Kinetic and molecular characteristics of allosteric pyruvate kinase from muscle tissue of the sea mussel Mytilus edulis L

Biochimica et Biophysica Acta, 309 (1973) 296-3o6 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - Printed in The Netherlands

BBA

66900

K I N E T I C AND MOLECULAR CHARACTERISTICS OF A L L O S T E R I C P Y R U V A T E K I N A S E FROM MUSCLE TISSUE OF T H E SEA MUSSEL

MYTILUS EDULIS L

D I R K A. H O L W E R D A AND A L B E R T U S D E Z W A A N

Laboratorium voor Scheikundige Dierfysiologie, 4 ° .[an van Galenstraat, Utrecht (The Netherlands) (Received N o v e m b e r 23rd, 1972)

SUMMARY

I. At low phosphopyruvate concentrations, fructose 1,6-diphosphate enhances the activity of pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.4o ) from the adductor muscle of sea mussels. At pH 7.6, [phosphopyruvate] = 0.2 mM and [ADP l = 5 mM, half-maximal stimulation is reached with [Fru-I,6-P2] = 2 #M ; at pH 6. 7 it is reached with [Fru-I,6-P21 = 15/,M. 2. Glucose 1,6-diphosphate enhances enzyme activity but less so than Fru1,6-Ps. Cyclic AMP stimulates to a even lesser extent. 3. The reaction rate is inhibited when the concentration of the co-substrate ADP is substantially higher than phosphopyruvate. The Lineweaver-Burk plots for ADP at fixed concentrations of phosphopyruvate show no cooperative effects, whilst similar plots for phosphopyruvate at fixed concentrations of ADP all show positive cooperativity. 4. Alanine strongly inhibits enzyme activity. At pH 6. 7, Iphosphopyruvate] = 0.2 mM and [ADPI ~-- 2 raM, 50% inhibition is reached with [alaninel -- o.i raM; at pH 7.6 it is reached at [alanineJ = I raM. o.i mM Fru-I,6-P 2 counteracts this inhibition giving a hyperbolic substrate-saturation curve. 5. ATP inhibition is strongly pH-dependant. At pH 6.7, [phosphopyruvatel -0.2 mM and EADPI = 2 raM, 50% inhibition is reached at [ATP l = 0.62 raM. At pH 7.6 there is a little influence of ATP. Fru-I,6-P 2 also cancels this inhibition. Other trinucleotides and AMP inhibit to a less extent. 6. The molecular mass of the enzyme was found to be 220 ooo 3_ lO%. On electrofocusing, one main peak is found with pI 6.70. Three smaller peaks have pI values of 6.44, 6.59 and 6.83.

INTRODUCTION

In a previous communication 1 we reported that pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.4o ) from the posterior adductor muscle of the sea mussel Mytilus edulis has allosteric properties.

ALLOSTERIC PYRUVATE KINASE IN

Mytilus edulis

297

in common with allosteric pyruvate kinases from other sources 2-4, fructose 1,6-diphosphate has a strong stimulating effect on enzyme activity. However, the effect of p H change proved to be contrary to the effect on pyruvate kinase from yeast 5, rat liver6, ~ or human erythrocytes 4. We now report kinetic characteristics with regard to the influence of ADP, ATP, fructose 1,6-diphosphate, alanine, succinate and some other metabolites on enzyme activity, and molecular characteristics such as molecular mass and electrophoretic behaviour. MATERIALS AND METHODS

Chemicals ADP and glycerol p.a. were purchased from E. Merck AG (Darmstadt, W. Germany), further biochemicals and enzymes were from Boehringer G m b H (Mannheim, W. Germany). Other chemicals were of analytical grade. Sephadex G-I5O and DEAE-Sephadex A-5o were obtained from Pharmacia AB (Uppsala, Sweden). Mussels (M. edulis L.) were supplied by the Institute of Mussel Research (Texel, The Netherlands).

