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The initial phase of actomyosin-adenosinetriphosphitase
VOL. 26 (X957)
SINGLE ELECTROPLAX PREPARATION I
I5
xo. The depolarization of the cell produced by the quaternary derivatives is due to an action upon the synaptic region. i i. Blocking and depolarizing action by quaternary compounds cannot be dissociated. Experiments with curare show t h a t they act exclusively upon the synaptic junction. x2. New evidence is offered t h a t the tertiary and quaternary compounds tested compete for the same receptor. REFERENCES x D. NACHMANSOHN, Harvey Lectures, (I953/I954). 2 D. NACX~MANSOH~,in JOHN F. FULTON, A Textbook o] Physiology, I7th Ed., W. B. Saunders Co., Philadelphia, I955, P. I92. s D. NACHMANSOHN, in Ergebnisse der Physiologie, Vol. 48, Springer Verlag, Heidelberg, I955, P. 575. 4 M. ALTAMIRANO, C. W. COAXES, H. GRUNDFEST AND D. NACHMANSOHN, J. Gen. Physiol., 37 (I953) 9 I5 M. ALTAMIRANO, W. L. SCHLEYER, C. W. COATES AND D. NACHMANSOHN, Biochim. Biophys. ACta, I6 (I955) 268. 6 W. L. SCHLEYER, Biochim. Biophys. Acta, I6 (I955) 396. M. ALTAMmANO, J. Cellular Comp. Physiol., 46 (x955) 249. 8 M. ALTAMmANO, Biochim. Biophys. Acta, 2o (x956) 323. 8 E. SCHOFFENmLS, Arch. intern, physiol, et biochem., 63 (I955) 36t. t o E. SCHOFFENmLS, Arch. intern, physiol, et biochem., 63 (I955) 513. n E. SCHOFFENmLS, Arch. intern, physiol, et biochem., 64 (x956) 57 I. 11 E. SCHOFFENmLS AND M. ALTAMmANO, Federation Prec., ~5 (I956) 536. Is T. H. BULLOCK, D. NACHMA~SOHN AND M. A. ROTHENBERG, J. Neurophysiol., 9 (I946) 9x4 M. A. ROTHENBERG. D. B. SeRXNSON AND D. NACHMANSOHN, J. Neurophysiol., II (I948) I I I . x5 D. LUDWIG AND M. C. BARSA, Biol. Bull., t i e (x956) 77" x8 G. E. PALADE, in O. H. GAEBLER, Enzymes: Units o[ Biological Structure and Function, Academic Press, Inc., New York, i956. l~ I. 13. WrLSON AND M. ALTAMmANO,in S. R. KOREY, Neurochemistry, Hoeber-Harper, New York, I956, p. x55. xs I. B. WILSON AND E. CABIn, J. Am. Chem. Soc., 78 (I956) 202.
Received May 2oth, i957
THE
INITIAL
PHASE
OF
ACTOMYOSIN-ADENOSINETRIPHOSPHATASE* Y U J I TONOMURA A~D SHOTARO KITAGAWA
Research Institute/or Catalysis, and Chemistry Department, Faculty o/Science, University o/Hohhaido, Sapporo (Japan)
I t h a s b e e n o b s e r v e d b y A. WEBER AND HASSELBACH 1 a n d KIELLEY AND KALCKAR 2 t h a t t h e r a t e of l i b e r a t i o n of P f r o m t h e a c t o m y o s i n - A T P s y s t e m is, i n e a r l i e r s t a g e s , several times higher than the constant value ultimately attained at a stationary state. A c c o r d i n g t o t h e f o r m e r a u t h o r s , t h i s p h e n o m e n o n is n o t d u e t o t h e a c c u m u l a t i o n of e n d p r o d u c t s n o r t o a fall i n s u b s t r a t e c o n c e n t r a t i o n ; i t a p p e a r s e v e n w h e n f r e s h A T P is a d d e d a f t e r t h e o r i g i n a l s u b s t r a t e h a s b e e n u s e d u p . * The following abbreviations have been used: ATP, adenosine triphosphate; ATPase, adenosinetriphosphatase; ADP, adenosine diphosphate; P, inorganic orthophosphate; EDTA, ethylenedlaminetetra~cetic acid; DNP, z,4-dinitrophenol: PCMB, p-chloromercuribenzoate.
