse, very acidic peptide fraction that contained most of the radioactivity.

These peptides were eluted into water and chromato-

graphed on a column of Sephadex G25. The acidic electrophoresis fraction yielded two peaks of radioactivity, a small one (GIA) which was excluded from the resin and a much larger one (G2A) which was included. The basic electrophoresis fraction yielded a single peak on G25 (G3B) which had approximately the same apparent molecular weight as G2A. The release of these radioactive glycopeptides from the high molecular weight material by treatment with the proteolytic enzyme confirmed that the product of the biosynthetic reaction was a glycoprotein. Biosynthesis of the polymer The chromatographic properties of the N-acetylglucosamine-containing lipid that was formed during biosynthesis of the polymer, and its extreme lability to acid (it had a half-life of 15 min at 100° in 0.01MHC1 in 50% MeOH) suggested that it might be a polyprenol phosphate

47 derivative of the type known to participate in the biosynthesis of many (22) bacterial polysaccharides

. In order to test this hypothesis, the

effect of added C^.,.-polyprenol monophosphate on lipid and polymer synthesis was examined.

Added polyprenol monophosphate stimulated

lipid synthesis, but only in the presence of the nonionic surfactant Triton X-100 (Fig. 1).

Fig. 1.

The dependence of stimulation of polyprenol phosphate upon Triton X-100.

R u c t i o n mixtures contained 0 . 1 ml membrane suspension, MgCl ( 5 ^ m o l ) , UDP-N[ C-acetyl]acetyl glucosamine (0. 1 ^ m o l , 2. 98 x 10 5 cpm), and Triton X100 at the indicated concentration. The total volume was 0. 13 ml. C,.,.-polyprenol phosphate (In m o l ) was added to each incubation tube f i r s t in chloroform;methanol 2:1 v/v, then the solvent was evaporated off under reduced p r e s s u r e before addition of the other reagents and mixing. Reaction was terminated by the addition of 0. 05 ml nbutanol and lipid synthesis was measured as described for T a b l e 1. Incubations for 1 h at 30°

In the presence of Triton X-100 C ^ . -polyprenol monophosphate stimulated incorporation of label from UDP-N-acetyl glucosamine into the lipid as shown in Fig. 2. The effect of a number of different polyprenol monophosphate preparations on lipid and polymer synthesis is shown in Table 3.

Only the

phosphate of the all-trans C- ^ isoprenol, solanesol failed to stimulate both lipid and polymer synthesis.

48

Fig. 2.

Stimulation of lipid synthesis by C ^ - p o l y p r e n o l monophosphate.

Incubation mixtures were as described for Fig. 1. All incubations contained Triton X-100 at a final concentration of 0. 3% v / v . Incubations were carried out at 30° for 1 h and lipid synthesis was assayed as described for Table 1.

Table 3 - Effect of polyprenol monophosphates on lipid and polymer synthesis no added lipid

heveaprenol phosphate

bactoprenol phosphate

solaneaol phosphate

cpm in polymer

8144

13263

11652

8102

cpm in lipid

6060

12297

12235

6091

Incubation mixtures were as described for Fig. 1. Approx. 3 nmol of the indicated polyprenol phosphate was added. This evidence suggested that the lipid was a polyprenol monophosphate or pyrophosphate N-acetylglucosamine that might be intermediate in the biosynthesis of the polymer.

In order to obtain further information

about the synthesis of the lipid the effects of UMP and UDP on its formation were examined.

It was found that UMP strongly inhibited

both polymer and lipid synthesis, while UDP inhibited the synthesis of polymer but not of lipid.

This result suggested that lipid synthesis

49 involves the t r a n s f e r to polyprenol monophosphate of N-acetylglucosamhe 1-phosphate f r o m UDP-N-acetylglucosamine, with the r e l e a s e of UMP. UDP-N-acetylglucosamine + lipid-P — ^ UMP + l i p i d - P - P - N - a c e t y l ^

glucosamine

UMP would inhibit this reaction by a m a s s - a c t i o n effect. T r a n s f e r of either N - a c e t y l g l u c o s a m i n e or its 1-phosphate f r o m lipid to polymer could subsequently occur. DISCUSSION Our p r e l i m i n a r y r e s u l t s show that in the m e m b r a n e of B. licheniformis t h e r e a r e enzymes that catalyse the incorporation of N-acetylglucosamine, N-acetylglucosamine 1-phosphate, or both, into an endogenous glycoprotein a c c e p t o r .

It is not yet known whether the product of this

c e l l - f r e e biosynthetic s y s t e m r e p r e s e n t s the cellular end-product although work is in p r o g r e s s to c h a r a c t e r i s e the glycoprotein of the cell.

The amino acid analysis of the glycoprotein does not indicate a l a r g e e x c e s s of acidic AA r e s i d u e in the protein moiety and the polyanionic nature of the glycoprotein may t h e r e f o r e be attributable to the glycan p a r t , and in p a r t i c u l a r to phosphate groups in the glycan.

The high

acidity of the phosphoglycoprotein and its high glycan content m a k e m e a s u r e m e n t s of its m o l e c u l a r weight by conventional physical techniques difficult.

The r o l e of lipid i n t e r m e d i a t e s in the biosynthesis of glycoproteins in (10) .. (23,24,25) , . yeasts and in s o m e m a m m a l i a n s y s t e m s has receivedJ c o n s i d e r a b l e attention recently, but work has been hindered by difficulties in c h a r a c t e r i s i n g the protein a c c e p t o r and the end products in t h e s e e x p e r i m e n t s .

The B. licheniformis s y s t e m d e s c r i b e d h e r e

50 appears to provide a simpler model for lipid-mediated glycoprotein synthesis, since the product has a simpler sugar composition and is freely soluble in water, and therefore more amenable to conventional purification. The cellular location of the end product and its role in the cell remain to be determined.

SUMMARY N-acetylglucosamine or N-acetylglucosamine-1-phosphate residues were found to be transferred from UDP-N-acetylglucosamine through a lipid intermediate into macromolecular material by a membranebound enzyme system from B. licheniformis. The product was watersoluble, contained protein and carbohydrate and was excluded from Sephadex G-75. After treatment with a proteolytic enzyme the material yielded glycopeptides which were included in Sephadex G-25 and were separated by paper electrophoresis.

The original glycoprotein

was strongly acidic, and it yielded two acidic glycopeptides and a weakly basic one, all of which contained N-acetylglucosamine.

The

preparation contained phosphate groups in the carbohydrate part of the glycoprotein. REFERENCES 1.

Odds, F. C. & Hierholzer (1973) J. Bacteriol. 114, 257-266.

2.

Smith, W. J. & Ballon, C. E. (1974) Biochemistry 13, 355-361.

3.

Sentandreau, R. & Northcote, D. H. (1969) Biochem. J. 115, 231-240.

4.

Biely, P., Farkas, V. & Bauer, S. (1972) F. E . B . S . Letters 23, 153-156.

5.

Everse, J. & Kaplan, N. O. (1968)]. Biol. Chem. 243, 60726074.

51 6.

Okada, S. & Weinbaum, G. (1968) Biochemistry 7, 2319-2825.

7.

Clarke, K . , Gray, G. W. & Reaveley, D. A. (1967) Biochem. J. 105, 755-758.

8.

Datta, A . , Otero, R. D . , Braunstein, S. N. & Franklin, R. M. (1973) Biochim. Biophys. Acta 311, 163-172.

9.

Sadowski, P. L. & Strobel, G. A. (1972) J. Bacteriol. 115, 668672.

10.

Kozak, L. P. & Bretthauer, R. K. (1970) Biochemistry 9, 11151122.

11.

Lloyd, K. O. (1972) Biochemistry U, 3884-3890.

12.

Gander, J. E . , Jentoft, N. H., Drewes, L. R. & Rick, P. D. (1974) J. Biol. Chem. 249, 2063-2072.

13.

Hancock, I. C. & Baddiley, J. (1972) Biochem. J. ¿27, 27-37.

14.

Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412.

15.

Segrest, J. P. & Jackson, R. L. (1972) Methods in Enzymol. 28, 54-62.

16.

Hanes, C. S. & Isherwood, F. A. (1949) Nature (London) 164, 1107-1109.

17.

Trevelyan, W. E . , Procter, D. P. & Harrison, J. S. (1950) Nature (London) 166, 444-445.

18.

Anderson, R. G . , Hussey, H. & Baddiley, J. (1972) Biochem. ]. 127, 11-25.

19.

Popjak, G . , Cornforth, J. W., Cornforth, R. H., Ryhage, R. & Goodman, D. S. (1962) J. Biol. Chem. 237, 56-62.

20.

Lahar, M . , Chin, T . H. & Lennarz, W. J. (1969) J. Biol. Chem. 244, 5890-5896.

