The interplay between covalent and non-covalent regulation of glycogen phosphorylase. The role of different effectors of phosphorylase b on the phosphorylase b to a conversion rate

BIOCHIMIE, 1979, 61, 633-643.

The interplay between covalent and non-covalent regulation of glycogen phosphorylase. The role of different effectors of phosphorylase b on the phosphorylase b to a conversion rate. M. MORANGE and H. BUC.

Ddpartement de Biologie Mol~culaire, Institut Pasteur, 7572I~ Paris Cedex 15 (France).

R6sum6.

d u s u b s t r a t prot6ique, l a g l y c o q ~ n e p h o s p h o r y l a s e b. Les effets diff6rentiels observ6s, et la cin6tique d ' 6 t a b l i s s e m e n t de l'effet inhibiteur de I'AMP sont en b o n a c c o r d a v e c la s e c o n d e interpr6tation : la k i n a s e s e m b l e r e c o n n a l t r e a v e c u n e efficacit6 diff6rente les d i v e r s e s conformations de la q l y c o g 6 n e p h o s p h o r y l a s e b.

La q l y c o q 6 n e p h o s p h o r y l a s e du m u s c l e de l a p i n p e u t 6tre activ6e de m a n i 6 r e non-covalente p a r I'AMP ou de m a n i ~ r e c o v a l e n t e sous l'action de la p h o s p h o r y l a s e kinase. Pour r e c h e r c h e r si la vitesse de c o n v e r s i o n de la q l y c o q ~ n e p h o s p h o r y l a s e b e n p h o s p h o . r y l a s e a est affect6e p a r l a c o n f o r m a t i o n de l ' e n z y m e non p h o s p h o r y l 6 e , n o u s a v o n s 6tudi6, h d e u x t e m p 6 r a t u r e s , l'action des effecteurs de l ' e n z y m e d 6 p h o s p h o r y l 6 e sur s a vitesse d e conversion (activateurs forts c o m m e I'AMP, activateurs faibles c o m m e I'IMP, substrats, et qlucose-6-phosphate, inhibiteur du syst6me). L'effet simultan6 des a c t i v a t e u r s et d e s s u b s t r a t s a 6t6 a u s s i 6tudi& Nous a v o n s o b s e r v 6 des effets tr6s m a r q u 6 ~ de ces diff6rents effecteurs & des concentrations physioloqiques. L'effet inhibiteur du qlucose-6p h o s p h a t e et le rSle a c t i v a t e u r d u q l y c o q ~ n e ont 6t6 confirm6s. Nous a v o n s montr6, de plus, q u e les a c t i v a t e u r s forts et faibles ont d e s effets inhibiteurs tr6s diff6rents, tandis q u e l'orthop h o s p h a t e , seul, ou ajout6 a u x a c t i v a t e u r s , a un effet inhibiteur tr~s prononc6. Darts c h a c u n de ces cas, l'effet 6tre dfi, soit h u n e action directe sur la p h o s p h o r y l a s e kinase, soit indirecte, p a r modification d e l a

observ6 peut du c o m p o s 6 b une action conformation

A b b r e v i a t i o n s used : Glc-I-P Glc-6-P

: Ghzcose-l-phosphate. : Glucose-6-phosphate.

Nucleotides are a b b r e v i a t e d according to reference (5). ~AMP : 1, Na-etheno-AMP. P E I cellulose : Polgeth?lleneimine celhzlose. Phosphorglase b : E.C. 2.~.1.1. P h o s p h o r y l a s e k i n a s e : E.C.2.7.1.38.

In vivo, la p h o s p h o r y l a s e est ins6r6e darts la particule de qlycoq~ne. Les diff6rents effecteurs qui, en se fixant sur l a q l y c o q ~ n e phosphor y l a s e b, modifient son t a u x de c o n v e r s i o n en p h o s p h o r y l a s e a, affectent a u s s i d ' a u t r e s enz y m e s contrSlant le m 6 t a b o l i s m e du qlycoq6ne.

En r a s s e m b l a n t ces o b s e r v a t i o n s , nous suqq6rons qu'il existe bien u n e 6troite coop6ration entre les r6gulations c o v a l e n t e s et non-covalentes du m6{abolisme du qlycoq~ne.

Summary. G l y c o q e n p h o s p h o r y l a s e b is c o n v e r t e d to q l y c o q e n p h o s p h o r y l a s e a, the c o v a l e n t l y activ a t e d form of the e n z y m e , b y p h o s p h o r y l a s e kinase. Glc-6-P, w h i c h is a n allosteric inhibitor of p h o s p h o r y l a s e b, a n d qlycoqen, w h i c h is a s u b s t r a t e of this e n z y m e , a r e a l r e a d y k n o w n to h a v e r e s p e c t i v e l y a n inhibitinq a n d activatinq effect u p o n the r a t e of c o n v e r s i o n from phosp h o r y l a s e b to p h o s p h o r y l a s e a b y phosphor y l a s e kinase. In the former case, this effect is d u e to the bindinq of qlucose-E-phosphate to qlycocjen p h o s p h o r y l a s e b. In order to investiqate w h e t h e r or not the rate of c o n v e r s i o n of q l y c o q e n p h o s p h o r y l a s e b to p h o s p h o r y l a s e a d e p e n d s on the conformatioh a l state of the b substrate, w e h a v e tested the action of the m o s t specific eftectors of q l y c o q e n

634

M. Morange and H. Buc.

phosphorylase b activity upon the rate of conversion from phosphorylase b to phosphorylase a at 0°C and 22°C : AMP and other strong activators, IMP and weak activators, Glc-6-P, glycogen, Glc-I-P and phosphate. AMP and strong activators have a very important inhibitory effect at low temperature, but not at room temperature, whereas the weak activators have a l w a y s a very weak, if even existing, inhibitory effect at both temperatures. W e confirmed the very strong inhibiting effect of Glc-6-P at both temperatures, and the strong activating effect of glycogen. We have shown that phosphate has a very strong inhibitory effect, whereas Glc-I-P has an activating effect only at room temperature and at non-physiological concentrations. The concomitant effects of substrates and nucleotides have also been studied. The observed effects of all these liqands m a y be either direct ones on phosphorylase kinase, or indirect ones, the ligand modifying the conformation of phosphorylase b and its interaction with phosphorylase kinase. Since we