Enzyme preparation The enzyme preparation used was the same as described in the previous paper 1. There was no loss of activity when the enzyme was stored in solution containing glycerol (30%, v/v) at 5 °C for several weeks. No influence of glycerol on kinetics was found.

Enzyme activity P y r u v a t e kinase activity was measured according to Bficher and Pfleiderer s. In the reaction medium the following concentrations were not varied: MgSO 4 (8.3 raM), KC1 (67 mM), N A D H (o.12 mM), lactate dehydrogenase (36 I.U.). Temperature was 25 °C. Other factors were variable and are indicated in each experiment. The enzyme was pre-incubated for 15 min at 25 °C. Change in absorbance at 34 ° n m was measured with a Zeiss PMQ I I spectrophotometer coupled with a Vitatron U R 400 recorder. The reaction was started b y adding the substrate phosphopyruvate. The activity is expressed as/,moles N A D H . min -1 ; the specific activity as the activity per mg protein. Protein was determined using the Lowry method.

Molecular mass The molecular mass was measured according to Andrews 9 with a Sephadex G-I5O column (1.2 cm × IOO cm) in o.I M Tris-HC1 buffer, p H 7.6. For calibration, enzymes with known molecular masses were used. These are mentioned under the figure.

Electrofocusing "Electrofocusing" was carried out to determine possible electrophoretic multiplicity and isoelectric points. A 44o-ml L K B electrofocusing column was used with a gradient of glycerol (o-6o%, w/v) in water, and p H ranges 3-1o and 5-8. Runs were performed at 4 °C for 48 h.

298 RESULTS

D.A. HOLWERDA, A. DE ZWAAN AND

DISCUSSION

Fructose x , 6 - d i p h o s p h a t e

The influence of increasing c o n c e n t r a t i o n s of fructose 1,6-diphosphate (Fru1,6-P2) on the initial reaction velocity at low p h o s p h o p y r u v a t e c o n c e n t r a t i o n a n d at two p H values is shown in Fig. I. At b o t h p H values, n e a r - m a x i m a l s t i m u l a t i o n

.c

<,

JJM Fru-l~- P2

Fig. I. The influence of the concentration of fructose 1,6-diphosphate on the reaction rate of the pyruvate kinase reaction at various pH values. [Phosphopyruvate] = o.2 raM; [ADP] = 2 mM; buffer: o.I M Tris-maleate.

is reached at [Fru-I,6-P2] = 5 ° #M, b u t there is a great difference in the F r u - I , 6 - P 2 c o n c e n t r a t i o n of h a l f - m a x i m a l s t i m u l a t i o n (K~). At p H 7.6 K~ is 2 #M, at p H 6. 7 it is 15/~M. The l a t t e r value is w i t h i n the range in which the c o n c e n t r a t i o n of F r u 1,6-P2 in rat liver fluctuates (5-20 /*M)1° b u t we do n o t k n o w in which range this i n t e r m e d i a t e fluctuates in the muscle d u r i n g anaerobiosis, nor do we know to what e x t e n t the p H will drop. F r o m the curves it can be seen t h a t at higher p H the effector site is readily accessible for F r u - I , 6 - P 2, while at lower p H there is a cooperative effect of the activator. The lower m a x i m a l s t i m u l a t i o n in the l a t t e r case is t h o u g h t to be a g e n u i n e p H effect r a t h e r t h a n a m a t t e r of c o n f o r m a t i o n of the enzyme. TABLE I T H E E F F E C T OF CYCLIC AMP, G L U C O S E PYRUVATE KINASE ACTIVITY