21.

Baddiley, J . , Blumsom, N. L. & Douglas, L. J. (1968) Biochem. J. U0, 565-570.

22.

Lennartz, W. J. & Scher, M. G. (1972) Biochim. Biophys. Acta 265, 417-441.

23.

Parodi, A. J. et al (1973) Carbohydrate Res. 26, 393-400.

52 24.

Maestri, N. & de Luca, L. (1973) Biophys. Biochem. Res. Commun. 53, 1344-1349.

25.

Waechter, C., Lucas, L. & Lennartz, W. J. (1974) Biophys. Biochem. Res. Commun. 56, 343-350.

53

Degradation of Thyrotropin Releasing Hormone (TRH). Its Inhibition by Pyroglu-His-OCH3 and the Effect of the Inhibitor in Attempts to study the Biosynthesis of TRH K. B a u e r M a x - V o l m e r - I n s t i t u t , Abteilung Biochemie, Technische U n i v e r s i t ä t

Berlin

I NTRODUCT I ON Due t o o u r i n t e r e s t

in t h e mechanism of p e p t i d e s y n t h e s i s

in general

a t t e n t i o n was drawn t o t h e r e c e n t l y d i s c o v e r e d group o f p e p t i d e s

our

produced

by t h e h y p o t h a l a m u s , t h e s o - c a l l e d r e l e a s i n g hormones which t r i g g e r r e l e a s e of t h e hypophyseal

hormones. We became e s p e c i a l l y

the

interested

s t u d y f i r s t t h e s m a l l e s t of t h e s e p e p t i d e s , t h e t h y r o t r o p i n

to

releasing

hormone ( F i g . 1 ) . Thyrotropin-re leasing-hormone (TRH)

pyroGlu In view of t h e s m a l l

His

Pro-NHi

s i z e of t h e p e p t i d e t h e b i o s y n t h e s i s c o u l d be c a r r i e d

o u t e n z y m a t i c a l l y a n a l o g o u s t o t h e s y n t h e s i s of g l u t a t h i o n s t r u c t u r a l s i m i l a r i t y of TRH w i t h t h e o t h e r s o f a r hormone, t h e

(1).

identified

The

releasing

l u t e i n i z i n g hormone r e l e a s i n g hormone ( I H - R H ) w i t h t h e

quence p y r o G l u - H i s - T r p - S e r - T y r - G l y - L e u - A r g - P r o - G l y - N I ^ l i k e l y that the s y n t h e s i s polypeptide a n t i b i o t i c s

strated for several

i s mediated by a muitienzyme s y s t e m as f o r t h e

( 2 ) . However,

s y n t h e s i z e d by a ribosomal

se-

makes i t more

it

i s a l s o p o s s i b l e t h a t TRH

is

mechanism as p a r t of a prohormone a s demon-

p o l y p e p t i d e hormones ( 3 , 4 , 5 , 6 ) . The p y r o g l u t a m y l

mation and t h e a m i d a t i o n a t t h e c a r b o x y - t e r m i n a I , which i s

for-

frequently

found in many b i o l o g i c a l l y a c t i v e p e p t i d e s , c o u l d be p a r t of t h e c l e a v a g e mechanism o r c o u l d be formed by s u b s e q u e n t r e a c t i o n s . The s i m p l e of TRH would make i t an ideal model s u b s t a n c e t o s t u d y t h e s e

structure

questions.

54 RESULTS Degradation of TRH In the meantime it has been reported that TRH is synthesized by a particulate free extract of freeze-dried porcine hypothalamic tissue (7,8). How14 ever, when we repeated the incubations as reported using C-proline as precursor and subjected tne methanolic extract from such an incubate to two dimensional thin layer chromatography many unidentified radiolabeI led products could be detected by radioautography, but the area of the cochromatographed marker TRH, which was visualized by the PAULY reaction, did not contain any radioactivity. Moreover with such a preparation we observed powerful degradation activity which made it impossible to find any biosynthetic activity. A rapid inactivation to TRH by animal and human plasma was first reported by BOWERS et a I. (9). The same laboratory reported recently (10) that the inactivation of TRH by serum is only due to a deamidation reaction. Our studies with TRH,

H-labelled in the proline moiety, show that this is

only the first step of TRH inactivation which is followed by proteolytic cleavage yielding free proline as the major radiolabelled degradation product (Fig.2). That pr line derives from the hydrolysis of deamido-TRH could be demonstrated by incubation of isolated deamido-TRH with serum followed by thin layer chromatography as before. These findings confirm the results of VALE et al. (11) who suggested that there must be an additional mechanism of TRH degradation since they observed a total

loss of the biological activity of TRH after incubation

with serum while deamido-TRH exhibits some of the biological activity of TRH. The high degradation activity of the tissue extract could not be explained by the serum contamination of these preparations. After

incuba-

tion of TRH-^H-proIine with a high speed supernatant from freeze-dried porcine hypothalamic tissue, as used in the report on the biosynthesis of TRH (7,8) the degradation pattern of Fig.3 was obtained.

55

I

3000

I

Start

•v A Proline

|

M .1!

t TRH

Front

Deamido-TRH

F i g . 2 . Degradation of TRH by Rat Serum. ^ 10 Ail of r a t serum was incubated f o r 60 min a t 37 C w i t h 0 . 5 / u C i TRH- Hp r b l i n e (40 Ci/mM) in 20 mI b u f f e r A ( T r i s - H C I pH 7 . 4 , c o n t a i n i n g 50 mM KCI and 2 mM DTT). The r e a c t i o n was stopped by the a d d i t i o n of methanol, the p r e c i p i t a t e removed by c e n t r i f u g a t i o n and the supernatant s u b j e c t e d t o t h i n layer chromatography on s i l i c a gel ( S i l p l a t e 22) using the s o l vent system CHCI3:CH30H:NH40H ( 1 2 5 : 7 5 : 2 5 ) . The separated r a d i o l a b e l l e d s p l i t products were l o c a l i z e d by scanning f o r r a d i o a c t i v i t y and i d e n t i f i e d by cochromatography w i t h the marker substances in several o t h e r s o l v e n t systems.

F i g . 3 . Degradation of TRH by Hypothalamic Tissue E x t r a c t . The i n c u b a t i o n was done as described in t h e legend of F i g . 2 using a high speed supernatant prepared from a 10$ homogenate of f r e e z e - d r i e d p o r c i n e hypothalamic fragments in b u f f e r A. As b e f o r e , t h e r a d i o l a b e l l e d s p l i t products were resolved by t h i n layer chromatography, l o c a l i z e d by scanning f o r r a d i o a c t i v i t y and i d e n t i f i e d by cochromatography w i t h marker substances in several s o l v e n t systems.

56 As r a d i o l a b e l l e d s p l i t products deamido-TRH, p r o l i n e and prolineamide could be detected. The p r o t e o l y t i c cleavage y i e l d i n g prolineamide comparable t o the reported r e l e a s e of the carboxy-termlnal

Is

glycineamide

from oxytocin by o x y t o c i n responsive t i s s u e ( 1 2 ) . When \ l - p r o l i neaml de was I s o l a t e d and incubated with such an e x t r a c t no deamidation could be observed. The free p r o l i n e d e r i v e s from the h y d r o l y s i s of deamido-TRH as demonstrated by the Incubation of ^H-deamldo-TRH with t h i s e x t r a c t . When a high speed supernatant was prepared from a homogenate of

fresh

rat hypothalamic o r c o r t i c a l t i s s u e we could only observe a deamidation reaction whi le the whole homogenates possess a l s o the p e p t i d a s e s

yield-

ing p r o l i n e and prolineamide as r a d i o l a b e l l e d s p l i t products (Table

1).

Table I : Degradation of TRH by Various P r e p a r a t i o n s % of r a d i o a c t i v i t y added as TRH- 3 H-prol Ine A

B

C

D

37

35

65

39

Deam i do-TRH

33

30

35

ProlIneamide

21

10

none

none

9

25

none

57

Preparation TRH

Prol1ne

4

TRH- H - p r o l l n e was Incubated with the p r e p a r a t i o n s l i s t e d as described in the legend of F i g . 2 . A: High speed supernatant of a homogenate prepared from f r e e z e - d r i e d hypothalamic fragments. B: Whole homogenate from f r e s h rat hypothalamic o r c o r t i c a l t i s s u e . C: High speed supernatant from B. D: Rat serum. A f t e r t h i n layer chromatography the r a d i o a c t i v i t y of the r a d i o l a b e l l e d s p i l t products were determined by s c i n t i l l a t i o n c o u n t i n g . Apparently these peptidases are membrane bound and become s o l u b l l i z e d during l y o p h y I i s a t l o n . So I t must be concluded t h a t the peptidase which s p l i t s p r o l i n e from deamido-TRH i s d i f f e r e n t from the comparable serum enzyme. That the s o l u b l e TRH-deamldating enzyme i s a l s o d i f f e r e n t from the serum enzyme w i l l be shown by I t s s p e c i f i c I n h i b i t i o n by the d l peptide e s t e r pyroGlu-His-OCH, (see below).