Introduction. Glycogen p h o s p h o r y l a s e b can be converted to p h o s p h o r y l a s e a, a covalently activated form of the e n z y m e by p h o s p h o r y l a s e k i n a s e [1]. Some authors have a l r e a d y studied the effects of differ e n t ligands of glycogen p h o s p h o r y l a s e b on the rate of c o n v e r s i o n from p h o s p h o r y l a s e b to phosphorylase a by p h o s p h o r y l a s e kinase. At room temperature, Glc-6-P has a strong sloveering effect on this conversion, w h e r e a s glycogen accelerates it [2-4]. Glc-6-P does not directly affect phosphorylase kinase, since its effect is not observed w h e n p h o s p h o r y l a s e b is replaced by the tetradecapeptide s u r r o u n d i n g the s e r i n e residue ~vhich is p h o s p h o r y l a t e d : Glc-6-P p r o b a b l y modifies p h o s p h o r y l a s e b c o n f o r m a t i o n , w h i c h is t h e n recognized differently by p h o s p h o r y l a s e kinase. I n a p r e c e d i n g p a p e r [5], vee have studied the effects of different n u c l e o t i d e s on glycogen phosphorylase b from r a b b i t skeletal muscle. We have s h o w n that all the nucleotides studied so far can be divided into two classes, d e p e n d i n g on t h e i r effects on the kinetics and p h y s i c o c h e m i c a l properties of the enzyme. The first class, c o m p r i s i n g AMP, m6AMP, clGAMP, cTAMP, eAMP, has a strong activating effect on p h o s p h o r y l a s e b, the second

BIOCHIMIE, 1979, 61, n ° 5-6.

h a v e no control experiments with a peptidic fraqrnent of phosphorylase b, the interpretation of our results rematns putative. However, the differential effects observed with different nvcleotides are in agreement with the simple conformational scheme proposed earlier. Therefore, it is suggested that phosphorylase kinase recognizes differently the different conformations of glycogen phosphorylase b. In agreement with such an explanation, it is shown that the inhibiting effect of AMP is mediated b y a slow isomerisation which has been previously ascribed to a quaternary conformational change of glycogen phosphorylase b. The results presented here (in particular, the important effect of glycogen and phosphate) are also discussed in correlation with the physiological role of the different ligands as regulatory siq~nals in the in v i v o situation where phosphorylase is inserted into the glycogen particle. Key words : Covalent and non-covalent regulations. Phosphorylase b kinase.

class formed by IMP, GMP, dAMP, gives only a weak activation to p h o s p h o r y l a s e b. These differential effects of the nucleotides have been i n t e r p r e t e d by the folloveing scheme, w h i c h is also consistent w i t h the k n o w n rates of isomer i s a t i o n of the enzyme at different t e m p e r a t u r e s : at love t e m p e r a t u r e , the d i m e r i c enzyme exists in three different c o n f o r m a t i o n s : E2 ~ E 1 ~ E3 E 2 and E 1 are in fast e q u i l i b r i u m , E 2 being stabilized by glucose-6-phosphate a n d E 1 by the veeak activators. E 3 is stabilized by the strong activators and the t r a n s i t i o n time from E~ to E a called T4 is of the order of several m i n u t e s [6]. A fourth state Q is formed by the association of two molecules of d i m e r i c enzyme in the E~ conformation. At room t e m p e r a t u r e , two m a i n differences exist w i t h the scheme p r o p o s e d at 4°C : the state Q is generally absent and the state E 3 is very poorly populated even in the presence of strong activators like AMP. It can be populated only in the presence of both orthophosphate and AMP. The t r a n s i t i o n from E 1 to E a is much more rapid t h a n at 4°C [5].

Glycogen phosphorylase-conversion of phosphorylase b to phosphorylase a. S i n c e it h a s a l r e a d y b e e n s h o w n t h a t p h o s p h o r y l a s e k i n a s e is v e r y s e n s i t i v e to s l i g h t c o n f o r m a _ t i o n a l c h a n g e s of its p h o s p h o r y l a s e b s u b s t r a t e , w e h a v e t e s t e d i n t h e p r e s e n t p a p e r t h e e f f e c t s of the different nucleotides associated or not with the substrates on the conversion rate from phosp h o r y l a s e b t o p h o s p h o r y l a s e a at 0 ° C ( i n i c e ) a n d 22°C with the hope that the two different conform a t i o n s of p h o s p h o r y l a s e b t r i g g e r e d b y t h e n u cleotides would be discriminated by phosphorylase k i n a s e . W e h a v e also t e s t e d t h e e f f e c t s of Glc-6-P a n d of t h e d i f f e r e n t s u b s t r a t e s a n d p r o d u c t s of p h o s p h o r y l a s e b o n t h i s c o n v e r s i o n r a t e i n o r d e r t o ,gain s o m e i n s i g h t o n t h e i n t e r a c t i o n between the covalent and non-covalent controls w i t h i n t h e g l y c o g e n p a r t i c l e [7].

Materials

and

Methods.

P h o s p h o r y l a s e b was p r e p a r e d according to Fischer a n d Krebs [8], except t h a t 2 - m e r c a p t o e t h a n o l was used instead of eystein d u r i n g t h e p r e p a r a t i o n . Removal of AMP f r o m the e n z y m e and m e a s u r e m e n t s of the enzyme c o n c e n t r a t i o n s arc p e r f o r m e d as described earlier [9]. The m o l e c u l a r weight of glycogen phosp h o r y l a s e b m o n o m e r is t a k e n equal to 94.000 daltons [10]. P h o s p h o r y l a s e k i n a s e is purified according to Cohen