1,6-DIPHOSPHATE

AND

FRUCTOSE

[Phosphopyruvate] -- 0.2 mM; buffer: o.i M imidazole-HC1, pH 7.6. Addition

Control o.i mM Glc-I,6-P~ 0. 3 mM Glc-I,6-P~ 4 rnM cyclic AMP o.i rnM Fru-i,6-Pz

Enzyme activity (%) [ A D P ] = 0. 5 r a m

[ADP] = 5raM

I oo 15o I7o 12o 18o

I oo 18o 250 13o 37°

1,6-DIPHOSPHATE

ON

ALLOSTERICPYRUVATE KINASE IN Mytilus edulis

299

Glucose x,6-diphosphate, cyclic A M P Other stimulating factors are listed in Table I. It has been found 11 with hepatic pyruvate kinase that the influence of glucose 1,6-diphosphate (Glc-I,6-P2) on the enzyme activity is of the same nature as the action of Fru-I,6-P v The effect of these two activators was also quantitatively the same. In our case, as in the case with erythrocyte pyruvate kinase tl the effect of Glc-I,6-P2 is less pronounced. Stimulation by cyclic AMP has also been found with pyruvate kinase from loach embryos TM. Haeckel et al. 5 reported inhibition with yeast pyruvate kinase.

Adenosine diphosphate In Fig. 2A, Lineweaver-Burk plots are shown. When the ADP concentration is substantially higher than the concentration of phosphopyruvate there is considerable inhibition of reaction velocity. It is important to note that the ratio of ADP to phosphopyruvate which leads to inhibition is not constant but decreases with increasing concentration of phosphopyruvate. No positive cooperative action of ADP is found. Similar results have been reported by Tanaka et al. TM with rat liver pyruvate kinase. It was found that the inhibition was not caused by ATP. With the erythrocyte enzyme 4 there might also be inhibition at high levels of the co-substrate, but this is not fully clear. From Fig. 2B it appears that the cooperative action of phosphopyruvate exists at all A D P concentrations. In the presence of o.I mM Fru1,6-P 2 inhibition by large concentrations of ADP does not occur (Fig. 3).

Alanine Strong inhibition is exerted by alanine (Fig. 4). Whereas V is not much influenced, the apparent Km is increased from 0.60 mM to 3.0 mM and the Hill coefficient from 1.3 to 1.75 (Fig. 5), indicating that alanine is an allosteric inhibitor. In the presence of o.I mM Fru-I,6-P 2 the inhibition is reversed and the saturation curve becomes a hyperbole (nil = I.O; apparent K m = 0.09 mM).

A

~PEP](mM)

B rAoP]:/

7 0.1

i i

-i

1.25 t q

.65

.= i ~2

I

I

I

I 3

g[AoP]

I

(mM)*l

5

y[PEP]

Fig. 2. (A) Lineweaver-Burk plots of the reaction rate of the pyruvate kinase reaction against ADP concentration at various phosphopyruvate concentrations. Buffer: o.I M imidazole-HC1 pH 7.6 (B) Variation of enzyme activity with phosphopyruvate at various ADP concentrations.

300

D.A.

o

HOLWERDA,

A. D E Z W A A N

----//

Ab0~ o

<

I

b,

L mM ADP

Fig. 3. The effect of ADP concentration on pyruvate kinase activity in the absence (O--O) and presence (0--(2)) of o. I mM Fru-I,6-P 2. [Phosphopyruvate] = o.2 raM; buffer: o.I M imidazoleHCI, pH 6.8. & 40