57 Inhibition of TRH-Degradation We had some hope to overcome the TRH degradative activity of the tissue preparations since it had been reported by GUILLEMIN's

laboratory

(11)

that the methylester of the N-terminal dipeptide of TRH, pyroGlu-His-OCH strongly

inhibits the inactivation of the biological activity of TRH by

serum. Following the effect of the inhibitor on the degradation of TRH by serum Fig.4 was obtained. At a 4 mM concentration of this material degradation was reduced by

50%.

100 r i ro

"a o

m M pyro-Glu-His-methyl ester

Fig.4. TRH recovery and degradation after incubation with rat serum dependent on inhibitor'concentration. PyroGlu-His-OCHj was added to the incubation of TRH-3H-proline with rat serum as in Fig.2. After thin layer chromatography the radioactivity in the TRH and proline zone was determined by scintillation counting. When the inhibitor was added to the high speed supernatant from freezedried porcine hypothalamic fragments a marked difference could be shown (Fig.5). While the activity of the peptidases yielding proline and prolineamide was inhibited at inhibitor concentrations comparable with the serum enzymes the deamidation reaction was less sensitive,

indicating

that the TRH-deamidating enzyme of the tissue is different from the serum enzyme.

58 TRH

100 X Q: i— fO

in a "O a> •o TJ o

20'!\ Proline omide Proline 3610

Deamido-TRH

//— J

I

40

L

I

60

130

I

A I 150

mM pyroGlu- His-methyl ester

F i g . 5 . TRH r e c o v e r y and d e g r a d a t i o n a f t e r i n c u b a t i o n w i t h an e x t r a c t of h y p o t h a l a m i c t i s s u e dependent on i n h i b i t o r c o n c e n t r a t i o n . The experiment was done as f o r F i g . 4 using t h e high speed s u p e r n a t a n t from a homogenate of f r e e z e - d r i e d p o r c i n e h y p o t h a l a m i c t i s s u e . Whether some of t h e s e TRH d e g r a d a t i n g enzymes may p l a y a r o l e g u l a t i o n of t h e b i o l o g i c a l inary origin

activity

results with p a r t i a l l y

of TRH w i l l

in t h e

be i n v e s t i g a t e d .

p u r i f i e d TRH-deamidating enzyme of

re-

Prelimtissue

i n d i c a t e t h a t t h i s enzyme i s s p e c i f i c f o r TRH.

Attempts t o Study t h e B i o s y n t h e s i s of TRH With t h e

i n f o r m a t i o n from t h e s e s t u d i e s we had some hope t o f i n d

thetic activity

f o r TRH by adding t h e d i p e p t i d e e s t e r t o an

biosyn-

incubation

of f r e s h r a t h y p o t h a l a m i c t i s s u e homogenate supplemented w i t h amino a c i d s , ATP, Mg++ and an ATP r e g e n e r a t i n g system ( s e e

legend of T a b l e

III).

Since

t h e d i p e p t i d e e s t e r does not p r e v e n t t h e deamidation of TRH by h y p o t h a l a mic t i s s u e , reaction

one should e x p e c t deamido-TRH r a t h e r than TRH i t s e l f

product.

as t h e

59 First a very effective purification procedure had to be worked out which reliably

identifies deamido-TRH, present only

in minute amount besides 14

many unidentified radioIabeI Ied metabolites. Using

C - p r o l i n e as t h e

radioIabeI Ied precursor for the incubation and adding "^H-deami do-TRH after the incubation as Internal

standard gave us the possibility to

determine by scintillation spectrometry the recovery during the purification and so to account for the variability which allows to correlate the data obtained for quantitative evaIuation. • As

listed in T a b l e

purification was achieved by five successive steps. T h e purity of the material

radiochemical

from t h e deamido-TRH zone3 of14the second thin

layer chromatogramm was proven by the constant isolated material was subjected to thin other solvent systems, thin of the isolated material Table

II the

H:

C ratio when the

layer chromatography

in several

layer electrophoresis and chemical

conversion

into T R H by esterif¡cation followed by

II: Purification Scheme for the

Isolation of

Deamido-TRH

Recovery

Purification

t

amidation.

factor^++'

Charcoal absorption CM-cellulose column Ce 11u1ose-phosphate paper chromatography TLC-Sol vent System A TLC-Sol v e n t System B

90-95 95-99

30 1 .25

85-90

20

70-80 70-80

2.0 1.1

Total

34-54

1900 fold

T h e incubation conditions are given in the legend of Table III. After incubation 3 H - d e a m l d o - T R H (90 000 dpm) and 50/ug synthetic deamido-TRH were added. After absorption on charcoal and intensive washing with 0.02 N H O A c the material was eluted by 95? pyridine, taken to dryness and applied on a short column of CM-cellulose which was eluted with water. T h e eluate was applied to cellulose-phosphate paper and developped twice with water. The material from the deamido-TRH zone was eluted with 2 N NH4OH and subjected to thin layer chromatography on silica gel (Silplate 22) in the solvent system A: C H C I 3 : C H 3 0 H : N H 4 0 H (125:75:25). T h e radioactive zone corresponding to deamido-TRH was eluted with 50? methanol and used for thin layer chromatography in the solvent system B: Phenol : H2O (75:25). After elution the radioactivity was determined by scintillation spectrometry. T h e recovery'*) is determined by t h e recovery of 3n-deamido-TRH which was added as internal standard and t h e purificat i o n ( + + ) is based on the 1 4 C - d p m added as roline and corrected for recovery.

60 The incorporation obtained was unexpectedly high (30 000 dpm, which accounts for 56 pmole or 20 ng of TRH) and we began to wonder about the mechanism of incorporation. Therefore we studied first the incorporation 14 of the other constituent amino acids. When C-histidine was added as radioIabeI led precursor to such incubation mixtures no incorporation

into

deamido-TRH could be observed demonstrating that the formation of deamidoTRH is not due to a de novo biosynthesis of the tripeptide. Table III surveys the results from further studies of this reaction which is not sensitive to inhibitors of ribosomal

synthesis such as puromycin,

cycloheximid or RNAse and is even not energy dependent. When we observed that the dipeptide es.ter is hydrolyzed by such an incubation we realized that the split is presumably due to the same peptidase that degrades deamido-TRH yielding free proline since it is well that peptidases show some esterase activity and explaining

known

inhibition of

the degradation by a competition for the enzyme. Therefore the hydrolysis of the dipeptide ester pyroGlu-His-OCHj could lead to the same enzyme bound intermediate ENZYME«»His-pyroGlu as the hydrolysis of deamido-TRH (pyroGlu-His-Pro-OH). By accepting proline in the reverse reaction the peptidase could incorporate proline into the dipeptide in amounts comparable to what we were finding. Such an enzymatic process would explain that for the reaction ATP is not only not necessary but is rather inhibitory, presumably since the M g + + chelating nucleotides reduce the concentration of free M g + +

required as

cofactor of the Qnzyme. The specific incorporation of proline, which is not greatly reduced in the presence of all the other amino acids added in comparable molar concentration, supports this explanation.

Further

evidence therefore can be seen in comparing Table I with Table

III.

While homogenates prepared from fresh rat cerebral cortex were also active in the formation of the tripeptide, a high speed supernatant from freeze-dried hypothalamic fragments had some activity and a high speed supernatant from fresh tissue was inactive. This is in parallel with the TRH degradation activity of the different preparations in respect to the formation of proline as radiolabelled split product (see Table I).

61 Table III: Deamido-TRH formed on Incubation with pyroGIu-HiS-OCH3 . 14. and C-prol1ne Preparation

Tripeptide formed (pmo I e)

Hypothalamic homogenate supplemented 56 + puromycin (100/ug/ml) 58 + cycloheximid (300/ug/ml) 50 + RNAse ( 20/Ug/mI) 56 - ATP, GTP and~P regenerating system 121 - pyroGIu-HiS-OCH3 0 + 17 ami no ac i ds 42 Homogenate of cerebral cortex 48 High speed supernatant of freeze-dried hypothalamic tissue 18 High speed supernatant of fresh tissue 0 Male rats were killed by decapitation, the tissue removed and immediately homogenized in ice cold buffer B (0.25 M sucrose, 50 mM Tris-HCI, ph 7.5, 150 mM NH4CI, 5 mM MgAcg and 2 mM DTT). To 300/UI of homogenate was added: 10/\jmole pyroG I u-H i S-OCH3, 1/limole ATP, 0.25/Umole GTP, 2.5/umole histidine, 2.5/umole glutamine and 25/uCi 1 4 C-proline (250 mCi/mM). The total volume was 0.5 ml. The mixture was incubated for 45 min at 37°C and the deamido-TRH formed was isolated as described in Table II.