et al. [11]. P h o s p h o r y l a s e b to a conversion is done in the following m e d i u m : one v o l u m e of the p h o s p h o r y l a s e b dissolved in glycylglycine buffer 50 mM, KC1 50 mM, pH 6.9 a t room t e m p e r a t u r e is mixed w i t h one volume of a d e n o s i n e t r i p h o s p h a t e 18 mM, m a g n e s i u m acetate 60 mM a t pH 7.00, a n d two volumes of Tris-glycerophosphate buffer 0.125 M pH 8.6. The reaction is started b y a d d i t i o n of a v a r i a b l e q u a n t i t y of p h o s p h o r y l a s e kinase. No extra calcium ions are added to this conversion m e d i u m . P h o s p h o r y l a s e b final c o n c e n t r a t i o n is a l w a y s equal to a b o u t 10 ~tM. F i n a l pH is 8.2. The conversion is followed b y m e a s u r i n g the increm e n t of activity of glycogen p h o s p h o r y l a s e assayed in the absence of AMP, w i t h respect to time. The conversion is stopped b y d i l u t i o n in glycylglycine buffer 50 mM. P h o s p h o r y l a s e a activity is m e a s u r e d according to Helmreich and Cori [12] at 28°C in 10 mM phosp h a t e a n d 2.5 per mille glycogen. Under these eondiEons, p h o s p h o r y l a s e a activity assayed in the absence of AMP is equal to p h o s p h o r y l a s e b activity assayed in the presence of AMP. In all the experiments, we determ i n e d the initial r a t e of conversion f r o m p h o s p h o r y lase b to p h o s p h o r y l a s e a b y p h o s p h o r y l a s e kinase, when the percent of conversion was less t h a n 40 per cent. The p h o s p h o r y l a s e kinase used in these e x p e r i m e n t s is p r o b a b l y activated (perhaps by proteolysis), since thc ratio of its activity assayed a t pH 8.2 a n d 6.8 is only of the order of 3 [11, 13]. E q u i l i b r i u m dialysis e x p e r i m e n t s were p e r f o r m e d according to Myer a n d S c h e l h n a n n [14] at 6°C, as already described [5].

BIOCHIMIE, 1979, 61, n o 5-6.

635

ATP, AMP, IMP, dAMP, GMP, cAMP, Glc-6-P, GIe-I-P a n d glycogen were purchased f r o m Sigma ; cleAMP a n d m6AMP f r o m T e r r a Marine Bioresearch. P h o s p h o glucomutase a n d glueose-6-phosphate dehydrogenase, used for p h o s p h o r y l a s e b activity m e a s u r e m e n t s , are p u r c h a s e d f r o m Boehringer. Nucleotide p u r i t y is checked b y t h i n l a y e r c h r o m a t o g r a p h y on PEI cellulose [15]. Tubercidine 5'monophosphate was k i n d l y synthetised for us b y Mrs. O. Stoven as in [5]. Radioactive AMP is p u r c h a s e d f r o m C o m m i s s a r i a t h l'Energie Atom i q u e (Saclay) a n d radioactive IMP f r o m A m e r s h a m Nuclear Center. These two c o m p o u n d s h a v e a very h i g h specific activity and are diluted for the experiments w i t h <
Results. A. INFLUENCE OF THE NUCLEOTIDES ON T/rE PHOSPHORYLASE b TO PHOSPHORYI~SE d[ CONVERSION RATE.

1. At O°C. W e h a v e s t u d i e d t h e e f f e c t s of a s e r i e s of n u c l e o t i d e s b e l o n g i n g e i t h e r to t h e c l a s s of s t r o n g a c t i v a t o r s o r to t h e c l a s s of w e a k a c t i v a t o r s . T h e r e s u l t s a r e p r e s e n t e d i n l a b i e I. O n e c a n s e e c l e a r l y t h a t t h e n u c l e o t i d e s of c l a s s I, l i k e AMP, have a strong slowering effect upon the convers i o n r a t e f r o m p h o s p h o r y l a s e b to p h o s p h o r y l a s e a, w h e r e a s a c t i v a t o r s o f c l a s s II, l i k e IMP, h a v e o n l y a w e a k e f f e c t u p o n t h i s r a t e of c o n v e r s i o n ( a l l t h e n u c l e o t i d e s w e r e u s e d at s a t u r a t i n g c o n c e n t r a t i o n s a c c o r d i n g t o [5]). W e h a v e v e r i fied b y e q u i l i b r i u m d i a l y s i s e x p e r i m e n t s p e r f o r m e d w i t h A M P a n d IMP, t h a t t h e d i s s o c i a t i o n c o n s t a n t s of t h e d i f f e r e n t n u c l e o t i d e s w e r e n o t d r a s t i c a l l y c h a n g e d b y t h e p r e s e n c e of A T P a n d Tris-glycerophosphate buffer. The saturating conc e n t r a t i o n s of n u c l e o t i d e s d e t e r m i n e d i n g l y c y l g l y c i n e b u f f e r m a y also b e c o n s i d e r e d as s a t u r a t i n g i n p r e s e n c e of A T P a n d g l y c e r o p h o s p h a t e buffer). W e h a v e s t u d i e d t h e d e p e n d e n c e of t h e slo~ver i n g e f f e c t of A M P t o w a r d s t h e c o n c e n t r a t i o n of t h i s n u c l e o t i d e . T h e r e s u l t is s h o w n o n f i g u r e 1 : t h e s l o w e r i n g e f f e c t of A M P is n o t c o m p l e t e , e v e n at v e r y s t r o n g c o n c e n t r a t i o n s ; it r e a c h e s a p l a t e a u v a l u e c o r r e s p o n d i n g to 10 to 15 p e r c e n t of r e s i d u a l c o n v e r s i o n r a t e . H a l f effect o f A M P is r e a c h e d f o r a c o n c e n t r a t i o n e q u a l to 0.02 mM. We have determined by equilibrium dialysis t h e b i n d i n g c o n s t a n t of g l y c o g e n p h o s p h o r y l a s e b f o r A M P i n t h e p r e s e n c e of t h e s a m e l i g a n d s as t h e o n e s u s e d f o r t h e c o n v e r s i o n of g l y c o g e n p h o s -

M. M o r a n g e a n d H . B u c .

636 a.

A

tu t~

.

L 0.2

0.1

0.3

3_5

hMP ] m M

FIG. 1. - - Dependence of the phosphorylase h to a conversion

rate towards AMP concentration.

The e x p e r i m e n t is p e r f o r m e d as described in Material and Methods. AMP is p r e i n e u b a t e d w i t h p h o s p h o r y l a s e b, ATP a n d glycerophosphate buffer at least 10 m i n u t e s before the b e g i n n i n g of the e x p e r i m e n t . The r e a c t i o n is followed d u r i n g a b o u t q u a r t e r of a n hour, period d u r i n g w h i c h it r e m a i n s linear. Vchen the nueleotide c o n c e n t r a t i o n is h i g h in the conversion medium, one observes a f t e r d i l u t i o n in the p h o s p h m ' y l a s e assay m e d i u m a slight p h o s p h o r y l a s e activity due to t h e a c t i v a t i o n of p h o s p h o r y l a s e b b y t h e r e s i d u a l AMP c o n c e n t r a t i o n present in this m e d i u m . An a p p r o p r i a t e correction h a s been done to take into account t h i s effect.

