The reaction of phosphages with ATP: Creatine phosphotransferase

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 65500

T H E REACTION OF PHOSPHAGENS W I T H A T P : C R E A T I N E

PHOSPHOTRANSF ER AS E

327

ELIZABETH JAMES* AND J. F. MORRISON Department of Biochemistry, John Curtin School of Medical Research, Australian National Uni- versi(v, Canberra (Australia)

(Received April 26th, 1966)

SUMMARY

I. A kinetic investigation has been made of the effect of various phosphagens on the reverse reaction catalysed by creatine kinase (ATP:creatine phosphotransferase, EC 2.7.3.2).

2. The results indicate that phosphoglycocyamine functions as a substrate of the enzyme with a maximum velocity only o.18% of that obtained with phosphocreatine. Phosphotaurocyamine and phosphoarginine were found not to act as substrates although they could combine with the enzyme as inhibitors.

3. Values have been obtained for the true kinetic constants associated with the reaction of each phosphagen with both the free enzyme and the enzyme-MgADP complex.

INTRODUCTION

Kinetic investigations of the specificity of creatine kinase (ATP: creatine phosphotransferase, EC 2.7.3.2 ) with respect to the guanidino substrate have been confined to a limited number of guanidino compounds. Further, the results have been qualita- t ive in that no a t tempt has been made to determine true kinetic constants. However, it has become apparent that the enzyme has a narrow specificity. Thus it has been reported that N-ethylglycocyamine 1 and glycocyamine ~ (I) are phosphorylated by ATP in the presence of creatine kinase at much slower rates than that observed with creatine (N-methylglycocyamine) under the same conditions. In addition, it has been found that creatinine, arginine and histidine 8, as well as taurocyamine 4 (II) are inactive as substrates.

NH 2 NH~ / /

NH=C HN=C \ \

NH--CHz---C00H NH--CH~--CH~--S0sH (I) (II)

* Present address: Department of Biochemistry, University of Sheffield, England.

Bio~him. Biophys. Aeta, 128 (1966) 327-336

3 2 8 E. JAMES, J. F. MORRISON

The present work was undertaken to obtain more quantitative data about the reaction of guanidino compounds with creatine kinase and forms part of a general study which is designed to gain further insight into the catalytic mechanism. Studies were made of the reverse reaction because of the sensitivity of the method for esti- mating free guanidines and because of the greater affinity of the enzyme for phosphagens as compared with their corresponding non-phosphorylated derivatives. The results indicate that phosphoglycocyamine can function as a substrate, although with a very much lower maximum velocity than that obtained with phosphocreatine, while phosphoarginine and phosphotaurocyamine act only as inhibitors. Dissociation constants for the reaction of phosphagens with free enzyme and the enzyme-MgADP complex have been calculated on the basis that the reaction has a rapid equilibrium, random mechanismS, e.

THEORY"

On the assumption that the reaction is of the rapid equilibrium, random type, irrespective of the guanidino substrate, the initial velocity equation for the reaction at pH 8.0 in the presence of I.O mM free Mg 2+ may be expressed asS:

V A B V = (I)

KlaKb + K~B + KbA + A B

where A and B represent the concentrations of MgADP- and phosphagen, respectively; Kla represents the dissociation constant for the reaction of A with free enzyme; Ka and Kb are Michaelis (also dissociation) constants for the reaction of A and B with EB and EA, respectively and V represents the maximum velocity of the reaction. Eqn. I may be written in double reciprocal form as:

~ - - ~ t - - b - + ~ ~ - + T -b - + ~ (~)

with MgADP- (A) as the variable substrate and as:

~ = - C L - ~ - + 1 ~ + ~ -~-+~ (~)

with phosphagen (B) as the variable substrate. Klb represents the dissociation constant for the reaction of B with free enzyme and because of the rapid equilibrium condition, KiaKb ~- Kd£1b. I t follows from Eqns. 2 and 3 that secondary plots of the slopes and intercepts of the lines against the reciprocal of the concentration of the non-variable substrate will yield values for Klb, Kb, Kta and Ka.

I f an analogue of phosphocreatine can combine with both free enzyme and the enzyme-MgADP complex so as to form dead-end complexes, then the initial velocity Eqn. I becomes :

V A B v = (4) ( ° ) KiaKb I + Ktg + KaB + KbA I + + AB

where G represents the inhibitory phosphagen and K t g and Kig represent dissociation constants for its reaction with free enzyme and enzyme-MgADP, respectively.