SUMMARY In attempts to study the mechanism of biosynthesis of TRH very active degradation of TRH by serum and tissue preparations was observed. By addition of pyroGIu-His-0CH^ the complete breakdown of TRH could be prevented. In the hope that this might enable us to study the biosynthesis of TRH, 14 appropriate incubates of hypothalamic tissue homogenate with C-proline as precursor were supplemented with the dipeptide ester. After vigorous purification radioIabeI led deamido-TRH could be isolated. However, the formation of deamido-TRH is not related to the biosynthesis of TRH but is due to incorporation of proline by the reverse reaction of peptide hydrolysis. Acknowledgment This study was supported by a grant to Dr. F. Lipmann from the United States Public Health Service. It was done during my stay in the laboratory of Dr. Lipmann who initiated this project. I am most thankful to him as my teacher and mentor for the educational training, for all his

62 advice, encouragement and guidance. I thank Dr.R.O. Studer, Hoffmann-La Roche A.G., Basle, for the valuable gift of peptide intermediates. Dr. R. Guillemin and his colleagues at the Salk Institute I am very much obliged for the interest and encouragement and for the support during my stay in his

laboratory.

REFERENCES• (1) Mooz, E.D., Meister, A.: Tripeptide (glutathion) synthetase. Purification, properties, and mechanism of action. Biochemistry^, 1722-1734 (1967). (2) Lipmann, F., Gevers, W., Kleinkauf, H., Roskoski, R., Jr.: Polypeptide synthesis on protein templates: The enzymatic synthesis of gramicidin S and tyrocidines. Adv.Enzymology 35, 1-34 (1971). (3) Steiner, D.F., Oyer, P.E.: The biosynthesis of insulin and a probable precursor by a human islet cell adenoma. Proc.Nat.Acad.Sci .US._57, 473-480 (1967). (4) Kemper, B., Habener, J.F., Potts, jun., J.T., Rich, A.: Proparathyroid hormone: Identification of a biosynthetic precursor to parathyroid hormone. Proc.Nat.Acad.Sci.US.69, 643-647 (1972). (5) Cohn, D.V., MacGregor, R.R., Chu, L.L.H., Kimmel, J.R., Hamilton,J.W.: Calcemic fraction-A: Biosynthetic peptide precursor of parathyroid hormone. Proc.Nat.Acad.Sci.US.69, 1521-1525 (1972). (6) Tager, H.S., Steiner, D.F.: Isolation of gIucagon-containing peptide: Primary structure of a possible fragment of proglucagon. Proc.Nat.Acad. Sci.US.70, 2321-2325 (1973). (7) Mitnick, M.A., Reichlin, S.: Enzymatic synthesis of thyrotropin-releasing hormone: Biosynthesis by rat hypothalamic fragments in vitro. Science V72, 1241-1243 (1971). (8) Mitnick, M.A., Reichlin, S.: Enzymatic synthesis of thyrotropin-releasing hormone (TRH) by hypothalamic 'TRH synthetase'. Endocrinology 91, 1145-1153 (1972). (9) Bowers, C.Y., Redding, T.W., Hawley, W.D.: The effect of thyrotropinreleasing factor (TRF) in animals and man. Program of the 48th Meet, of the Endocrine Soc., Chicago, III., June 20-22, 1966, p.43 (abstract). (10) Nair, R.M.G., Redding, T.W., Schally, A.V.: Site of inactivation of thyrotrop in-releasing hormone by human plasma. Biochemistry 3621-24, (1971). (11) Vale, W.W., Burgus, R., Dunn, T.F., Guillemin, R.: In vitro plasma inactivation of thyrotropin-reIeasing factor (TRF) and related peptides. Its inhibition by various means and by the synthetic dipeptide PCA-HisOME. Hormones 2, 193-203 (1971).

Amphibian Cells in Culture: An Approach to Metamorphosis

Thomas Peter Bennett Biological Science, Florida State University, Tallahassee, Florida 32306, USA The liver of Rana catesbeiana undergoes metabolic differentiation during the metamorphosis of this animal from a tadpole into an adult frog. The biochemical criteria for this differentiation include the induction of synthesis of urea cycle enzymes and oertain serum proteins in addition to changes in many enzyme activities which have been well documented and have been reviewed (1-4). Recently, we (5,6) have reported cytological criteria for differentiation of liver cells during thyroxineinduced and natural metamorphoses . One may ask: "How does thyroxine control the cytological differentiation of a tadpole liver cell into a frog liver cell?" A similar question may be asked about the biochemical differentiation which occurs. In attempting to answer these questions, our first task was to establish cytological criteria by light and electron microscopy for liver cells of R. catesbeiana in vivo undergoing natural and thyroxine induced metamorphosis (5,6). These studies on liver from animals at various metamorphic stages were correlated with the morphological features of R. catesbeiana undergoing metamorphosis and are summarized in Figure 1. The nuclei of parenchymal cells are euchromatic in early metamorphic stages and become heterochromatic about the time of forelimb development and remain so (6). They become more irregular in shape, and the number of nucleoli increases. The mitochondria increase in size, and their cristae change from a broad lamellar to a smaller, more tubular appearance during the period from hindlimb development through metamorphic climax. The rough endoplasmic reticulum (RER) proliferates throughout the cytoplasm and the cisternae become dilated during this period. The Golgi conplex is associated with dense granules (indicating biosynthetic activity) during early stages of metamorphosis, appears less active during metamorphic climax, and returns to a more active appearance in froglet and adult animals. A conparison of the fine structural changes in liver cells during natural metamorphosis with those which occur during thyroxineinduced metamorphosis (5) reveals that the alterations are qualitatively similar but often differ quantitatively in the two types of metamorphoses (see Figure 1). Further, the organelle features of liver cells in thyroxine-induced metamorphosis represent a oonbination of the organelle features in cells

64 Class

1

2

3

%J

/ • • T V .

Nucleus

r.-

4

« /

Tsess

E

Mitochondria

RER

Golgi

iHjl UP US it .' A 1 1 >'""

Appeoranct

Stage Leg Length Tail Length

IX-Xill

0058-016

8

5

"t *

XVII-XX

038-061

XXI-XXIV

073-096

Frog let Frog CO

Figure 1. Schematic diagrams shewing the salient changes in the organelles of parenchymal cells frcm four developmental classes of Rana catesbeiana. The gross appearance of representative members of each class is shown along with the morphological stages according to Taylor and Kollros (16) and leg length to tail length (L/T) ratios. Based on data in (5) and (6). frcm animals in Classes 3 and 4. The nuclear features observed following thyroxine treatment of premetamorphic (Class 1) tadpoles resemble those of Class 3 animals; the extent of heterochromatization and the amount of perinuclear KER are similar. The mitochondria Show marked increases in size during both types of netarrcirphoses but are considerably larger in induced metamorphosis with volumes about 2.5 times those of Class 4 mitochondria The proliferation and cisternal features of KER in induced metamorphosis most resemble those observed in Class 3 animals. The increased nunber of Golgi regions in induced metamorphosis is unique; however, the appearance of individual Golgi resembles that observed in Class 4 animals. From these findings, "the picture which emerges is "that thyroxine-induoed metamorphosis , under conditions which are frequently used in biochemical studies, results in cytological features found late in natural metamorphesis. However, in thyroxine-induced metamorphosis the extent of the changes do not precisely parallel those found in natural metamorphosis. In preliminary reports Tata (7), Cbhen (8) and Spiegle and Spiegle (9) have made seme of these cytological observations on, respectively, R. catesbeiana and R. pipiens•