TABLE

I.

E f f e c t of d i f f e r e n t nucleotides on the rate of c o n v e r s i o n f r o m p h o s p h o r y l a s e b to p h o s p h o r y l a s e a by p h o s p h o r y l a s e kinase at 0°C. Nucleotides of class I

Nucleotides of class II

Nucleotide

Concentration used (raM)

P

Nucleatide

AMP

1.13

11

IMP

2.8

77

c 7AMP

0.7

41

dAMP

1.45

80

el 6AMP

2.5

24

GMP

3.7

63

~AMP

2.46

41

m 6AMP

2.5

32

Concentration used (raM)

P

P h o s p h o r y l a s e b c o n c e n t r a t i o n is 10 ~M. p is the percentage of the i n i t i a l r a t e of b to a convers_:on observed in the presence of the ligand (here the nucleotide) over the same q u a n t i t y m e a s u r e d in the absence of added ligand. For the classification of the nucleotides in the two classes, see reference [5].

phorylase b to phosphorylase ditions, the half saturation 0.18 m M (fig. 2).

a. U n d e r l h e s e c o n v a l u e is e q u a l to

T h i s s l o w e r i n g e f f e c t of A M P o n t h e c o n v e r s i o n r a t e is n o t i m m e d i a t e b u t d e p e n d s u p o n t h e t i m e during which the enzyme has been preincubated w i t h t h e n u c l e o t i d e b e f o r e t h e a d d i t i o n of p h o s p h o r y l a s e k i n a s e (cf. fig. 3). T h e d e c r e a s e i n t h e i n i t i a l v e l o c i t y w i t h r e s p e c t to t h e t i m e of i n c u b a t i o n c o r r e s p o n d s to a n e x p o n e n t i a l p r o c e s s , t h e

BIOCHIMIE, 1979, 61, n ° 5-6.

r a t e c o n s t a n t of w h i c h is e q u a l t o a b o u t s e c -1 f o r a n A M P c o n c e n t r a t i o n of 60 t~M.

4.10 a

This relaxation time can not be explained by a l a g i n t h e a c t i v i t y of t h e k i n a s e i t s e l f ; it is n o t o b s e r v e d if p h o s p h o r y l a s e is i n c u b a t e d w i t h w e a k a c t i v a t o r s i n s t e a d of s t r o n g a c t i v a t o r s . It is also i n d e p e n d e n t of t h e g l y c o g e n p h o s p h o r y l a s e b c o n centration. It has been checked that the low concentrations of e n z y m e w h i c h is u s e d , as w e l l as t h e h i g h coil-

Glycogen phosphorylase-conversion of phosphorylase b to phosphorylase a. c e n t r a t i o n of a d d e d A T P , p r e c l u d e t h e c r i s t a l l i s a t i o n of g l y c o g e n p h o s p h o r y l a s e b at O°C i n t h e p r e s e n c e of n u c l e o s i d e m o n o p h o s p h a t e .

201 1 SITE

£ n ~x IE

g i.J

/

2. A t 22°C. As t h e t e m p e r a t u r e is r a i s e d , t h e s l o ~ v e r i n g eff e c t of t h e n u c l e o t i d e s d e c r e a s e s ( t a b l e II). W h e n added alone, weak and strong activators appear to h a v e a s m a l l e f f e c t o n t h e c o n v e r s i o n r a t e at room temperature. Furthermore, the slowering e f f e c t of A M P t a k e s p l a c e w i t h i n a f e w s e c o n d s , as s h o w n i n f i g u r e 4. No d i f f e r e n c e c a n b e s e e n w h e t h e r A M P is p r e i n c u b a t e d w i t h p h o s p h o r y lase b o r is a d d e d t o g e t h e r w i t h p h o s p h o r y l a s e kinase.

]~.

10.t

1

637

INFLUENCE

2

TO

OF

GIc-6-P

PHOSPHORYLASE

ON THE

a

PHOSPHORYLASE

CONVERSION

3

RATE.

C FREE AMP ] mM

Fro. 2. - - B i n d i n g o f A M P on glycogen p h o s p h o r y lase b at 6°C in presence of A T P - M a g n e s i u m and Tr'isglycerophosphate buffer. The e x p e r i m e n t is p e r f o r m e d as described in Material and Methods. The final p h o s p h o r y l a s e b c o n c e n t r a t i o n being 165 I~M the ATP c o n c e n t r a t i o n 4.5 raM, the m a g n e s i u m c o n c e n t r a t i o n 15 mM-, in 0.03 M Tris-glycerophosphate buffer. (Final c o n c e n t r a t i o n s for the dialysis h a v e been a d j u s t e d to be identical to the ones used in t h e conversion assay).

A. A t 0°C. W e h a v e m e a s u r e d t h e s l o w e r i n g effect of Glc6-P o n t h e c o n v e r s i o n r a t e f r o m p h o s p h o r y l a s e b to p h o s p h o r y l a s e a, a n d d e t e r m i n e d t h e d e p e n d e n c e of t h i s e f f e c t t o w a r d s Glc-6-P c o n c e n t r a t i o n . T h e r e s u l t is p l o t t e d o n f i g u r e 5. O n e c a n see t h e s t r o n g i n h i b i t o r y e f f e c t of Glc-6-P o n t h e r a t e

tu id

PREiNCUBATION TIME (ran)

FIG. 3. - - Change of the conversion rate w h e n the incubation time of glycogen p h o s p h o r y l a s e b w i t h A M P is oaried prior to the addition of phosphorylase kinase. E x p e r i m e n t is p e r f o r m e d at 0°C in presence of 60 IxM AMP, all the o t h e r conditions being identical to the ones used in figure 1. In t h i s case, t h e conversion is not strictly l i n e a r w i t h t i m e a n d the i n i t i a l slope is determined by extrapolation. X corresponds to the conversion r a t e m e a s u r e d in absence of AMP. The final p o i n t (O) h a s been t a k e n at 15 minutes. TABLE II. E f f e c t o f t h e d i f f e r e n t n u c l e o t i d e s o n t h e b to a c o n v e r s i o n r a t e at 22°C. Nucleotides of class 1

Nucleotides oi class I1

Name

Concentration used

P

AMP

2.8 mM

50

p is defined as in t a b l e I.

BIOCHIMIE, 1979, 61, n ° 5-6.