Relerences p. eL
16
Y. TONOMURA, S. KITAGAWA
VOL. 9.6
(x957)
It may be expected that more intensive investigation of this phenomenon will throw some light on the nature of actomyosin-ATPase and also on the process of dephosphorylation of ATP in living muscle. However, it is almost impossible to evaluate quantitative properties of this effect by the usual kinetic procedures, bemuse the phenomenon disappears in a rather short time. Furthermore, as was pointed out by MORALES et al. 3, the effect might also be explainable as a displacement of P left over by the previous reaction, or by the presence of a labile impurity in the ATP preparation. In view of these facts, attempts were made to construct an apparatus with which the rate of the ATPase reaction during an extra-short time could be measured, 82p_ labelled ATP being used to follow the course of the reaction. In this way it was confirmed that the initial, additional burst of P liberation is due to a higher catalytic activity of ATPase in an early stage of the reaction than at the stationary state. Several quantitative aspects of the initial phase of ATPase are also presented.
EXPERIMENTAL
Materials Myosin B ( n a t u r a l a c t o m y o s i n ) w a s p r e p a r e d f r o m r a b b i t skeletal m u s c l e according to s t a n d a r d practice in t h i s laboratory 4. T h e p r o t e i n solution w a s s t o r e d a t I ° C a n d n o t u s e d later t h a n t w o w e e k s a f t e r p r e p a r a t i o n . I n a l m o s t all e x p e r i m e n t s , a S i g m a p r e p a r a t i o n of " c r y s t a l l i n e " A T P ( d i s o d i u m salt) w a s used. I n s o m e e x p e r i m e n t s , t h e A T P u s e d w a s purified on a c o l u m n of D o w e x - I b y t h e u s u a l chloride cycle e l u t i o n 5. T h e d e p h o s p h o r y l a t i o n r a t e s were, however, n o t affected, w i t h i n e x p e r i m e n t a l a c c u r a c y , b y t h e m e t h o d of purification. A T P labelled w i t h 81p w a s p r e p a r e d according to t h e HEMS AND BARTLEY procedureS; A T P , K H s n P O 4 , Mg ++ a n d a - k e t o g l u t a r a t e were i n c u b a t e d w i t h pig h e a r t h o m o g e n a t e for 4 ° m i n . Labelled A T P t h u s p r o d u c e d w a s isolated b y t h e m e t h o d described b y I ~ PAGE7, a n d purified o n a c o l u m n of D o w e x - i b y m e a n s of t h e u s u a l chloride cycle elution. E D T A , D N P a n d P C M B were o b t a i n e d c o m m e r c i a l l y .
A l~paratus and p~'ocedures T h e a p p a r a t u s for t h e m e a s u r e m e n t of t h e r a p i d initial p h a s e of t h e A T P a s e r e a c t i o n (Fig. i) w a s c o n s t r u c t e d according to | h e i n s t r u c t i o n s of Dr. Y. OGURA a n d Mr. T. NAKAMURA of T o k y o U n i v e r s i t y . E i g h t e e n m l of t h e e n z y m e solution, c o n t a i n i n g a n a p p r o p r i a t e a m o u n t of m y o s i n B, o . 6 M KC1, t h e buffer solution a n d a n a c t i v a t o r , were p o u r e d into a conical flask (A). Stirring of t h e solution b y m e a n s of a m a g n e t i c stirrer (B) w a s c o m m e n c e d j u s t before t h e s t a r t of t h e reaction. A t t h e lower e n d s of t w o glass t u b e s (C a n d C'), i .2 c m in d i a m e t e r a n d 26 c m in l e n g t h , celluloid p l a t e s were a t t a c h e d b y silicone grease ( K G X - 5 7 o o * ) ; 2 m l of A T P solution w a s p u t into one t u b e a n d 2 m l of 3o % perchloric acid solution into t h e other. T h e glass t u b e s were fixed so t h a t their lower e n d s were s i t u a t e d j u s t n e a r t h e s u r f a c e of t h e e n z y m e solution. A glass rod (D or D'), o. 7 c m t h i c k a n d 28 c m long, e q u i p p e d w i t h a celluloid b r i m , w a s inserted into each glass t u b e a n d h u n g b y its b r i m on a d u r a l u m i n plate (E or E'). B y m e a n s of e l e c t r o m a g n e t s (F a n d F'), t h e s e plates could be m o v e d h o r i z o n t a l l y a t a k n o w n t i m e interval, t h u s c a u s i n g t h e glass r o d s (D a n d D') to fall, w h i c h resulted in t h e celluloid plates b e c o m i n g d e t a c h e d f r o m t h e glass t u b e s (C a n d C'). A t first A T P solution w a s r~leased into t h e e n z y m e solution b y t h e fall of rod D, t h u s s t a r t i n g t h e reaction; t h e n , after a k n