/

Z~o

o

b~ 3(

S

E

/ :~ 1C

/

m M Phosphopyruvate

Fig. 4. Reaction rate of the pyruvate kinase reaction against concentration of phosphopyruvate. [ADP] = 2 mM; buffer: o.i M Tris-HC1, pH 7.6. 0 - - 0 , with 2 mM alanine; O---O, with 2 mM alanine and o.i mM fructose 1,6-diphosphate; A - - A , control. At [Phosphopyruvate~ = t 5 mM (not shown in the figure) the velocities without and with Fru-I,6-P 2 in the presence of 2 mM alanine were identical and used as V in the Hill plot (Fig. 5). T h e i n h i b i t i o n b y alanine, w h i c h is also k n o w n w i t h r a t l i v e r p y r u v a t e kinase14,15, is r e l e v a n t in t h e case of t h e m u s s e l e n z y m e as this a m i n o a c i d is t h e first to e m e r g e a n d , w i t h s u c c i n a t e , t h e m a i n e n d - p r o d u c t of g l y c o l y s i s w h e n t h e m u s s e l lies d r y 16. T o g e t h e r w i t h l o w e r i n g o f p H b y a c c u m u l a t i o n o f o r g a n i c acids, an i n c r e a s e of a l a n i n e concentration would suppress the mussel pyruvate kinase activity, thus favouring t h e c a r b o x y l a t i o n of p h o s p h o p y r u v a t e , p r o v i d e d t h a t t h e F r u - I , 6 - P 2 c o n c e n t r a t i o n is low e n o u g h u n d e r t h e s e c o n d i t i o n s . Fig. 6 s h o w s t h e effect of i n c r e a s i n g c o n c e n t r a t i o n s of a l a n i n e on e n z y m e a c t i v i t y at p H 7.6 a n d 6. 7. A t t h e h i g h e r p H v a l u e , w h e n t h e e n z y m e is in t h e activ a t e d f o r m , t h e a f f i n i t y o f t h e e n z y m e for a l a n i n e is low. A t t h e l o w e r p H v a l u e t h e i n h i b i t i o n c u r v e has a n o r m a l shape, i n d i c a t i n g t h a t t h e e n z y m e exists in a f o r m

ALLOSTERIC PYRUVATE KINASE IN Mytilus edulis

3Ol

¢ /o o

~s~ LX/

/

log [PEP]lmM)

,H=1.0

A/

~ nil=l,3

..! 1.75•

nil=

/

•

Fig. 5. Hill plot of the reaction rate against phosphopyruvate concentration. 0 - - 0 , with 2 rnM alanine; O - - O , with 2 mM alanine and o.i mM fructose 1,6-diphosphate; A - - A , control. Conditions as for Fig. 4.

10G

o

--5~

o

o

i o

|

2

mM Alanine

3

o

4

Fig. 6. Effect of alanine concentration on the reaction rate of the pyruvate kinase reaction at various pH values. [Phosphopyruvate] = o.2 mM; [ADP] = 2 mM; buffer: o,i M Tris-HC1. readily accessible for the ligand. A t this pH, 5 o % i n h i b i t i o n is already reached with o.I mM alanine.

Adenosine triphosphate and other nucleotides The influence of A T P is strongly p H - d e p e n d a n t . At p H 7.6 there is no m a i o r change in V a n d i n a p p a r e n t Kra with 2 mM A T P in the reaction m e d i u m (Fig. 7)The Hill plot shows that, in the presence of A T P , the cooperative effect towards p h o s p h o p y r u v a t e increases: the Hill coefficient becomes 2.0. The same has been reported 17 for p y r u v a t e kinase type I I from rat k i d n e y cortex. At the lower p H value the e n z y m e a c t i v i t y decreases r a p i d l y with increasing c o n c e n t r a t i o n s of ATP,

302

D. Ao HOLWERDA, A. DE ZWAAN

0t //

=

~ ~

SY

~

I

-d~e

v

log[PEP]lmM )

.
ot /f 1

mM Phosphopyruvate

2

5

Fig. 7- The influence of A T P on the reaction rate of the p y r u v a t e kinase reaction against p h o s p h o p y r u v a t e c o n c e n t r a t i o n : Michaelis-Menten plots and Hill plots. [ADP] = 2 raM; buffer: o.I M Tris-HC1, p H 7.6. O---O, control; © - - © , plus o.i mM F r u - i , 6 - P z ; ,ik--&, plus 2 mM A T P ; A - - A , plus 2 mM A T P and o. i mM F r u - i ,6-.P v F o r the Hill plot, the velocities at [ P h o s p h o p y r u vate] = 5 mM were t a k e n as V.

o o

o

100 o °

o o

o

o

o

pHZ5

7d .