Name

Concentration used

P

IMP

2 . 8 mM

88

GMP

2.4

mM

125

M. Morange and H. Buc.

638

c o n c e n t r a t i o n , the i n i t i a l velocity reaches a plateau c o r r e s p o n d i n g to 20 p e r cent of the value observed i n absence of i n h i b i t o r . The Glc-6-P concentration, at w h i c h half of the effect is observed, is equal to 0.42 mM, nearly the same value as the one observed at 4°C.

rr °

¢J 40

•

I

ii

I

5

10

o

15BllmM

TIME (ran)

~mM [ GIc-6-P

Appearance of p h o s p h o r y l a s e a a c t i v i t y as a f u n c t i o n of t i m e in absence (©) or in presence of AMP, t h i s l i g a n d b e i n g a d d e d 10 m i n u t e s ( 0 ) or i m m e Fro. 4. - -

d i a t e l y (11) b e f o r e t h e a d d i t i o n of p h o s p h o r y l a s e k i n a s e a t 22°C.

FIG. 6. - - Dependence of the p h o s p h o r y l a s e b to a

conversion

rate

22oC.

C.

INFLUENCE ON T H E

towards

Glc-6-P

OF PHOS~PHATE, GLYCOGEN AND

PttOSPHORYLASE

CONVERSION

concentration

b

at

GIc-I-P

TO PHOSPHORYLASE

fl

RATE.

A. At. O°C.

\ I lmM

I 2ram

I 3raM

[ Gh'6-P ]

Dependence of the p h o s p h o r y l a s e b to p h o s p h o r y l a s e a conversion rate towards Glc-6-P concentration at O°C.

Orthophosphate alone has a relatively strong i n h i b i t o r y effect on the rate of conversion. At saturation, the residual c o n v e r s i o n rate is only 29 per cent of the control and the phosphate concen t r a t i o n at w h i c h half of the i n h i b i t o r y effect is r e a c h e d is equal to 8 mM. I n the p r e s e n c e of either 3.70 mM AMP, or 3.5 mM GMP, the i n h i b i tory effect at s a t u r a t i n g phosphate c o n c e n t r a t i o n is slightly stronger (table III).

FIG. 5. - -

w

w

S a m e e x p e r i m e n t a l c o n d i t i o n s a s i n f i g u r e 1. T h e r e s i d u a l c o n c e n t r a t i o n of g l u e o s e - 6 - p h o s p h a t e a f t e r d i l u t i o n i n t h e a s s a y m e d i u m is too l o w to i n t e r f e r e w i t h t h e e n z y m a t i c a s s a y of p h o s p h o r y l a s e .

of c o n v e r s i o n from p h o s p h o r y l a s e b to phosphorylase a. The c o n c e n t r a t i o n of Glc-6-P necessary to r e a c h half of the slo'wering effect is equal to 0.4 mM. This effect is immediate. We confirmed at 0°C the results o b t a i n e d by Tu a n d Graves [3] at 30°C, s h o w i n g that Glc-6-P increases the Km of p h o s p h o r y l a s e k i n a s e for glycogen p h o s p h o r y lase b (results not p r e s e n t e d here).

i

Q:

88 lOmM

50raM [ PHOSPHATE 3

2. At 22°C.

FIfi. 7. - - Dependence of the p h o s p h o r y l a s e h to p h o s p h o r y l a s e a conversion rate towards p h o s p h a t e concentration at O°C.

The same effect of Glc-6-P is f o u n d at 22°C (fig. 6). I n h i b i t i o n is not complete. At high Glc-6-P

P h o s p h a t e r e s i d u a l c o n c e n t r a t i o n in the final phosp h o r y l a s e a s s a y m e d i u m is t oo l o w to h a v e a n y effect i n t h e a s s a y of p h o s p h o r y l a s e a.

BIOCHIMIE, 1979, 61, n ° 5-6.

Glycogen phosphorylase-conversion

o f p h o s p h o r y l a s e b to p h o s p h o r y l a s e

On the c o n t r a r y , glycogen has an a c t i v a t i n g effect u p o n the rate of c o n v e r s i o n from p h o s p h o rylase b to p h o s p h o r y l a s e a. T h e factor of activation is h i g h e r than 3 at 0°,C, and the glycogen c o n c e n t r a t i o n c o r r e s p o n d i n g to the half activation is equal to 2.65 p e r m i l l e (fig. 8 and table III).

E f f e c t of the snbslrates of glycogen phosphorylase b on the b to a conversion rate in presence or in absence of nacleotide at O°C. " Snbstrales alone : Concentration used

2. At 22°C. O r t h o p h o s p h a t e has also a strong i n h i b i t o r y effect u p o n the c o n v e r s i o n rate f r o m p h o s p h o rylase b to p h o s p h o r y l a s e a at this t e m p e r a t u r e . In the p r e s e n c e of 5ram AMP, this i n h i b i t i o n is i n c r e a s e d w h e r e a s in the p r e s e n c e of 6.20 mM GMP, t h e i n h i b i t i o n due to 50 mM p h o s p h a t e is l o w e r t h a n in the p r e s e n c e of p h o p s h a t e alone.

p

Phosphate

50 mM

29

Glycogen

8.33 gll

300

Substrates and nucIeot~des together : Substraie concentration

Nuclenlide concentration (raM)

0

4

AMP -3L glycogen

8.3 g/l

2.70

100

AMP -~- phosphate

3.7

22

0

4

63

GMP -[- glycogen

8.3 g/l

2.50

175

3.50

26

50 mM

We h a v e c o n f i r m e d the result p r e v i o u s l y obtained by Krebs et aL I4! w i t h a c t i v a t e d p h o s p h o rylase kinase, that glycogen activates p h o s p h o rylase kinase by d e c r e a s i n g the Km of this e n z y m e for p h o s p h o r y l a s e b. At 22°C, in the p r e s e n c e of 8.33 p e r mille glycogen, the Michaelis constant for p h o s p h o r y l a s e b is equal to 0.012 mM, w h e r e a s it is g r e a t e r t h a n 0,04 mM in t h e absence of glycogen. In this last case, the m e a s u r e of t h e Michaelis constant is m a d e difficult by the high

12

GMP alone

GMP -~- phosphate

50 mM

Glycogen has an a c t i v a t i n g effect on the conv e r s i o n rate, w h i c h is g r e a t c r at 22°C than at 0°C f o r the same p h o s p h o r y l a s e b c o n c e n t r a t i o n .

p

AMP alone

p is defined as in table I.

I 1"/*

I

i

..