65 Because of individual differences among tadpoles in "their time of response to thyroxine and tissue variability, the development of organ or cell cultures of their tissues is of considerable importance for approaching the elucidation of the mechanism(s) of thyroxine-induced changes in biochemical and cytological patterns during metamorphosis. In order to develop a model system for e>ploring the basis for natural and thyroxine-induced differentiation of tadpole tissues, with the ultimate objective of understanding the mechanism(s) of thyroxine action in this process, it was necessary to define the techniques for preparing and conditions for maintaining tadpole liver cells in vitro over a period of 6 days in a close approximation to their native morphological and biochemical states. This period is sufficient to observe in vivo effects of thyroxine (4,5) and should be suf-. ficient for in vitro study of the nechanism of thyroxine action. The extensive literature about culturing liver cells (reviewed in 10) suggested that very careful morphological studies on cells in culture must precede or be closely correlated with biochemical studies. Thus our initial studies were cytological and eirployed light and electron microscopy to verify the condition of cultured tadpole liver (10, 11). This was done since it is clear that, if liver cells or liver tissue are placed in sterile culture medium, stable enzyme activities may be measured for days and weeks, even though cells may be dying, lysing and releasing their contents into the sterile medium (12). The results of our studies (10) demonstrated that the normal premetamorphic fine structure features of parenchymal cells and their organelles are well preserved for a period of six days in culture. However, on being put into culture medium and during the early period (up to about 30 hours) in culture, parenchymal cell nuclei become, according to the terminology of Fawcett (13) and Porter and Bonneville (14), heterodhromatic; later they return to their so called euchromatic state. Our results (15) also suggested that in our case the "trauma," often mentioned in connection with cell culture work, that occurs when tissue adapts to in vitro conditions is reflected in defined fine structural changes (i.e., chromatin condensation) in the cultured cells. After an adjustment period these changes are reversed completely or in part. Here we report preliminary biochemical studies on tadpole liver in organ culture. Since we are interested ultimately in an organ cultured liver system that will facilitate the analysis of thyroxine-induced changes in protein biosynthesis, we are particularly interested in establishing biochemical standards, such as wet weight, protein, RNA and DNA content, and in establishing morphological correlates to these changes when possible. The present biochemical study supports our cytological observations (10, 15) that our techniques for preparing and maintaining tadpole liver cells in vitro over a period of six days is

66 satisfactory for keeping the cells in close approximation to •their native state. METHODS AND MATERIALS All animals used were Class 1, that is Stage IX-XIII of Taylor and Kbllros (16) R. catesbeiana tadpoles (Connecticut Valley Biological Supply Go.., Southampton, Massachusetts). These tadpoles have short hind limbs but no forelinibs and are said to be in late premetamorphosis. The culture medium used in these experiments was based on the formulation of Wolf and Quiiriby (17) and was purchased from the Grand Island Biological Supply Company, Grand Island, N. Y. This medium was modified to contain 100 mg glucose per liter except as indicated in individual experiments. Organ cultures were prepared as previously described (10). The "wet weight" of tissue was determined, after it had been blotted, by weighing it on a tared glass cover slip using a Mettler (Model B1250-1) analytical balance (Mettler Corp., Princeton, N. J.). The dry weight was determined after the tissue had been dried to oonstant weight at 120°C and placed in a desiccator containing Drierite. For the determination of DNA and RNA, about 2 irgs of combined pieces of liver from culture tubes were harvested, blotted dry, and accurately weighed. The tissue was homogenized in 2 ml distilled water and the homogenate was made up to 5% vol/vol with trichloroacetic acid (TCA). The mixture was centrifuged for 10 minutes at 4-000 rpm in an International Clinical Centrifuge Model-CL (International Equipment Company, Boston, Itos.). The precipitate was washed two times with cold 10% TCA and once with 95% ethanol. The precipitate was then resuspended in 5% TCA and heated at 95°C for 15 minutes. The mixture was centrifuged as before, and aliquots of the supernatant were removed for the standard diphenylamine assay for DNA or for the orcinol assay for RNA (18). Protein was determined by biuret and Folin-phenol reagent methods according to the method described by Bennett (19). C11+-lysine and C11+-valine (specific activity 336 mC/mmole and 228 mC/nmole,respectivelyobtained from New England Nuclear, Boston, Mass.) and H3-6-thymidine (10,000 mC/ mmole), and H 3 5-uridine (2.0 C/mmole), obtained from Schwarz Bioresearch, Orangeburg, N. J.,'were used in incorporation studies. Tissue was harvested and transferred to incubation tubes containing 2 ml. Niu-Twitty solution (20), supplemented with 10 moles glucose. The final concentration of radioactive label in the incubation medium is indicated in the figure legends. After the incubation time indicated in the figures, the reaction was terminated by the addition of 0.10 nmoles of unlabeled compound

67 followed by the napid removal of the tissue. The tissue was blotted, transferred to a tared cover glass slip, and the "wet weight" of the tissue determined on "the analytical balanoe. The tissue was then processed by a modification of Palmiter's (21) methods. It was placed in 3 ml of acetone at 0°C, in which it was insoluble, washed twice with 1 ml portions of cold 5% trichloroacetic acid (TCA) and then washed twice with 3 ml of a mixture of ethanol-ether (3:1), and, finally, "the remaining tissue was dissolved in Soluene (Packard Instrument Co. , Dcwners Grove, 111.). One ml of Liquifluor (New England Nuclear, Boston, Mass.) was added to the sarrple in scintillation vials which had been previously cooled to 20°C. After 8 hours of dark-adaptation, each vial was counted using the automatic Beckman Liquid Scintil-i lation System (Model IS-250). Ornithine transcarbanylase (OTC) activity was determined according to the method described by Brown and Cohen (22). Some ejiperiments are "short term" (3 to 4 days) in duration, whereas others are "long term" (6 days). Although both long and short term microscopic experiments oould be made with tissue from one tadpole liver, biochemical experiments, which require more tissue for an assay, were designed to consider either long term or short term effects. Only seldom oould sufficient tissue be obtained from one animal to secure statistically valid data for some of the subtle points of short term studies as well as the more general observations of long term studies. RESULTS Water, Protein, UNA and RNA Content Several biochemical parameters of cultured tissue were studied over a 3 day to 4 day period to establish the condition of liver cells after varying times in culture. Representative short term and long term experiments are summarized in Tables 1 and 2. There is essentially no change in the dry weight/wet weigjrt ratio during increasing times in culture. Although these data do not exclude the possibility of absolute changes in the wet or dry weight of cells, they suggest "that no marked changes in hydration or dehydration of the tissue fragments occur during the period in culture. No change occurs in the amount of protein of cultured tissue over a similar period in culture (Tables 1 and 2); in contrast, a decrease in the amount of DNA and RNA occurs. In both cases the decrease is more gradual after about 40 hours in culture "than during earlier periods. The overall decrease in DNA content is approximately 30 per cent; 40 per oent for RNA. The decrease in ENA and RNA content appears to have leveled off by four da^s and remains constant througih 6 d^rs.

68 Table 1 BIOCHEMICAL CONSTITUENTS IN SHORT TEEM CULTURE

Hours in Culture

Dry Weight Wet Weight

0 20 i+0 60 80

0.20 0.18 0.21 0.23 0.19

Biochemical Parameter ygProtein ygENA ygENA 80+10 85 + 11 97 + 8 85+6 80+9

7.5+0.5 6.9 + 0.46.5 + 0.5 6.3+0.6 5.7+0.7

3.1+0.2 2.7 + 0.3 1.7 + 0.5 1.8+0.3 1.9+0.4

Except for the ratio of dry weight to wet weight, all paraneters are expressed per mg wet weight of tissue. Mean + Standard Error. Table 2

Hours in Culture 0 36 72 108 144

BIOCHEMICAL CONSTITUENTS IN LONG TEEM CULTUEE Dry Weight Wet Weight 0.19 0.21 0.20 0.17 0.18

Biochemical Parameter ygProtein ygENA ygENA 94+9 80+7 105+11 88+10 98+7

7.2+0.6 6.4+0.5 6.0+0.4 5.2+0.3 5.2+0.4

2.9+0.3 2.1+0.4 1.8+0.5 1.9+0.3 1.8+0.3

Except for the ratio of dry weight to wet weight, all parameters are expressed per mg wet weight of tissue. Mean +_ Standard Error.

Ornithine Transcarbamy lase (OTC) Activity Numerous enzymatic changes are known to occur in the liver of tadpoles during metamorphosis and are thus of interest for in vitro culture studies. In particular, the metamorphic change from annonotelism to ureotelism involves the introduction of the urea cycle enzymes (2) (23). Since this change in protein biosynthesis was of interest to us for future studies, the activity of one of these enzymes, ornithine transcarbamylase (OTC), was followed in time course experiments over 96 hours. Long term experiments involved assays at more extended time intervals for ' a total period of six days.