>2

"6

25

I

I

-

2 mM ATP

3

i

4

Fig. 8. E f f e c t o f A T P c o n c e n t r a t i o n on t h e r e a c t i o n r a t e o f t h e p y r u v a t e kinase r e a c t i o n a t v a r i o u s pH

values.

[Phosphopyruvate]

=

0. 5 mM;

~ADP]

=

z mM;

buffer:

o.i

M

Tris-HC1.

ALLOSTERIC PYRUVATE KINASE IN Mytilus edulis

3o3

leading to a s a t u r a t i o n curve of n o r m a l shape (Fig. 8). The curves h a v e m u c h resemblance to those o b t a i n e d w i t h r a t liver 7 a n d e r y t h r o c y t e 4 p y r u v a t e kinase, with an opposite p H d e p e n d a n c y . S t r o n g c o o r d i n a t i o n of Mg 2+ b y A T P m i g h t be responsible for the inhibition. I n t h a t case f u r t h e r a d d i t i o n of Mg 2+ would be e x p e c t e d to c o u n t e r a c t the a c t i o n of A T P . I n Table I I however it is seen t h a t t h e i n h i b i t i o n b y A T P does n o t d e p e n d on t h e Mg z+ c o n c e n t r a t i o n . This m e a n s t h a t A T P inhibition is at least p a r t l y caused b y a direct action on the e n z y m e (conformation). I n Table I I I the influence of o t h e r nucleotides on the e n z y m e a c t i v i t y is given. I t is seen t h a t A T P is b y far the m o s t p o t e n t i n h i b i t o r u n d e r the conditions used. TABLE II E F F E C T O F i g 2+ C O N C E N T R A T I O N ON THE INHIBITION OF PYRUVATE K I N A S E B Y ATP EPhosphopyruvate3 = 0. 5 mM; [ADP] = 2 mM; buffer: o.i M imidazole-HC1, pH 7.6. THE

d ddition

Enzyme activity (AA 340nm/min)

Control (8. 3 mM Mg~+) + 4 mM ATP + 4 mM A T P + 8 mM Mg2+

o.124 0.063

+ 8 m M Mg 2+

0.070 o.118

TABLE III THE

EFFECT

OF NUCLEOTIDES

(4

raM) O N

PYRUVATE

KINASE

ACTIVITY

EPhosphopyruvate] = 0. 5 mM; EADP] -- 5 mM; buffer: o.I M Tris-HCl, pH 6.8.

Addition

Enzyme activity (%)

Control ATP GTP ITP AMP

ioo
Miscellaneous factors The following i n t e r m e d i a t e s a n d o t h e r c o m p o u n d s h a v e only little influence on t h e r e a c t i o n v e l o c i t y (conditions as in T a b l e I I I ) : a s p a r t i c acid a n d succinate b o t h 4 mM, fructose 1-phosphate, fructose 6 - p h o s p h a t e , glucose 1-phosphate, glucose 6phosphate, glyceraldehyde 2-phosphate, glyceraldehyde 3-phosphate and dihydroxyacetone p h o s p h a t e - a l l a t o.I mM. Molecular characteristics The m o l e c u l a r m a s s has been s t a t e d (Fig. 9) as 22o ooo ± 1 0 % , B l u m e et al. TM r e c e n t l y listed m o l e c u l a r masses of p y r u v a t e kinases from several sources. The nonallosteric t y p e s from muscle tissue a p p e a r to h a v e a higher m o l e c u l a r mass (240 o o o 250 ooo) t h a n t h e allosteric t y p e s from y e a s t 19, liver a n d e r y t h r o c y t e s (around 200 ooo). The effect of u r e a on t h e m o l e c u l a r m a s s a n d t h e a c t i v i t y of possible subunits is u n d e r i n v e s t i g a t i o n .