10°/o

[ GLYCOGEN]

Fl(~. 8.

Dependence of the phosphorylase b to phosphorylase a conversion rate towards glycogen concentration at O°C. Glycogen residual concentration in the assay medium has no interference w i t h phosphorytase a assay.

BIOCHIMIE, 1979, 61, n ° 5-6.

--

639

In the p r e s e n c e of 2.70 mM AMP, the a c t i v a t i n g effect of 8.3 p e r mille glycogen is totally abolished. This is not true if 2.5 mM GMP are used i n s t e a d of AMP. In this case, the k i n e t i c a c t i v a t i n g effect o f glycogen is not c o m p l e t e l y a b o l i s h e d (from 300 p e r cent to 175 p e r cent ; see table III). G l u c o s e - l - p h o s p h a t e has no effect on the c o n v e r sion rate f r o m p h o s p h o r y l a s e b to p h o s p h o r y lase a at 0°C, even at a c o n c e n t r a t i o n of 0.1 M.

TABLE III.

Nameof the compound

a.

640

M. M o r a n g e a n d H. Buc.

phosphorylase b concentrations r e a c h the p l a t e a u (fig. 9).

necessary

to

z

=

30C

rn

z 20(:

m

~ lo(

O.tmM [ PHOSPHORYLASEb ]

Fro. 9. - - Determination of the Michaelis constant of phosphorylase kinase for phosphorylase b at ~2°C in the absence (0) or in the presence (©) of 8.33 per mille glycogen ; b to a conversion rate is expressed in arbitrary units.

In the p r e s e n c e of 2.7 mM AMP, the a c t i v a t i o n of p h o s p h o r y l a s e k i n a s e b y 8.3 p e r mille glycogen is r e d u c e d f r o m 300 p e r cent to 215 p e r cent, w h e r e a s in the p r e s e n c e of 2.5 mM GMP, the activ a t i o n of p h o s p h o r y l a s e k i n a s e b y 8.3 p e r m i l l e glycogen is u n c h a n g e d (table IV). Glc-I-P, the p r o d u c t of the r e a c t i o n w h i c h has no effects on p h o s p h o r y l a s e b to a c o n v e r s i o n rate at 4°C, h a s a s t r o n g a c t i v a t i n g effect at 22°C, at c o n c e n t r a t i o n s w h i c h are p r o b a b l y too h i g h to be physiological.

Discussion and Conclusions. The rate of c o n v e r s i o n of glycogen p h o s p h o r y l a s e b to a b y p h o s p h o r y l a s e k i n a s e i:,: affected b y the activators, i n h i b i t o r s a n d substrates of the p r o t e i n w h i c h is c o v a l e n t l y m o d i f i e d . These effects m a y be due to t h r e e different causes : BIOCHIMIE, 1979, 61, n ° 5-6.

a) The p r o t e i n substrate, glycogen p h o s p h o r y lase b, m a y be p h o s p h o r y l a t e d at different rates d e p e n d i n g on its t e r t i a r y or q u a t e r n a r y conformation. A c c o r d i n g to this h y p o t h e s i s , the effector m e r e l y d i s p l a c e s glycogen p h o s p h o r y l a s e f r o m one c o n f o r m a t i o n to a n o t h e r one, each of these states h a v i n g a different i n t r i n s i c rate of p h o s phorylation. b) The effector, b o u n d on glycogen p h o s p h o r y l a s e b, l o c a l l y i n t e r f e r e s 'with the k i n a s e action. This h y p o t h e s i s is not u n l i k e l y due to the p r o x i m i t y of the site to be p h o s p h o r y l a t e d 'with the AMP and Glc-6-P sites. In this case, i n h i b i t i o n of k i n a s e action s h o u l d be d i r e c t l y p r o p o r t i o n a l to the s a t u r a t i o n of p h o s p h o r y l a s e b w i t h the effector. c) The effectors might d i r e c t l y b i n d on phosp h o r y l a s e kinase. This t h i r d e x p l a n a t i o n is plausible for the ATP analogs (AMP, GMP) or for glycogen w h i c h could a n c h o r the k i n a s e on the glycogen p a r t i c l e . I n this case, the effector should also affect the k i n a s e action in a c o n t r o l experim e n t w h e r e t h e p r o t e i n s u b s t r a t e is r e p l a c e d b y a small p e p t i d e d e r i v e d f r o m p h o s p h o r y l a s e b and c o n t a i n i n g the s e r i n e to be p h o s p h o r y l a t e d . By this a p p r o a c h , Graves has u n a m b i g u o u s l y s h o w n that Glc-6-P w a s affecting p h o s p h o r y l a s e b itself. W e have not u s e d h e r e t h i s elegant p r o c e d u r e , a n d therefore, it is less easy to c o m p l e t e l y d i s c a r d a d i r e c t action of AM,P, GMP, glycogen or orthop h o s p h a t e on the kinase. H o w e v e r , the f o l l o w i n g a r g u m e n t s p l e a d in favour of an i n d i r e c t effect of the l i g a n d s v i a the f o r m a t i o n of the glycogen p h o s p h o r y l a s e b-effector complex. 1. The l i g a n d c o n c e n t r a t i o n s at w h i c h i n h i b i t i n g effects of n n c l e o t i d e s on the c o n v e r s i o n rates are o b s e r v e d are v e r y s i m i l a r to t h e d i s s o c i a t i o n constants of t h e n u c l e o t i d e - p h o s p h o r y l a s e b comp l e x e s [5]. This a r g u m e n t applies to t h e other effectors w h i c h have been tested ,(except glucose1-phosphate a n d p e r h a p s glycogen). 2. A strong a r g u m e n t in f a v o u r of an i n d i r e c t effect of the n u c l e o t i d e s and s u b s t r a t e s on phosp h o r y l a s e k i n a s e is b r o u g h t about b y the compar i s o n of the effects of the n u c l e o t i d e s b e l o n g i n g to the tv¢o classes d i s c r i m i n a t e d b y p h o s p h o r y lase b at 4°C. The s t r o n g a c t i v a t o r s of glycogen p h o s p h o r y l a s e b have a v e r y s t r o n g i n h i b i t o r y effect u p o n the c o n v e r s i o n rate from p h o s p h o r y lase b to p h o s p h o r y l a s e a, 'whereas the w e a k activ a t o r s (belonging to class If) have al~vays a weak e r i n h i b i t o r y effect than the n u c l e o t i d e s of class I, all the e x p e r i m e n t s b e i n g done at saturation in nucleotides at 0°C [5]. This d i s c r i m i n a -