Biochim. Biophys. Acta, 128 (1966) 327-336

REACTION OF PHOSPHAGENS WITH CREATINE KINASE 329

When the inhibition is studied with MgADP- (A) as the variable substrate, Eqn. 4 may be rearranged in reciprocal form as:

- - = ff -~- I + ----'~-- I + 7 + ~ I + ~-- I + (~)

Thus at non-saturating concentrations of phosphocreatine (B), the inhibition would be non-competitive with the slopes and vertical intercepts of the lines varying in a linear fashion with the concentration of G. Secondary plots of the slopes and vertical intercepts against the concentration of G would yield apparent values for -/17ig and Kig, respectively, and the true values of these constants may be obtained by calculation from the relationships:

Kig = Apparent Kig { K l b

and f / Kb

Klg = Apparent K i g / ~ ~

using appropriate values for Kib, Kb and B. I f phosphocreatine (B) is the variable substrate, Eqn. 4 may be written in reci-

procal form as:

~_-p-tl+~,g+7 1+~ B+T I+7f (6)

so that irrespective of the concentration of MgADP- (A), the inhibition would be linear competitive. At non-saturating concentrations of MgADP-, the apparent inhibitor constant, obtained from a replot of the slopes of the lines against the concen-

tration of G, would be a complex function equal to (A + Kl~) / L ~ + Kx~--

For comparison with the directly determined value for a particular phosphagen inhibitor, the value of this complex function may be calculated using the values for Kig and KIg obtained from the non-competitive inhibition (Eqn. 5) as well as the appropriate values of A and Kta.

I t should be mentioned that the Michaelis constant (Kb) for a substrate and K,~ for an inhibitor are analogous in that each represents the dissociation constant for the reaction of a phosphagen with the enzyme-MgADP complex. Similarly, Ktb for a substrate and Kig for an inhibitor represent dissociation constants for the reaction of a phosphagen with free enzyme.

EXPERIMENTAL

Materials

Phosphoarginine was isolated from crayfish muscle as a barium salt by the method of MARCUS AND MORRISON ~ and the barium salt of monophosphotaurocyamine was prepared according to the procedure of MORRISON, ENNOR AND GRIFFITHS s. After analysis, the barium salts were converted to sodium salts using columns of Zeo-Karb 225 (Na+ form) as previously described 9.

Phosphoglycocyamine was prepared by the phosphorylation of glycocyamine (14 g) with POC13 under conditions similar to those described for the phosphorylation


330 E. JAMES, J. F. MORRISON

of taurocyamiue by MORI~ISON, ENNOR AND GRIFFITHS 8. The purification procedure of these authors was followed up to the stage of adjusting the combined extract to pH 7-5 after which the t reatment was as follows: To the extract were added 4 vol. of 95 % (v/v) ethanol. The resulting precipitate was collected by filtration after 24 h and dissolved in 250 ml of water. One vol. of ethanol was then added and after the mixture had stood for 2 h, the precipitate was removed by filtration and the supernatant treated with a further 2 vol. of ethanol. After standing for 24 h, the precipitate was collected by filtration, dissolved in 800 ml of water and i M barium acetate (25 ml/1) was added. The solution was adjusted to pH 8.2 and the small precipitate which for- med was removed by centrifugation. The supernatant was passed through a column (2 cm × 25 cm) of Zeo-Karb 225 (Na + form) to remove barium and the effluent treated with 2 vol. of ethanol to yield a crystalline precipitate of sodium phosphoglycocyamine. This was collected by centrifugation, washed with ethanol and ether and dried in vacuo over anhydrous calcium chloride. The product (I .I g) was pure as judged by elementary analysis (Found: C, 12. 7 % ; H , 4.5 %; N, 13.9%; H~O, 17.9%. Calculated for C3HsOsN3P(Na)2. 3 H20:C, 12.2%; H, 4.1%; N, 14.2%; H20, 18.3%).

Solutions of the sodium salts of the above phosphagens were standardized by estimation of the free guanidine concentration 1° after complete hydrolysis of the compounds in I M HC1 at IOO ° for IO min 8. Other reagents, including the preparation of creatine kinase, were as previously described 5.