o w n t i m e interval, t h e reaction w a s s t o p p e d b y allowing rod D ' to drop a n d release t h e perchloric acid solution. T e s t s w i t h d y e s h a v e s h o w n t h a t w i t h this a p p a r a t u s m i ~ i n g is n e a r l y c o m p l e t e o.o 5 sec after t h e fall of t h e glass rod. Fig. 2 s h o w s a c o m p l e t e w i r i n g - d i a g r a m of t h e circuit w h i c h r e g u l a t e s t h e t i m e i n t e r v a l b e t w e e n t h e a c t i o n s of t h e t w o e l e c t r o m a g n e t s . B y s w i t c h i n g B on, t h e e l e c t r o m a g n e t s a c t a t a k n o w n t i m e i n t e r v a l b y t h e cooperative f u n c t i o n of a c o n d e n s e r C 4 a n d a relay discharge t u b e 2D21, t h e t i m e interval being d e t e r m i n e d b y t h e c a p a c i t a n c e of C 4 a n d t h e r e s i s t a n c e of R s. T w o sets of four resistances were used, w h i c h fixed reaction t i m e s a t i .4, 2.8, 4.2 a n d 5.6 sec a n d 4.7, 9.4, i I . 7 a n d I4.2 sec respectively. W h e n t h e r e a c t i o n t i m e w a s m o r e t h a n 2o sec, t h e d e p h o s p h o r y l a t i o n r a t e w a s d e t e x m i n ~ i * K i n d l y supplied b y Mr. K. HAYASHX of S h i n - e t s u K a g a k u Co.
Re/erences p. 2I.
VOL. 26 (1957)
INITIAL PHASE OF ACTOMYOSIN-ATPASE
17
by a usual method. Throughout the present study experiments were carried out in 0.6 M KCi and 7 m M Ca ++, and at pH 6. 7 (tris-maleate buffer, o.o 3 Air) and 24-25 ° C, unless otherwise stated. .Inorganic phosphate was isolated from the ~ reaction mixture by the isobutanol extraction proE ~'~-~, ] cedure s and determined according to the MARTIN AND DOTY methods. The radioactivity was measured F" F, by a Riken Model z6 G-M counter. The content of protein was calculated by multiplying by a factor of 6 the nitrogen content determined by the microKjehldahl method.
T
_R,
R2
:o
IIM~ II
v,i C ,. E Fig. I. Apparatus for measuring rapid reactions. A, conical flask; B, agitator driven by a rotating magnet; C and C', glass tubes conraining substrate and perchloric acid solution respectively; D and D', glass rods; E and E', duralumin plates; F and F', electromagnets.
1~
Fig. 2. A circuit for regulating the time interval between the action of two electromagnets. R 1, 3 kQ; R s, i k.Q; R s, to be adjusted; R 4, i o o Q ; Cp 2o/~F; Cs, i o / , F , WV 350; Cs, io/~F, WV 15o; C4, io/~F; V1, 12 F; Vs, VR 135; V~, 2D2I; M t and Ms, electromagnets (R = i k.Q); T, transformer, 25o V, 6o mA.
RESULTS
Initial phase o/adenosinetriphosphatase WEBER AND HASSELBACH1 noted that the rate of P-liberation in the first 15 sec was twice as great as its rate at the stationary state. But, as shown in Fig. 3, the genuine initial rate was found to be about 4-6 times as high as the stationary one. The quantity of the initial, additional burst, i.e., the intercept obtained by extrapolating the linear P-liberation to zero time, was under our standard condition 1.3-1. 9 /~g P/mg protein. Since the weight unit for the ATP binding site is 2- 4. lO 5 4,9,1o the initial burst of P per one ATP binding site becomes 9-25 molecules. The initial burst was increased b y addition of D N P and PCMB, because, as will be described below, these substances enhanced both the initial and the stationary rate of Pliberation. For example, in the presence of 5 m M D N P and 7 m M Ca ++, the burst was about 6.5/~g/mg protein. In other words, when 0.8 m M ATP was added to a reaction medium containing 2.5 mg myosin B/ml and 7 m M Ca ++, the initial burst was found to be about 18% of the terminal P of ATP added, and when the same quantity of ATP was incubated with 1.2 mg myosin B/ml, 7 m M Ca ++ and 5 m M DNP, it came up to even 30% of the terminal P. This fact shows clearly that the initial phase of P-liberation is not due to the presence of a labile impurity in the A T P ' . In one experiment s'P-labelled ATP, of which the radioactivity was diluted with carrier ATP to I,OOO counts/min//~g terminal P, was incubated with myosin B, and the quantity of liberated P was determined b y both MARTIN AND DOTY'S method * BERNHARDla has recently noted that the acid-catalyzed hydrolysis of "c~'stalline" ATP also exhibits an initial, very rapid phase. But, in this case, the initial burst of P-liberation was only about 1-2 % of the terminal P of the added ATP. Re/erences p. 2z.