304

D. A. HOLWERDA, A. DE ZWAAN

60

v =

-~ so

.... 40

~3

,

-

\

i i

5

5.5

Log molecular mass

Fig. 9- Elution volume from the Sephadex G-iso column of marker enzymes and of mussel pyruvate kinase against molecular mass. (I) Malate dehydrogenase; (2) glucose 6-phosphate dehydrogenase; (3) xanthine oxidase; PyK, mussel pyruvate kinase. pH

B

A

c

o2 r,1 <

<

20

40

60

80

20

40

60

80

fraction number

Fig. io. Elution pattern of mussel pyruvate kinase from the electrofocusing column. (A) Enzyme fraction with specific activity 4 (/~moles N A D H . m i n 1. mg-1 protein). (B) Enzyme fraction with specific activity 2i. 0.75 vol% Ampholine carrier Ampholyte pH 5-8; other conditions as in the text. T w o f r a c t i o n s of p y r u v a t e k i n a s e of d i f f e r e n t p u r i t y - - s p e c i f i c a c t i v i t i e s 4 a n d 21, r e s p e c t i v e l y - - h a v e b e e n s u b j e c t e d to " e l e c t r o f o c u s i n g " . I n a p r e l i m i n a r y e x p e r i m e n t , u s i n g a p H g r a d i e n t 3 - 1 o , it was e s t a b l i s h e d t h a t all p y r u v a t e k i n a s e a c t i v i t y s e t t l e d w i t h i n a n a r r o w r a n g e b e t w e e n p H 6 a n d 7. I n t h e e x p e r i m e n t s h o w n in

ALLOSTERIC PYRUVATE KINASE IN M y t i l u s e d u l i s

3o5

Fig. IO a p H g r a d i e n t 5 - 8 was used. W i t h b o t h e n z y m e fractions applied, an i d e n t i c a l elution p a t t e r n was o b t a i n e d . The f r a c t i o n w i t h specific a c t i v i t y 4 has one m a i n p e a k a t p H 6.65, a n d at least t h r e e smaller p e a k s at p H values of 6.39, 6.54 a n d 6.80 (Fig. i o A ) . T h e fraction with specific a c t i v i t y 21 has one m a i n p e a k at p H 6.75, a n d a t least t h r e e smaller p e a k s at p H values of 6.49, 6.64 a n d 6.86 (Fig. l o B ) . W e t h i n k t h a t t h e v a r i o u s peaks are closely r e l a t e d to each o t h e r a n d m i g h t be aggregates of i d e n t i c a l s u b u n i t s or m o n o m e r s w i t h different a g g r e g a t i o n numbers. I n c o n t r a s t w i t h our results for the sea mussel, o y s t e r p y r u v a t e kinase 2° shows a p a t t e r n with two m a i n p e a k s at p H values of 5.6 a n d 6.5 a n d because of this m a r k e d difference in p I it is likely t h a t two d i s t i n c t p y r u v a t e kinases are present. A n allosteric p y r u v a t e kinase in muscle tissue of the sea mussel m a y be of physiological significance in t h e r e g u l a t i o n of glycolysis d u r i n g anoxia. The mussel has to be able to w i t h s t a n d anaerobic c o n d i t i o n s due to its h a b i t a t , the i n t e r t i d a l zone. The m a j o r e n d - p r o d u c t s of anaerobic glycolysis are alanine, succinate a n d some g l u t a m a t e i n s t e a d of l a c t a t O 6. A l a n i n e is the initial m a j o r e n d - p r o d u c t , b u t d u r i n g p r o l o n g e d periods of a n o x i a t h e conversion of p h o s p h o p y r u v a t e to alanine is g r a d u a l l y p r e v e n t e d in f a v o u r of an increased p r o d u c t i o n of succinate a n d g l u t a m a t e . The f o r m a t i o n of succinate t a k e s place b y c a r b o x y l a t i o n of p h o s p h o p y r u v a t e to oxala c e t a t e followed b y some reverse steps of the K r e b s cycle. I n i t i a l a c c u m u l a t i o n a n d the g r a d u a l decrease of p H caused b y the f o r m a t i o n of organic acids m i g h t e x p l a i n w h y p h o s p h o p y r u v a t e becomes available for succinate p r o d u c t i o n , because these c o n d i t i o n s suppress p y r u v a t e kinase a c t i v i t y . The m a i n factors g o v e r n i n g t h e fate of p h o s p h o p y r u v a t e seem to be the p H a n d the c o n c e n t r a t i o n of the s u b s t r a t e itself a n d of the effectors fructose 1,6-diphosp h a t e , alanine a n d A T P . F o r A r e n i c o l a it has been d e t e r m i n e d ~1 t h a t at low tide, when o x y g e n s u p p l y falls of, the p H w o u l d fall to a level of a b o u t 6.6. O t h e r d a t a are n o t y e t available. A precise e v a l u a t i o n has to a w a i t t h e q u a n t i t a t i v e d e t e r m i n a t i o n of several i n t e r m e d i a t e s a n d e n d - p r o d u c t s , a n d off the change off p H d u r i n g anaerobic m e t a bolism.