Methods Measurement of creatine kinase activity. Reaction mixtures, in a total volume of

I.O ml, contained: N-ethylmorpholine-HC1 buffer (pH 8.o), o.I M; EDTA, o.oi mM; substrates (and in some cases an inhibitory phosphagen) at the concentrations indica- ted in the figures and sufficient MgC12 to give the required concentrations of MgADP--- while maintaining the concentration of free Mg ~+ constant at I.O mM. After the addition of all components, except enzyme, the tubes were incubated for 3 min at 30 °. The amount of creatine kinase added corresponded to 0.54 #g of protein, except that 43 #g was used with phosphoglycocyamine as substrate. Each experiment was run for two time periods to ensure that initial velocities were being measured. For experiments involving inhibitors they were between 0.5 and 1.5 min, while with phosphoglycocyamine as substrate, reactions were run for either 3 and 6 min or 4 and 8 rain.

The reaction was stopped and the release of free guanidino compound determined as described by MORRISON, O'SULLIVAN AND OGSTON 11. Colour development was allowed to proceed for 15 min for the estimation of creatine and 3o min for the estimation of glycocyamine. Under the assay conditions, all free guanidines gave essentially the same absorbance of 0.65o for o.I/~mole when measured at 535 m/~ in a cell of I -cm light path. Reagent blanks, containing all components except enzyme, were incubated and treated in the same manner as the experimental tubes so as to allow for the contamination of the phosphagens by small amounts of the corresponding free guanidines. When the higher concentration of creatine kinase was used, enzyme was also added to the reagent blanks after the addition of stopping reagent since it significantly increased the blank readings.

Calculation of substrate concentrations. The concentrations of total MgCI~ and total ADP which were necessary to give the required concentrations of MgADP- and free phosphagen while maintaining free Mg z+ at a fixed concentration were calculated


REACTION OF PHOSPHAGENS WITH CREATINE KINASE 33I

as outlined previously u. The values of the stability constants were taken to be 4000 M -1 for MgADP-, 12 M -1 for Mg-phosphocreatine and ioo M -1 for Mg-phospho- argininen, lz. In the absence of values for Mg-phosphoglycocyamine and Mg-phosphotaurocyamine, it has been assumed that they were 12 M -x and IOO M -1, respectively. All the Mg-phosphagen complexes have been considered to be inert.

Analysis of data. The values and standard errors of the kinetic constants were obtained by analysis of the pr imary data using the Sequen, Comp or Noncomp computer programmes of CLELAND 18 (Eqns. 7, 8 or IO of ref. 13) in conjunction with an IBM 162o computer. Except where otherwise stated, the lines of the pr imary plots were drawn using the constants obtained by analysis of each set of data with the Hyper programme (Eqn. I of ref. 13). Secondary plots were drawn using the constants obtained by analysis of the data with the Line programme (Eqn. 3 of ref. 13). Weight- ed mean values for the apparent kinetic constants and their s tandard errors were calculated as described s while the standard errors of the true kinetic constants were determined using the formulae given by JAMES AND MORRISON 14.

RESULTS

Initial vdocity studies with phosphoglycocyamine as substrate The results illustrated in Fig. I in the form of a double reciprocal plot show

that phosphoglycocyamine acts as a substrate for the creatine kinase reaction. With both MgADP- (Fig. ia) and phosphogiycocyamine (Fig. ib) as the variable substrate, families of straight lines are obtained and each has a common point of intersection which lies to the left of the ordinate and approximately on the abscissa. Thus it may be concluded that the presence of one substrate on the enzyme has little or no effect on the combination of the other. The similarity of the values for the Michaelis and dissociation constants of the two substrates is shown in Table I I I .

The maximum velocity of the reaction (Table III) is less than one-five hundredth of that obtained with phosphocreatine and this finding is similar to the results of

12..5 12.5 I ¢°) r . / Oh)

~ .~.__ 10"3 o 10-3 . *

-6 -4 -2 0 2 4 6 -3 -2 -1 0 0.4 (raM ~) 15.0 (rnM -1)

pgADP-] ~nosphoolycocya min'~

Fig. I. (a) Effect of phosphoglycocyamine on the initial velocity of the reaction with MgADP- as the variable substrate. The concentrations of phosphoglycocyamine were: A, 15 mM; O, io raM; A, 7-5 raM; O, 6 mM. (b) Effect of MgADP- on the initial velocity of the reaction with phosphoglycocyamine as the variable substrate . The concentrations of MgADP- were: A, o.4 re_M; O, o.2 raM; A, o.133 raM; 0 , o.i raM; D, o.o8 raM; I , o.o67 mM. v is expressed as/~mole~ of glycocyamine per/~g of creatine kinase per mill. The lines were drawn using the constants obtained by analysis of the da ta with the Sequen computer programme.