18
Y. TONOMURA, S. KITAGAWA
VOL. 2 6
(1957)
and by its radioactivity. As indicated in Fig. 4, the rates of P-liberation measured b y these two methods coincided with each other. Therefore, it can be concluded that the initial burst of P originates from the terminal P of ATP and not from P absorbed on the protein TM by the previous reaction, because it has been demonstrated by KOSHLAND, BUDENSTEIN AND KOWALSKY13 that the exchange of KH282PO4 with ATP is not catalyzed by myosin. (x)
(
s,ooo~ ~
°
o~
E o
,x /
3b
~b
,~ec
9b
50
Fig. 3- Initial phase of P-liberation. 0.8 m M ATP, o . 6 M KCI, pH 6.7, 24 ° C. O, 2.5 mg myosin B]ml, 7 m M Ca++; ×, o.6 mg myosin B/ml, io m M EDTA.
These lation point phase
100
~
150
Fig. 4- Rate of liberation of P, determined both by colorimetry (O) and radioactivity (×). o.96 mg myosin B/m], I.I m M 8*P-labelled ATP, o.6M KC1, 9 m M Ca++, p H 6. 7, 24 ° C.
two series of experiments show clearly that the change in the dephosphoryrate is due to a change in the catalytic activity of myosin itself. From a kinetic of view, the transition of ATPase activity from the initial to the stationary can be folanulated in terms of a monomolecular reaction: d [M]s d'----'~ = k [M]i
where [M~s and [M] ~ represent the concentration of myosin B in the stationary and the initial phase respectively*. The full line A in Fig. 3, which was calculated by putting 0.2 and 0.035 tzg P/mg protein/sec for the initial and the stationary activity respectively, and 0.o83 sec -1 for k, showed rather satisfactory agreement with the experimental values.
Dependence o/initial adenosinetriphosphatase activity on substrate concentration, pH and temperature As in the case of the stationary ATPase 4,14, the relation between the reaction velocity (v) of the initial ATPase and the concentration of ATP ([S]) was given by the Michaelis-Menten formula. In Fig. 5 the Michaelis constant in the initial phase was determined according to the LINEWEAVER-BURK procedure 15 and found to be 1.3" Io-4M, which was slightly lower than that (1. 9. Io-4M) in the stationary phase under the same conditions le. In Fig. 6 the activities of the initial and the stationary ATPase as a function of pH are compared. The two activity-pH curves are very similar to each other and * We shall refer to ATPase in these two states as "stationary" and "'initial" ATPase, respectively. Re/erences p. 2I.
VOL. 26 (1957)
INITIAL PHASE OF ACTOMYOSIN-ATPASE
19
both have two optima at pH 6.6 and 9.5. At pH 8.2 the dephosphorylation rate in glycine-KOH buffer was compared with the rate in tris-maleate buffer. The two rates were identical within the experimental error, The temperature coefficient of the rate was measured between 5 and 25 ° C and the activation energies of the initial and the stationary ATPase were calculated to be iI.O and I3.7 kcal, respectively. 1.0
E' (J,
°(~5 >*
/
/
t !
I
,
,
0
10
20
o
~o
6.b
7b
s~
9b p.~ab
V(s) (VM) x 10-3
Fig. 5. LINEWEAVER-BURKplot of reciprocal velocity of initial ATPase (I/V) against reciprocal substrate concentration (I/[S]). o.6M KC1, 7 mM Ca++, pH 6.7, 24° C.
Fig. 6. Rate of initial P-liberation, as a/unction of pH. Reaction time, 2.8 sec. 0.8 mM ATP, o.6M KCI, 7 mM Ca++, 24° C. x, Ixis-maleate buffer; 0, glycine--KOHbuffer. The dotted line indicates the activity-pH curve of the stationary state.