REFERENCES I 2 3 4 5 6 7 8 9 IO ii 12 13

Zwaan, A. de and Holwerda, D. A. (1972) Biochim. Biophys. Acta 276, 43o-433 Hess, B., Haeckel, R. and Brand, K. (1960) Biochem. Biophys. Res. Commun. 24, 824-831 Taylor, C. B. and Bailey, E. (1967) Bioehem. J. lO2, 32c Staal, G. E. J., Koster, J. F. ,Kamp, H., van Milligen-Boersma, L. and Veeger, C. (1971) Biochim. Biophys. Acta 227, 86-96 Haeckel, R., Hess, B., Lauterborn, W. and Wi~ster, K.-H. (1968) Z. Physiol. Chem. 349, 699- 714 Bailey, E., Stirpe, F. and Taylor, C. B. (1968) Biochem. J. lO8, 427-436 Rozengurt, E., Jim6nez de Asua, L. and Carminatti, H. (1969) J. Biol. Chem. 244, 3142-3147 Bt~cher, Th. and Pfleiderer, G. (1955) in Methods in Enzymology (Colowick, S. P. and Kaplan, N. O., eds), Vol. I, pp. 435-436, Academic Press, New York Andrews, P. (1964) Biochem. J. 91, 222-233 Williamson, J. R., Jakob, A. and Scholz, R. (1971) Metabolism 20, 13 26 Koster, J. F., Slee, R. G., Staal, G. E. J. and Berkel, Th. J. c. van (1972) Biochim. Biophys. Acta 258, 763-768 Milman, L. S. and Yurowitzki, Y. G. (1967) Biochim. Biophys. Acta 146, 3Ol-3O4 Tanaka, T., Harano, Y., Sue, F. and Morimura, H. (1967) J. Biochem. Tokyo 62, 71-91

300 14 15 16 17 I8 19 20 2t

D . A . HOLWERDA, A. DE ZWAAN

Seubert, W., Henning, H. V., Schoner, W. and L'age, M. (I968) Adv. Enzyme Reg. 6, 153-187 Llorente, P., Marco, R. and Sols, A. (197 o) Eur. J. Biochem. 13, 45-54 Zwaan, A. de and Marrewijk, \V. J. A. van (1973) Comp. Biochem. Physiol., 44 B, 429-439 Costa, L., Jim6nez de Asua, L., Rozengurt, E., Bade, E. G. and Carminatti, H. (1972) Biochim. Biophys. Acta 289, 128-136 Blume, K. G., Hoffbauer, R. W., Busch, D., Arnold, H. and 1,6hr, G. W. (197 I) Biochim. Biophys. Acta 227, 364-372 Bischofberger, H., Hess, B., R6schlau, P., Wieker, H.-J. and Zimmermann-Telschow, H. (197 o) Z. Physiol. Chem. 351, 4Ol-4O8 Mustafa, T. and Hochachka, P. ~V. (1971) J. Biol. Chem. 246, 3196-32o3 Mangum, C. P. and Shick, J. M. (1972) Comp. Biochem. Physiol. 42A, 693-697