Biochim. Biophys. Aaa, 128 (1966) 327-336


(o) 25[ (b) 25[

1/" 15

1

-~ - 5 -3 -, o , 3 5 - ; -5 -3 -, o ~ ~ ; 0.4 (raM-') :20.0 (rnM--l)

EVIgADP] ~hosphocreatine]

Fig. 2. Inh ib i t ion of t he reac t ion b y p h o s p h o t a u r o c y a m i n e wi th (a) M g A D P - as t he var iable s u b s ~ a t e a n d phosphocrea t ine he ld c o n s t a n t a t 5 mM and (b) phosphocrea t ine as t he var iab le s u b s t r a t e and M g A D P - held c o n s t a n t a t o . i raM. The concen t ra t ions of p h o s p h o t a u r o c y a m i n e were :A, none; C), 7 raM; A, 14 raM; 0 , 21 mM. v is expressed a s / , m o l e s of creat ine per t*g of creat ine k inase per min .

TANZER AND GILVARG 2. They found that, at the same substrate concentrations, the rate of phosphorylation of creatine is 240 times greater than that of glycocyamine.

Inhibition of the reaction by phosphotaurocyamine and phosphoarginine Neither phosphotaurocyamine nor phosphoarginine were able to function as

substrates for creatine kinase under the conditions used to demonstrate enzymic activity with phosphoglycocyamine. Thus no taurocyamine or arginine was rele- ased in IO min when these phosphagens, at a concentration of 20 mM, were incubated with 43 /*g of enzyme per ml in the presence of 0.8 mM MgADP-. However, interaction with the enzyme did occur since each of the compounds acted as non-competitive inhibitor with respect to MgADP- and as a competitive inhibitor with respect to phosphocreatine (Figs. 2 and 3). The results are, therefore, in accord with Eqns. 5 and 6 as also is the fact that secondary plots of the slopes and vertical

Ca 1 / C b 4C

2C

1C

- 5 -4 - 3 -2 -1 0 1 2 3 , 5 - 5 -~, -3 - 2 -1 0

I

S 0.4 (raM-l) 10.O (raM-q)

EMg ADP3 ~o,pho~t~,e] Fig. 3. Inh ib i t ion of t he reac t ion b y phosphoa rg in ine w i t h (a) M g A D P - as t he var iable s u b s t r a t e and phosphoc rea t ine held c o n s t a n t a t 5 m M mad (b) phosphoc rea t ine as t he var iable s u b s t r a t e and M g A D P - held c o n s t a n t a t o. 4 raM. The concen t r a t ions of phosphoa rg in ine were: A, none ; O, 5 raM; A, io raM; O, 2o raM. v is expressed a s / , m o l e s of creat ine pe r /~g of c rea t ine k inase per min.



T A B L E I

APPARENT AND TRUE DISSOCIATION CONSTANTS FOR THE REACTION OF PHOSPHAGENS WITH CREATINE KINASE

Each apparen t inhibit ion cons tan t for phosphotaurocyamine is the weighted mean of values obta ined by analysis of three sets of data, including t h a t of Fig. 2a, using the Noncomp compute r p rogramme. Those for phosphoarginine were obtained in a similar manner f rom two sets of da ta including tha t of Fig. 3a. The values for the t rue cons tan ts were calculated by means of the relat ionships derived f rom Eqn. 5. Kib and Kb were taken to be 8.6 4- I. 3 and 2.9 + o.3 mM, respectively ~.

Inhibitor Apparent IQ~ Apparent K]g Kt¢ (raM) Kza (mM) (mM) (raM)

Phosphotaurocyamine 23 .0 4- 4.4 14-5 4- 3.5 35.6 4- 4.1 13.1 4- 2.i Phosphoarginine I2.7 4- 1.9 8.o 4- 1.9 36.9 -4- 4.7 I3.5 4- 2.3

(b) 15

(o) 12.5

SIoPoer x 10 IC

Vertical inlercept

Slope x 10 or 10

Vertica~ intercept

-'0 0 I~) L:~

f 3(~ -40 -30 -20 -10 0 -30 -20 ;o A

Phosphotavrocyamine concn.(rnM) Phosphoarg~nlne concn. (mMl

Fig. 4. (a) Secondary plots of the slopes and vertical intercepts of Fig. 2a against the concen- t ra t ion of phospho taurocyamine and (b) secondary plots of the slopes and vertical intercepts of Fig. 3a against the concentrat ion of phosphoarginine. [:], slope; m, vertical intercept.

intercepts of Figs. 2 and 3 against the concentrations of the inhibitory phosphagen are linear (Fig. 4).