Glycogen p h o s p h o r y l a s e - c o n v e r s i o n of p h o s p h o r y l a s e b to p h o s p h o r y l a s e a. tion b e t w e e n the two types of nucleotides is easily e x p l a i n a b l e if we assume that the two conf o r m a t i o n s of glycogen phosphorylase b b r o u g h t about b y these two classes of nucleotides are differently recognized by p h o s p h o r y l a s e kinase. 3. Another a r g u m e n t i n favour of a conformational effect of AMP on glycogen p h o s p h o r y l a s e b d u r i n g the c o n v e r s i o n is the fact that its i n h i b i tory effect requires for its complete establishment a r e l a x a t i o n time s i m i l a r to the one observed for the b i n d i n g of AMP on glycogen p h o s p h o r y l a s e b This slow e s t a b l i s h m e n t of the i n h i b i t i o n is not observed w i t h GMP or IMP ; conversely, we have s h o w n that the b i n d i n g of these two nucleotides is very rapid, even at 4°C [5]. These e x p e r i m e n t s establish, therefore, that the rate of i n h i b i t i o n of AMP parallels the rate of c o n v e r s i o n of E 1 to E 3 w h i l e i n h i b i t i o n due to nucleotides of class II can fully t~ke place on c o n f o r m a t i o n E r It is less easy to establish w h e t h e r the i n h i b i ting effect of AMP is due to local i n t e r f e r e n c e with the k i n a s e action (hypothesis b) or to the shift from one q u a t e r n a r y c o n f o r m a t i o n to another one (hypothesis a) or to both effects. An a r g u m e n t in favour of hypothesis a is the fact that the percentage of the i n h i b i t i n g effect is not p r o p o r t i o n a l to the s a t u r a t i o n f u n c t i o n but precedes it (compare figure 1 to figure 2). This b e h a v i o u r is expected in the case of a concerted t r a n s i t i o n . A single molecule of AMP, w h e n b o u n d to glycogen p h o s p h o r y l a s e b, displaces the w h o l e d i m e r to c o n f o r m a t i o n E 3 a n d i n h i b i t s the kinase action. This a r g u m e n t has, hoveever, to be taken w i t h caution here since the assay system is subjected to some c r i t i c i s m (ef. below). The p a t t e r n o b t a i n e d for the effects of these two classes of nucleotides seems to be more complex at 22~°C. However, the d i s c r i m i n a t i o n between ~he two classes of nucleotides always exists since nucleotides of class I are weak i n h i b i t o r s of the c o n v e r s i o n rate, w h e r e a s nucleotides of class II though they b i n d to p h o s p h o r y l a s e have no longer an effect at this t e m p e r a t u r e . The synergistic effects observed b e t w e e n orlhophosphate a n d the nucleotides are also "worth discussing. It has b e e n s h o w n that, at lcrw temperature, o r t h o p h o s p h a t e a n d nucleotides of class I stabilize the same q u a t e r n a r y c o n f o r m a t i o n E a. At h i g h t e m p e r a t u r e , they are both r e q u i r e d to trigger the E 1 to E a t r a n s i t i o n . The i n h i b i t i n g effect of phosphate, at high a n d at low tempera-

BIOCHIMIE, 1979, 61, n ° 5-6.

641

tures, and more specifically the r e i n f o r c e m e n t of the i n h i b i t o r y p o w e r of nucleotides of class I at 22°C, are in good agreement w i t h the hypothesis that the q u a t e r n a r y state E 3 is a bad substrate for p h o s p h o r y l a s e kinase. 4. Glucose-6-phosphate was already k n o w n to have an i n h i b i t o r y effect u p o n the b to a conversion rate by acting on the p r o t e i n substrate [2, 3, 4]. We have c o n f i r m e d this effect at 0°C and 22°C and show that it is immediate. 5. The two substrates glycogen a n d phosphate have a n t a g o n i s t i c effects u p o n the b to a conversion rate, at 0°C as well as at 22°C. The activating effect of glycogen w h i c h was already k n o w n is due to a l o w e r i n g of the Michaelis c o n s t a n t of p h o s p h o r y l a s e kinase for glycogen p h o s p h o r y lase b (fig. 10). This activating effect of glycogen can c o u n t e r a c t the effects of the nucleotides (tables III and IV).

TABLE IV.

E[fect o[ the substrates of glycogen phosphorylase b on the rate of b to a conversion in presence or in absence of nucleotides at 22°C. Substrate Nucleotide concentration concentration (mMI

p

Orthophosphate

50 mM

0

20

Glycogen

8.33 g/1

0

300

AMP alone

0

2.8

48

AMP -~- glycogen

8.33 g/l

2.7

215

AMP -~ phosphate

55 mM

5

GMP alone

15

0

2.3

100

GMP -q- glycogen

8.33 g/l

2.5

325

GMP -[- phosphate

50 mM

6.20

43

p is defined as in table I.

The c o n s t a n t at w h i c h half of the i n h i b i t i o n is observed w i t h p h o s p h a t e (6 mM) is not very differ e n t from the Miehaelis c o n s t a n t of glycogen p h o s p h o r y l a s e b for phosphate (5 mM) at 4°C. The phosphate c o n c e n t r a t i o n in the muscle is of the same order of m a g n i t u d e [16]. The same is not true for the activating effect of glycogen on p h o s p h o r y l a s e b to a c o n v e r s i o n rate at 33"C. The c o n c e n t r a t i o n of glycogen at w h i c h half of the a c t i v a t i n g effect is observed (5 per mille at 33°C) is relatively different from the Michaelis c o n s t a n t

642

M. Morange and H. Buc. d i s c a r d a significant i n f l u e n c e of this autoactivation process, since we have not observed any lag u n d e r our e x p e r i m e n t a l conditions.

of glycogen p h o s p h o r y l a s e b for glycogen (0.045 per mille at 25°C, d e t e r m i n e d i n presence of AMP). This result m a y p e r h a p s be c o r r e l a t e d w i t h the two different glycogen sites p r e s e n t on glycogen p h o s p h o r y l a s e b as seen by Xray diffraction studies [17]. However, this effect of glycogen could be due to a direct i n t e r a c t i o n betw,een p h o s p h o r y l a s e kinase and glycogen [18].