Ornithine Transcarbamylase Activity in vitro J7.6)

0

17

48

72

96

Time in Culture (hrs.) Figure 2. Ornithine trans carbanr/lase a c t i v i t y in cultured l i v e r t i s s u e . At the time indicated, t i s s u e fragments were harvested and assayed a s described in METHODS. Figure 2 summarizes the fluctuations in OTC a c t i v i t y during the early periods in culture. There i s a marked decrease in a c t i v i t y during the f i r s t 17 hours with subsequent a c t i v i t i e s remaining s t a b l e . This s t a b i l i z e d condition i s shown in Table 3 for assays, of cultured t i s s u e over a period of 6 days. Table 3 ORNITHINE TRMSCAKBAMYLASE (OTC) ACTIVITY IN LONG TERM CULTURES Hours in Culture 0 36 72

108

144

OTC Activity* (pmoles Citrulline/30 min mg protein) 7.3 5.0 5.4 5.9 5.6

+ 0.8 + 0.6 + 0.5 +0.7 + 0.5

*Mean +_ Standard Error. Incorporation of Amino Acids, Uridine and' Thymidine In order to obtain additional information about the condition of organ cultured l i v e r , experiments were designed to measure the overall a b i l i t y of c e l l s to transport and incorporate radioactive amino acids and nucleotides into macromolecules. The incorporation of C 11+ -lysine and C 1Lf -valine was measured by incubating pieces of t i s s u e , a f t e r they had been cultured f o r either 0, 20, 44 or 68 hours, with the two radioactive amino

70 acids under the conditions outlined in METHODS. After incubating the tissue with radioactive amino acids, etc. for the time indicated for a representative experiment shewn in Figure 3, the tissue was removed and TCA precipitable radioactivity was determined according to METHODS. As shown in this figure, the average rate over three hours and level of incorporation of radioactive lysine and valine into TGA-insoluble material after three hours decreases slightly during the first 20 hours in culture and then remains at a relatively constant level during the culture period.

i I"

I Î

£

ft.

0

1 2

Hours

Hours

Figure 3. Incorporation of radioactive lysine and valine into TCA insoluble material. In (A) through (D) tissue was harvested after the number of hours in culture (0, 20, 44, 68) that are indicated. Tissue was incubated as described in METHODS FOR 0, 1, 2 and 3 hours before the reaction was terminated with TCA. The CFM per mg tissue were determined as described in'METHODS. Each tube contained 0.5 ycuries each of -lysine and -valine. 3 The average rates of incorporation of H -uridine at varying times in culture for a representative experiment are shown in Figure 4. The rate of incorporation appears to decrease during the first 20 hours; the incorporation rate for tissue cultured 68 hours is decreased to about 75 per cent of the initial value. In Uie case of H3-thymidine incorporation, Figure 5, there was very little total incorporation by freshly excised tissue. A low average rate of incorporation was observed during the first 20 hours in culture. However, by 44 hours (Figure 5C) the level of incorporation increased and the rate of incorporation increased to a sli^vtly higher level through 68 hours in culture.

71

Figure 4. Incorporation of radioactive uridine into TCA insoluble material. Methods as in Figure 3, and in METHODS. Each tube contained 10 ycuries of H^-uridine.

£ r I Hours

Hours

Figure 5. Incorporation of radioactive thymidine into TCA insoluble material. Methods as in Figure 3 and in METHODS. Each tube contained 10 ycurles of H3-thymidine'.

72 Table 4 INCORPORATION OF AMINO ACIDS, URIDINE AND THYMIDINE IN LONG TERM CULTURES Hours in Culture

0 36 72 108 144

-Lysine and C -valine (CFM/Hr/mg Tissue) 1

450 435 510 409 500

+ + + + +

80 40 75 60 75

H3-Uridine

H3-Thymidine

(CFM/Hr/mg Tissue)

(CPM/Hr/mg Tissue)

370 300 185 135 185

+ + + + +

45 50 15 10 30

155 208 310 358 369

+ + + + +

10 35 10 45 70

Average rates were calculated as in Figures 3 through 5. Mean + Standard Error. As shown in Table 4, the long term cultures display a stable rate of incorporation of radioactive lysine and valine. The rate of uridine incorporation decreases with time and appears to stabilize by 72 hours in culture at a level of about 70% that of the initial value. Thymidine incorporation appears to be stabilized at an increased rate after about 72 hours in culture.

DISCUSSION The conditions have been elaborated for the in vitro culture of liver fragments from premetamorphic tadpoles• Our culture methods (10,15) make use of a standard aell culture medium for anphibia (17) which has been modified to contain less glucose (10). They enable us to maintain approximately fifty to seventy-five experimental tubes, each containing two pieces of tissue from a single tadpole for experimental periods of six days. According to morphological criteria reported elsewhere (10), the parenchymi cells approximate the cytological conditions of cells immediately after excision. In addition, several biochemical criteria have now been used to establish the extent to which the molecular and metabolic condition of the cultured tissue parallels the in vivo situation. These criteria include measurements of biochemical constituents of the tissue and gross metabolic studies using radioisotopes. The protein oontent of tadpole tissue is stable over short and long term culture periods. The values obtained are in the range of those reported by Wixom et al. (23) and Atkinson et al. (24)

73 for animals of a similar developmental stage. The stability of the protein content provides us with an additional parameter on which to base future in vitro measurements. The fact that amino acid incorporation shews only a slight decrease during the early period in culture and then stabilizes during later periods in culture is consistent with the protein content data. The incorporation increase may reflect a renewal of protein synthesis after a slightly depressed period during the "trauma" associated with being excised and put into culture. As cells become established in culture, growth nay occur, as reflected in increased incorporation of amino acids, but may not be measured by grosser methods, e.g. protein determination and weij^rt measurements, which may mathematically mask such growth effects. Ornithine transcarbanylase activity is a biochemical indicator for the onset of metamorphosis in R. catesbeiana (2,23). Although it is true that changes in protein content would affect "the specific activities reported here, our data on protein content, wet and dry weights indicate that this does not happen. The demonstration of stable activity levels of this enzyme in organ cultures of R. catesbeiana liver, after an initial decrease in activity, therefore satisfies an important criterion in the development of a culture system for the in vitro study of thyroxine action. The decrease in OTC activity during early periods in culture is subject to many interpretations. Unfortunately, there are no reports of the turnover rate of OTC in the literature and one can only speculate that the decrease in activity we observed may reflect a slow-down in biosynthesis of this enzyme during the "trauma" period, often mentioned in connection with cell culture studies. The patterns for RNA and DNA content, which show a decrease in the former after about 20 hours and a gradual decrease in the latter, are more complex than those discussed above for other biochemical features. Alone, these data would suggest that some cell death is occurring, as has been discussed in detail by Majno (12). However, the fact that a marked increase in thymidine incorporation occurs from 44 to 68 hours suggests that a more complex situation exists. One possibility is that cell division is occurring, perhaps, in a small population of parenchymal or, even, in other included (e.g. leucocytes, reticulocytes, etc.) cells. The nature of the data, however; i.e. DNA content being a gross measurement and thymidine incorporation being a sensitive measurement subject to the limitations discussed below, make it virtually impossible to estimate the proportion of cells dying to those proliferating. Further, increased incorporation of labeled thymidine does not necessarily imply an increase in DNA synthesis since incorporation depends on the intracellular concentration of thymidine as well as the actual rate of thymidine incorporation. An alternative interpretation would be that the