The apparent kinetic constants obtained by computer analysis of the non- competitive inhibition data of Figs. 2 and 3 and the true constants determined by calculation are listed in Table I. These indicate that phosphotaurocyamine and phos-

T A B L E I I

COMPARISON OF THE CALCULATED AND EXPERIMENTAL VALUES FOR THE APPARENT COMPETITIVE INHIBITION CONSTANTS OF PHOSPHAGENS

Each value for the apparen t inhibit ion constants is the weighted mean of the values obtained by analysis of two sets of data, including those of Figs. 2b and 3 b, using the Comp compute r programme. The calculated values were determined by means of the relationship derived f rom Eqn. 6 using appropr ia te values for KI~ and Kig (Table I) and a value of o. i 7 4- o.o2 mM for Kt# .

Inhibitor Apparent K~ (raM)

Experimental Calculated

Phosphotaurocyamine 13.2 + I . i 14.o -4- 2.6 Phosphoarginine " 13.3 4- 0. 7 11.2 -Jr- 1. 7


3 3 4 E. JAMES, J. F. MORRISON

phoarginine combine equally well with the enzyme-MgADP complex and while there is no significant difference between the reaction of phosphotaurocyamine with free enzyme and the enzyme-MgADP complex, the presence of MgADP- on the enzyme appears to hinder slightly the reaction of phosphoarginine.

A comparison of the values for the complex inhibition constants as obtained experimentally from the competitive inhibition data of Figs. 2 and 3 with those determined by calculation shows that there is good agreement between the two sets of values (Table II).

A summary of the true kinetic constants associated with the reactions of phosphagens with free enzyme and the enzyme-MgADP complex is set out in Table III.

T A B L E I I I

SUMMARY OF THE VALUES FOR KINETIC CONSTANTS ASSOCIATED WITH THE REACTION OF PHOS- PHAGENS WITH VARIOUS FORMS OF CREATINE KINASE

The cons tan t s for phosphocrea t ine and for M g A D P - in the presence of phosphoc rea t ine are t a k e n f rom ref. 5. Those for phosphog lycocyamine and M g A D P - in the presence of phospho- g lycocyamine are we igh ted m e a n va lues ob t a ined f rom two sets of da ta , inc lud ing t h a t of Fig. i , b y ana lys i s w i t h the Sequen c o m p u t e r p rogramme. The cons tan t s for p h o s p h o t a u r o c y a m i n e and phosphoarg in ine are t a k e n f rom Table I.

Compound Michaelis Dissociation constant Maximum velocity constant (i, moles guanidine

K~ (mM) KI (raM) per ktg of enzyme per rain)

Phosphocrea t ine 2.9 4- 0. 3 8.6 4- 1.3 Phosphog lycocyamine 5-3 4- 0.8 5.6 4- I. 4 P h o s p h o t a u r o c y a m i n e 14. 5 4- 3.5 13.1 -[- 2.1 Phosphoarg in ine 8.o 4- 1.9 13.5 4- 2.3 M g A D P - in the presence of:

Phosphoc rea t i ne o.o 5 4- o .oi o.17 4- o.o2 P h o s p h o g | y c o c y a m i n e o.o9 4- o .o i o . io + o.o2

0.204 4- 0.005 37.1o -'~ 4-4- 2.1o -s

DISCUSSION

In determining the kinetic constants relating to the reaction of phosphoglycocyamine with creatine kinase, it has been assumed that the mechanism of the reaction is still of the rapid equilibrium, random type when phosphocreatine is replaced by phosphoglycocyamine 5. This assumption would appear to be reasonable in view of the very much lower maximum velocity obtained with the latter guanidino substrate and the finding that there are no marked differences in the dissociation constants for the reaction of free enzyme with MgADP- in the presence of phosphocreatine or phosphoglycocyamine. However, it is planned to further investigate this point. The question of mechanism does not arise in connection with the inhibition experiments since it has been established that with phosphocreatine and MgADP- as substrates, the reaction has a rapid equilibrium, random mechanism 5.6. Thus it follows that the kinetic constants determined for the reaction of phosphotaurocyamine and phosphoarginine with free enzyme and the enzyme-MgADP complex are true dissociation constants.