In a d d i t i o n to the effects already m e n t i o n e d in the literature [3, 18], w e have s h o w n that the rate of c o n v e r s i o n from phosphorylase b to phosphorylase a is m o d u l a t e d b y nucleotides, phosphate a n d glycogen at physiological c o n c e n t r a t i o n s . It appears that one of the q u a t e r n a r y c o n f o r m a t i o n s of glycogen p h o s p h o r y l a s e b, stabilized by strong activators a n d substrates, is a bad substrate for p h o s p h o r y l a s e kinase. These effects have to be taken into c o n s i d e r a t i o n if one w a n t s to u n d e r s t a n d h o w covalent a n d n o n - c o v a l e n t regulations are coupled in the glycogen particle. Other effects are k n o w n to take place on other elements of the glycogen system. AMP b y b i n d i n g on p h o s p h o r y lase a i n h i b i t s p h o s p h o r y l a s e phosphatase [23] w h e r e a s glucose activates it [24, 251. I n the presence of AMP, it seems therefore, that the system of covalent m o d i f i c a t i o n is made less efficient, w h i c h is r e a s o n a b l e since the catalytic properties of the p h o s p h o r y l a t e d a n d d e p h o s p h o r y l a t e d species are almost e q u i v a l e n t i n the presence of this nucleotide. On the other h a n d , it is plausible that, in vivo, glucose a n d glucose-6-phosphate act in concert. W h e n e v e r these ligands are in excess, they can respectively activate the dephosphorylation of p h o s p h o r y t a s e a, i n h i b i t the p h o s p h o r y lation of p h o s p h o r y l a s e b and, f u r t h e r m o r e , convert both glycogen phosphorylases into inactive species (cf. table V). Last, glycogen has multivalent effects: it is k n o w n that the b i n d i n g of glycogen on glycogen synthetase i n h i b i t s the dep h o s p h o r y l a t i o n of this e n z y m e [261. Therefore, glycogen decreases the rate of f o r m a t i o n of active

These effects observed w i t h phosphate and glycogen might play a relatively i m p o r t a n t role in the regulation of the p h o s p h o r y l a s e system in the glycogen particles. On the c o n t r a r y , the effects observed 'with Glc-l-P take place at a Glc-I-P conc e n t r a t i o n far too high to play a physiological role i n the cell [16]. 6. One restrictive p o i n t u p o n our e x p e r i m e n t s must be m e n t i o n e d : we have m e a s u r e d the conversion from p h o s p h o r y l a s e b to p h o s p h o r y l a s e a solely by following the activity of glycogen phosphorylase b i n the absence of AMP, a n d not by m e a s u r i n g the i n c o r p o r a t i o n of radioactive phosphate into p h o s p h o r y l a s e a. The p r e s e n c e of h y b r i d species, composed of p h o s p h o r y l a t e d and n o n - p h o s p h o r y l a t e d p h o s p h o r y l a s e b subunits [19, 20~ of u n k n o w n specific activity may complicate the q u a n t i t a t i v e int.erpretation of our results. Another source of c o m p l i c a t i o n is due to the fact that p h o s p h o r y l a s e kinase can u n d e r g o an ¢ autoactivation >> process i,n the presence of ATP and Mg% This autoactivation is affected by glycogen and orthophosphate [4, 21, 221. However, this p h e n o m e n o n , an a u t o p h o s p h o r y l a t i o n of the kinase [18, 211, takes place at n e u t r a l pH [13~ in the presence of calcium ions a n d is not very efficien i n glycerophosphate buffers [22]. We can

TABLE V.

Some modulations of glycogen phosphorylase kinase and glycogen phosphorglase phosphatase which have been observed in vitro. Complexes between phosphorylase and its activators

Complexes involving glycogen

Complexes between phosphorylase and its inhibitors

Ph a

Ph b

Ph b

+

Synthetase b

Ph a

Ph b

+

AMP

AMP Jr- PO4H--

glycogen

glycogen

glucose

Glc-6-p

Good subslrate for kinase

Bad substrate for phosphatase

Good substrate for phosphatase

Poor substrate for kinase

+

Bad substrates for phosphatase and kinase respectively V Slowering down of the interconversion rate

+

+

+

f

I

Autoregulation of glycogen biosynthesis

Inhibition of glycogen degradation

Complexes involving phosphorylases a and b and their activators substrates or inhibitors are listed in the first row. The observed effects are given in the second row. The third row gives a plausible functional implication.

BIOCHIMIE, 1979, 61, n ° 5-6.

Glycogen phosphorylase-conversion of phosphorylase b to phosphorylase a. glycogen s y n t h e t a s e a n d i n c r e a s e s the rate of form a t i o n of a c t i v e g l y c o g e n p h o s p h o r y l a s e . It acts, t h e r e f o r e , as a r e g u l a t o r o f its o w n b i o s y n t h e s i s . Acknowledgements.

We acknowledge the C.N.R~. (L.A. 270), the D.G.R.S.T. (grant MRM 76.7.1191) and the I.N.S.E.R.M. (grant n ° 77.8~). We t h a n k also Mrs. G. S a u d e m o n t [or her very e f f i c i e n t technical assistance. REFERENCES. 1. Krebs, E. G. & Fischer, E. H. (1962) in <(Methods in E n z y m o l o g y , vol. 5, 373-376. 2. Graves, D. J., Martensen, T. M., Tu, J. I. a Tessmer, G. M. (1973) in (), pub. SpringerVerlag, N.Y., 53-61. 3. Tu, J. I. & Graves, D. J. (1973) Biochem. Biophys. Res. Corn.re., 53, 59-65. 4. Krebs, E. G., Love, D. S., Bratvold, G. E., Trayser, K. A., Meyer, W. L. & Fischer, E. H. (1964) Biochemistry, 3, 1022-1033. 5. Morange, M., Garcia-Blanco, F., Vandenbunder, B. & Bue, H. (1976) Ear. J. Biochem., 65, 553-563. 6. Remy, P. ~ Buc, H. (1970) FEBS Lelt., 9, 152-155. 7. Heilmeyer, L. M. G. Jr, Meyer, F., Haschke, R. H. & Fischer, E. H. (1970) J. Biol. Chem., 245, 66496656. 8. Fischer, E. H. & Krebs, E. G. (1958) J. Biol. Chem., 231, 65-71. 9. Buc-Caron, M. H., Faure, F., Oudin, L. C., Morange, M., Vandenbunder, B. &Buc, H. (1974) Biochin~ie, 56, 477-489.

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