74 endogenous pool of thymidine d i l u t e d t h e i s o t o p e during t h e e a r l y p e r i o d s i n c u l t u r e and, on b e i n g d e p l e t e d , p e r m i t t e d high r a t e s of thymidine i n c o r p o r a t i o n . F u r t h e r , thymidine i n c o r p o r a t i o n could r e f l e c t a s u s t a i n e d p e r i o d of mitochondrial DNA s y n t h e s i s . Although q u a l i t a t i v e morphological s t u d i e s on t a d p o l e l i v e r d u r i n g metamorphosis ( 2 5 , 5 , 6 ) i n d i c a t e t h a t no l i v e r o e l l d i v i s i o n o c c u r s , r e c e n t biochemical work i n F r i e d e n ' s l a b o r a t o r y (24) suggests t h a t enhanced l i v e r n u c l e a r DNA b i o s y n t h e s i s does o c c u r . T h i s , however, i s p a r t i a l l y i n c o n f l i c t with t h e r e p o r t of Cohen's group (23). The decrease i n t h e r a t e of u r i d i n e i n c o r p o r a t i o n during e a r l y p e r i o d s i n c u l t u r e i s c o n s i s t e n t with o t h e r biochemical and morphological d a t a (15) r e l a t i n g t o t i s s u e trauma when adapting t o c u l t u r e c o n d i t i o n s . The s t a b i l i z a t i o n of t h e r a t e of u r i d i n e i n c o r p o r a t i o n a f t e r a p e r i o d i n c u l t u r e s u g g e s t s : 1) t h a t a r e d u c e d , b u t f u n c t i o n a l , l e v e l of RNA s y n t h e s i s has been achieved o r 2) a l t e r a t i o n s i n u r i d i n e p o o l - s i z e , e t c . , as mentioned above f o r thymidine. I n t h e p r e s e n t work, emphasis has been p l a c e d upon determining t h e gross metabolic f e a t u r e s and biochemical c o n s t i t u e n t s of pre-metamorphic t a d p o l e l i v e r i n c u l t u r e . The assessment of t h e s e s t a t e s f o r t i s s u e a l s o r e s t s on morphological c r i t e r i a (10). The p a t t e r n s f o r biochemical c o n s t i t u e n t s during p e r i o d s i n c u l t u r e as w e l l as t h e p a t t e r n s f o r i n c o r p o r a t i o n of r a d i o a c t i v e amino a c i d s , u r i d i n e , and thymidine s t r e n g t h e n our morphological conclusions about "the t i s s u e . I n a d d i t i o n , i n some i n s t a n c e s they provide a foundation f o r f u r t h e r biochemical s t u d i e s and, i n o t h e r i n s t a n c e s , a s t a r t i n g p o i n t . The s t u d i e s of Wixom e t a l . (23) and Atkinson e t a l . (24) provide b a s i c i n f o r m a t i o n about "the i n vivo f e a t u r e s of s e v e r a l of t h e s e parameters during metaniorphosis. Although "there i s undoubtably some c e l l death o c c u r r i n g i n our system, p e r h a p s , as r e f l e c t e d i n "the decreases i n some biochemical a c t i v i t i e s , t h e s i t u a t i o n i s d r a s t i c a l l y d i f f e r e n t from t h a t which one sees i n a (tying c e l l p o p u l a t i o n as reviewed by Majno (12). There a r e of course innumerable b i o chemical c r i t e r i a ; e . g . , determination of l i v e r s p e c i f i c a n t i g e n s , r e s p i r a t o r y c h a r a c t e r i s t i c s , e t c . , t h a t need t o be e s t a b l i s h e d , as w e l l as such c y t o l o g i c a l f e a t u r e s as m i t o t i c i n c i d e n c e . These c r i t e r i a a r e needed t o s t r e n g t h e n o u r confidence i n t h e t a d p o l e l i v e r c u l t u r e system a s a model f o r i n v e s t i g a t i n g t h e mechanism of thyroxine-induced d i f f e r e n t i a t i o n of t a d p o l e l i v e r . SUMAKY Primary e x p l a n t s from Rana c a t e s b e i a n a t a d p o l e l i v e r have been maintained i n organ c u l t u r e f o r s i x days. S e v e r a l biochemical parameters of the c u l t u r e d l i v e r c e l l s have been s t u d i e d during t h e time i n c u l t u r e i n o r d e r t o e s t a b l i s h : 1) changes i n wet w e i g h t , dry w e i g h t , as w e l l as p r o t e i n , RNA and ENA c o n t e n t s ; 2) r a t e s of i n c o r p o r a t i o n of amino a c i d s , u r i d i n e and thymidine; 3)

75 changes in ornithine transcarbanylase activity. The effects of changes in the culture conditions on these biochemical characteristics are presented. It is a pleasure to oontribute this article on occasion of the Lipmann Fest since these studies were begun in the Lipmann Laboratory following ny Ph.D. studies there. I thank the Gesellschaft fur Biologische Chemie for their hospitality in Berlin. I acknowledge my students Henry Kriegstein and Shulamith Shafer, along with ny assistants Susan Fisher and Janice Glenn, who have contributed at various tines to these studies.

REFERENCES 1. Bennett, T. P., and Frieden, E.: Metamorphosis and biochemical adaptations in amphibia. In "Comparative Biochemistry" (M. Florkin and H. S. Mason, eds.), Vol. 4, pp. 483-556. Academic Press, New York (1962). 2. Cohen, P. P.: Biochemical aspects of metamorphosis: transition from ammonotelism to ureotelism. Harvey Lect. Ser. 60, 119-154 (1966). 3. Frieden, E.: Biochemistry of amphibian metamorphosis. In "Metamorphosis" (W. Etkin and R. I. Gilbert, eds.), pp. 349-398. Appleton-Century-Crofts, New York (1968). 4. Frieden, E., and Just, J.: Hormonal responses in amphibian metamorphosis. In "Mechanisms of Hormone Action" (G. Litwack, ed.). pp. 1-52. Academic Press, New York. (1970). 5. Bennett, T. P., Glenn, J. S., and Sheldon, H.: Changes in the fine structure of tadpole (Rana catesbeiana) liver during thyroxine-induced metamorphosis. Develop. Biol. 22_, 232-248 (1970). 6. Bennett, T. P., and Glenn, J. S.: Fine structural changes in liver oells of Rana catesbeiana during natural metamorphosis. Develop. Biol. 22_, 535-560 (19 70). 7. Tata, S.R.: The formation and distribution of ribosomes during hormone-induced growth and development. Biochem. J. 104, 1-15 (1967). 8. Cohen, P. P.: Biochemical differentiation during amphibian metamorphosis. Science, 168_, 533-543 (1970). 9. Spiegle, E. S. and Spiegle, M.: Some observations on the ultrastructure of "the hepatocyte in the metamorphosing tadpole. Exp. Cell Res., 61, 103-112 (1970). 10. Bennett, T. P. and Kriegstein, H.: The morphology of tadpole (Rana catesbeiana) liver under varying conditions of organ culture. Anat. Rec. 176, 461-474 (1973). 11. Bennett, T. P.: Morphological and biochemical studies on tadpole liver in organ culture. Abstract 44. The American Society for Cell Biology. 11th Annual Meeting (1971).

76 12. Majno, G.: Death of liver tissue. In The Liver, Morphology, Biochemistry, and Physiology, (Ch. Roullier, ed.). Vol. II, pp. 267-313. Academic Press, New York (1964). 13. Fawcett, D. W.: "An Atlas of Fine Structure. The Cell, its Organelles and Inclusions." pp. 2-28. W. B. Saunders Company, Philadelphia, Pennsylvania (1966). 14. Porter, K. R. and Bonneville, M.: "Fine Structure of Cells and Tissues", 3rd Edition, pp. 20-25. Lea and Febiger, Philadelphia, Pennsylvania (1970). 15. Kriegstein, H. and Bennett, T. P.: Chromatin condensation and nucleolar segregation induced in nuclei of parenchymal cells of Rana catesbeiana tadpoles. Exptl. Cell Res., 80, 152-158 (1973). 16. Taylor, A. C. and Kbllros, J. J.: Stages in the normal development of Rana pipiens larvae. Anat. Ree., 94, 7-23 (1946). 17. Wolf, K., and Quimby, M. C.: Amphibian cell culture: permanent cell line from the bullfrog (Rana catesbeiana). Science 144, 1578-1580 (1964). 18. Schneider, W. C.: Determination of nucleic acids in tissues by pentose analysis. In "Methods in Enzymology", (Colowick, S. P. and Kaplan, N. 0., eds.), 3, pp. 680684. Academic Press, New York (1957). 19. Bennett, T. P.: Membrane filtration for determining protein in the presence of interfering substanoes. Nature 213, 1131-1132 (1967). 20. Jacobson, A. G.: Amphibian cell culture, organ culture, and tissue dissociation. In "Methods in Developmental Biology", pp. 531-542. Thomas Y. Crowell Conpany, New York (1967). 21. Palmiter, R. D.: Early macromolecular synthesis in cultured mammary tissue from mid-pregnant mice. Endocrinology 85, 747-751 (1969). 22. Brcwn, G. W. and Cohen, P. P. : Comparative Biochemistry of Urea Synthesis. J. Biol. Chem., 2jS4, 1769-1774 (1959). 23. WixDm, R. L., Reddy, M. K., Cohen, P. P.: A concerted response of the enzymes of urea biosynthesis during thyroxine-induced metamorphosis of Rana catesbeiana. J. Biol. Chem. 247, 3681-3692 (1972). 24. Atkinson, B. G., Atkinson, K. H. , Just, J. J., and Frieden, E.: DNA synthesis in Rana catesbeiana tadpole liver during spontaneous and triod thyronine-induced metamorphosis . Develop. Biol. 2£, 162-175 (1972). 25. Kaywin, L.: A cytological study of the digestive system of anuran larvae during accelerated metamorphosis . Anat, Ree. 64, 413-441 (19 36).

The Hexokinase-Mitochondrial Binding Theory of Insulin Action Samuel P. Bessman, M.D. University of Southern California School of Medicine Department of Pharmacology, Los Angeles, California 90033 I should like to discuss a proposal for the mechanism of action of insulin which I first presented at a session chaired by Dr. Fritz Lipmann in lOSl"'" and to review it over the more than 20 years of philosophic and experimental evolution through which it has gone. The proposition simply is that insulin causes the binding of hexokinase to the mitochondrial membrane in such a way that the acceptor relation between energy generation and glucose utilization is made more efficient. An enhancement of efficiency of oxidative phosphorylation by an insulin mediated molecular rearrangement in the cell would result in increased delivery of energy at mitochondrial sites associated with the anabolic functions of the cell. These functions include increased permeability of membranes, increased synthesis of large molecules such as protein, fat and glycogen, increased turnover of RNA, detoxication of drugs, and synthesis of small molecules such as glutathione.