The similarity of the interactions of all four phosphagens with free creatine kinase (Table III) suggests that the phosphorylated guanidino group, which is corn-

Biockim. Biophys. Acta, 128 (1966) 327-336


mon to the four compounds, plays an important role in the binding. I t would also appear that the binding is due in part to the phosphoryl group since the affinity of the enzyme for phosphorylated guanidino compounds is approximately twice that of the corresponding non-phosphorylated derivatives. The differences in the values for the dissociation constants are probably not significant, although possibly phosphotaurocyamine does combine more weakly with free enzyme than the other phosphagens. This effect cannot be due to the length of the carbon chain because the molecule is intermediate in length between phosphoarginine and the other two phosphagens. I t may, however, be related to the presence of a sulphonic rather than a carboxyl group.

There is no significant difference between the abilities of phosphotaurocyamine and phosphoarginine to combine with the enzyme-MgADP complex and of phosphotaurocyamine to combine with the free enzyme. On the other hand, there is possibly a stronger reaction of phosphoarginine with free enzyme than with the enzyme- MgADP complex. When the results obtained with phosphocreatine are also considered, it would seem that the presence of MgADP- on the enzyme can enhance, possibly hinder or have no effect on the reaction of phosphagens with creatine kinase.

The kinetic data indicate that the ternary complexes, enzyme-MgADP- phosphocreatine and enzyme-MgADP-phosphoglycocyamine, can form. But whereas the presence of MgADP- (or phosphocreatine) on the enzyme enhances the combination of phosphocreatine (or MgADP-), similar effects are not obtained with phosphoglycocyamine as substrate. Such results might well mean that the N-methyl group of phosphocreatine is required to cause a conformational change at the active site of the enzyme which is necessary to give rise to the most desirable spatial relationship between enzyme and the substrates 15. Such a conclusion is in accord with the finding that the maximum velocity of the reaction with phosphocreatine is some 550 times greater than with phosphoglycocyamine. Further changes in the structure of phosphocreatine do not hinder the formation of ternary enzyme complexes for they are obtained with both phosphotaurocyamine and phosphoarginine. However, these changes are apparently sufficient so as to preclude the correct positioning of the enzyme and substrate molecules in order for the activation step of the reaction to take place.

The results of these investigations draw attention to the importance of distin- guishing between the specificity of an enzyme with respect to its ability to catalyse an overall reaction and to react with substrate analogues. The data with phosphoglycocyamine provide a good example of a reaction where the relatively low reaction velocity is not related to the interaction of the substrate molecule with the enzyme, but solely to the rate of the activation step.

ACKNOWLEDGEMENTS

The authors are indebted to Mrs. M. LABUTIS for skilled technical assistance. E. JAMES is the holder of a General Motors-Holden's Postgraduate Research Fellow- ship. The work was supported in part by Grant TW-98-o2 from the National Insti tutes of Health, U.S. Public Heal th Service.



R E F E R E N C E S

i A. H. ENNOR, H. ROSENBERG AND M. D. ARMSTRONG, Nature, I75 (1955) i2o. 2 M. L. TANZER AND C. GILVARG, J. Biol. Chem., 234 (1959) 32Ol. 3 S. A. KUBY, L. NODA AND H. A. LARDY, J. Biol. Chem., 21o (1954) 65. 4 A. H. ENNOR AND J. F. MORRISON, Physiol. Rev., 38 (1958) 63I. 5 J- F. MORRISON AND E. JAMES, Biochem. J., 97 (1965) 37- 6 J. F. MORRISON AND W. W. CLELAND, J. Biol. Chem., 241 (1966) 673. 7 F. MARCUS AND J. F. MORRISON, Biochem. J., 92 (I964) 429 • 8 J. F. I~0RRISON, A. H. ENNOR AND D. E. GRIFFITHS, Biochem. J., 68 (1958) 447- 9 M. L. UHR, F. MARCUS AND J. F. MORRISON, J. Biol. Chem., in t he press.

IO }{. ROSENBERG, A. H. ENNOR AND J. F. MORRISON, Biochem. J., 63 (1956) I53. I I J. F. MORRm0N, W. J. O'SULLIVAN AND A. G. OGSTON, Biochim. Biophys. Acta, 52 (196I) 82. 12 W. J. O'SULLIVAN AND D. D. PERRIN, Biochemistry, 3 (1964) 18. 13 W. W. CLELAND, Nature, 198 (1963) 463 . 14 E. JAMES AND J. F. MORRISON, J. Biol. Chem., in t h e press. 15 D. E. KOSHLAND, Proc. Natl. Acad. Sci. U.S., 44 (1958) 98.

BiocMm. Biophys. Aaa, i28 (1966) 327-336

Documents

The reaction of phosphages with ATP: Creatine phosphotransferase