It is of interest to note that

these physiologic results of an enhanced efficiency of mitochondrial function would seem to be those caused by insulin.

In ad-

dition, a number of physiologic phenomena which are at present inexplicable may be clarified by the felicitous interaction between hexokinase and the mitochondrial membrane. For example, the tissue most sensitive to insulin action is the fat cell and this cell has its mitochondria very closely approximated to the cell membrane, pressed there by the hydrophobic fat globules contained within the cell. This would render the mitochondria more accessible to direct contact with the exogenously administered insulin.

78 A second phenomenon which is physiologically inexplicable at the present time on any other basis is the insulin-like result of muscular activity. Although exercise hormones have been proposed to explain the increased anabolic activity of muscle action during exercise, even in the diabetic, no such hormone has ever been demonstrated.

On the other hand, the requirement for an acceptor

explains both this phenomenon and even the more interesting one of the binding of ATP and ADP to the enzymes of muscle contraction. At rest, the creatine of muscle is almost entirely in the form of phosphocreatine. When the muscle becomes active, the phosphocreatine breaks down to liberate free creatine with no 2

change in needed ATP until the exhaustion of the creatine supply . Nevertheless, the oxidation in muscle increases with activity. The question then arises, "How is the muscle mitochondrion 'informed' of the need for increased oxidation when exercise has taken place?". If the ATP of the muscle does not get converted to significant amounts of ADP, which then diffuse to the mitochondrion for the classical respiratory control phenomenon, what is the actual information transfer between the contracted fiber and the mitochondrion? The evidence to interpret this question was supplied first by Klingeriberg who showed that the mitochondria of heart, skeletal muscle, and brain had a bound creatine kinase.

3

The second information on the subject was the work of Fonyo , in our laboratory, who showed that creatine itself can produce the same phenomenon of respiratory control as ADP in heart sarcosomes. It now becomes clear that the signal from the contracting muscle fiber is free creatine diffusing to the mitochondrion causing the regeneration of the creatine phosphate at the mitochondrial site which restores the creatine phosphate content of the cytosol. This replenishes ATP by transphosphorylation of myosin in situ. The ADP formed by muscle contraction does not have to be displaced by ATP.

It is interesting that this ex-

79 planation fits the known fact that the binding of ADP to the contracting muscle fiber is stronger than the binding of ATP. The intermediary role of phosphocreatine in replenishing in situ the ATP which has broken down in contraction and in transporting new phosphate energy from the mitochondrion is thus an example of a parsimonious mechanism for producing efficiency in the presence of a limited amount of nucleotide. Otherwise, the muscle fiber would be dependent upon the diffusion process for moving the limited ATP from the mitochondrion and the ADP to the mitochondrion. This permits the rapid regeneration of muscle contraction and the substitution of creatine phosphate as an energy carrier molecule with minimal pathways of degradation for ADP and ATP for which the cytosol contains many active sites which use these molecules. Creatine is an excellent "messenger" mol'ecule for it undergoes no reactions at all in the cytosol except transphosphorylase. A third physiologic phenomenon which is explained by the molecular reorganization role of insulin in connecting hexokinase to mitochondria is the well-known fact that the brain is insensitive to insulin although it uses more glucose than any other tissue. The hexokinase of brain is for the most part attached to the mitochondria as shown by Crane and Sols^ a long time ago. If the role of insulin is to attach hexokinase to mitochondria, then the lack of sensitivity of the brain to insulin is clearly the result of the fact that the role of insulin in connecting hexokinase is no longer necessary in brain. It is interesting to note that the brain is a relatively primitive tissue and that mitochondria also have hexokinase attached to them in malignant tissues such as Hela cells. A number of experiments of different types have been done over the past 20 years to validate certain aspects of this theory.

80 In the first place we would have to demonstrate that insulin does not achieve its major anabolic effects by improving the permeag

bility of cell surfaces. This was done by Dr. Toyoda , in our laboratory, who measured the movement of radioactive leucine into the cell water and into the protein of surviving diaphragm sections as it was affected by insulin. Figure 1 shows the data obtained from this experiment and clearly demonstrates that under the influence of insulin there is less free leucine in a tissue space under the action of insulin. This difference in cellular free leucine from the control is accounted for by the extra incorporation of leucine into protein. This demonstration of the intracellular effect of insulin 7 on protein synthesis was corroborated by Dr?. Paul De Schepper who showed that diaphragm preloaded with radioactive leucine at 4°C, at which temperature no protein synthesis occurs, would then incorporate this intracellular radioactive leucine into protein more efficiently with insulin than the control. This was true even though there were Effect of Insulin on L-Leucine l - C ' 4 Incorporation and Uptoke in Function of Tim*.

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Time ( d a y s ) Fig. 1. Effect of actinomycin D on a primary infection. Six pupae of S. cynthia injected with living E. coli, strain D31. Samples of 5 yl of hemolymph were removed at different times and the number of viable cells of D31 was determined. The dotted line indicates the lowest detectible level of bacteria. of pupae were challenged with an injection of living P. aeruginosa . The results in Fig. 2 show that in the vaccinated pupae Pseudomonas was rapidly eliminated from the hemolymph while 9 in the control they grew up to a density of more than 10 viable cells/ml. Thus, a primary infection with Ent. cloacae protected the pupae from a secondary infection with P. aeruginosa. It should also be noted that the defense system induced eliminated P. aeruginosa faster than the living vaccine, an observation in agreement with our earlier findings in Drosophila (6). In vitro experiments with hemolymph from immunized pupae The fact that we use living bacteria as "substrate" has hindered the characterization of what seems to be at least in part an enzyme reaction. However, some of the properties so far known (7) are summarized in Table I. Especially the inhibition by lipopolysaccharide (LPS) has turned out to be a handy tool

107

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Incubation lime(min) Fig. 3. Effect of LPS (50 yg/ml) on the killing of a gram positive and gram negative bacteria. With E.coli the reaction mixture contained 1%, with B. subtilis 5% hemolymph from a pupa vaccinated with strain D31. Reaction mixtures with LPS, filled symbols; reaction mixtures without LPS, unfilled symbols. That insect blood can lyze bacteria has been observed

several

times and the responsible lysozyme was recently purified and characterized

(9). We have therefore compared also lysis of

Micrococcus lysodeikticus and E.coli, strain D31 and again found that LPS from D31 interferes only with the defense against the latter. Taken together the results in Fig.3 and 4 suggest that there are separate mechanisms for the defense against Gram positive and Gram negative bacteria. Preliminary experiments indicate that both defense systems are inducible but only the defense against Gram negative bacteria was inhibited by actinomycin D. We have in Umea previously studied the mutational steps by which ampicillin resistance is developed in E.coli. In several cases we have found mutants with alterations in the outer membrane and one group of strains have turned out to carry muta-

109

Fig. 4. Effect of LPS (50 yig/ml) on the lysis of a gram positive and gram negative bacteria. The reaction mixtures contained 1.6% of hemolymph from a pupa vaccinated with strain D31. Reaction mixtures with LPS, filled symbols; reaction mixtures without LPS, unfilled symbols. tions affecting the biosynthesis of LPS (10). It was therefore natural for us to try such a set of mutants. As illustrated in Fig. 5 we found that the immunity system in S. cynthia could distinguish our different LPS mutants. The more carbohydrates that were lost from the LPS molecule, the more sensitive did the bacteria become to the killing action of the hemolymph. Elsewhere we have shown that the lipopolysaccharide becomes a more potent inhibitor when increasing carbohydrate parts are lost from the molecule (7). As an extension of this result we tried lipid A liberated from LPS by mild acid hydrolysis. Despite the fact that lipid A is very poorly soluble in water there was a pronounced inhibition of the initial rate of killing (Fig. 6). Taken together the inhibition by LPS or lipid A and the increased susceptibility of LPS mutants indicate that at least one component in the hemolymph forms a strong complex with LPS. We are currently trying to isolate such a complex

110

0

2

i

6

Incubation lime

8

10

(min)

Fig. 5. Susceptibility of different LPS mutants of E.coli. The reaction mixtures contained 1 % of hemolymph from a pupa vaccinated with strain D31. Left part a schematic structure showing the LPS composition of the strain used (from work by Boman and Monner).

using different isotopes and fractionation procedures. Discussion In a scientific context the claim that something is interesting usually means that convincing arguments exist for the scientific and practical importance of a problem. We have here presented this reasoning in our introduction. In addition, the word interesting can also cover personal motivation as well as the excitement involved in the work. For some scientists a problem is often most stimulating

111

u