Effect o/modifiers It is well-known that the activity of (acto)myosin-ATPase is remarkably enhanced by EDTA 17-2° in the presence of high concentrations of KC1 and also by DNP2U 2 and PCMB ~3 in the presence of Ca ++. DNP and PCMB increase both the initial and the stationary rates to an almost identical degree. For example, in the presence of 7 m M Ca ++ and at pH 7.4, the initial and the stationary rates were increased 54 and 46% respectively b y the addition of 4 - I o - 5 M PCMB]g myosin B, and the transition of the rate was not changed substantially. On the other hand, EDTA inhibited completely the transition of the dephosphorylation rate, in the presence of io m M EDTA the initial rate agreeing perfectly with the stationary rate (line B in Fig. 3). FRIESS, MORALES AND BOWENz9 have concluded that the activating effect of EDTA is mediated by Mg++ intrinsically bound to the protein and KIELLEY AND BRADLEY23 have suggested that PCMB activates myosin-ATPase b y its combination with SH groups, which are involved in the binding of intrinsic Mg++ to the protein. Accordingly, from the above observations it may be suggested that intrinsic Mg++ is not a necessary factor for the transition of the dephosphorylation rate. On the contrary, the EDTA effect on the transition may preferably be interpreted as resulting from its chelation with "free" Mg++, with which myosin B preparations are contaminated (the concentration of Mg++ present as contamination in our myosin B was about 2.5 moles/Is 5 g protein*C). Re/erences p. 2x.
20
Y. TONOMURA, S. KITAGAWA
VOL. 2 6
(1957)
DISCUSSION
As already described, the estimation of the quantity of the initial, additional burst of P-liberation and the measurement of the rate of the dephosphorylation of 32p. labelled ATP have clearly shown that the initial, very rapid phase of the dephosphorylation is due to a higher ATPase activity in the early phase than in the stationary state. For the gradual decrease of the catalytic activity, any of the three following mechanisms might be possible. The first is a change of the protein induced by formation of the enzyme-substrate complex. If the transition originates in this complex formation, then the line I/V v e r s u s I/[S] of the initial phase must approach asymptotically to the line of the stationary one, as IS] becomes smaller than the Michaelis constant. Such a tendency could, however, not be recognized. The second possibility is the physical change of myosin B induced by ATP; this accompanies changes of viscosity, light scattering, etc., of the solution .5. This is also improbable, because the initial rapid phase can be observed also in myosin-ATPasO and, as stated above, in the presence of I0 m M EDTA and 0.8 m M ATP the transition is completely prevented, in spite of the fact that a nearly maximum change of scattering intensity occurs2°. Thirdly, if the reaction mechanism of ATPase consists of two steps, such as : M + ATP ~ M.ADP + P (I) and M.ADP --+ M + ADP (2) and the second step is much slower than the first one, I mole P per I mole of the ATPase site will be produced rapidly at the initial stage. Such a phenomenon has already been noted in the hydrolysis of p-nitrophenyl ethyl carbonate catalyzed by chymotrypsin 2s, 27. However, this mechanism cannot be of importance for the present case, since, as already mentioned, there are 9-25 times as many moles of P from the initial, additional burst as there are moles of ATP binding site, and, as discussed by WEBER AND HASSELBACH1, the transition of the rate of P-liberation is too slow to be interpreted by such a mechanism. Thus, several possible mechanisms for the transition of the rate of P-liberation have been excluded by the present experiments. It has been suggested that the transition of ATPase activity is induced by splitting ATP at the ATPase site several times and "free" Mg ++ may be a cofactor necessary to the transition. However, the intricate molecular mechanism still remains to be determined. It is highly probable that the remarkable, reversible and very rapid decrease of ATPase activity may be one of the mechanisms regulating the rate of the dephosphorylation of ATP in living muscle, and, as pointed out b y WEBER AND HASSELBACH 1, the difference between living muscle and muscle models m may be attributed to the difference in the states of their ATPase. Therefore, a more advanced study of the initial phase of ATPase is urgently needed. ACKNOWLEDGEMENTS
We should like to express our gratitude to Prof. J. HORIUTI (Research Inst. for Catalysis) and Prof. H. TAMIYA (Tokyo Univ.) for their support and encouragement and to Dr. Y. OGURA (Tokyo Univ.) and Prof. T. KWAN (Research Inst. for Catalysis) for their valuable advice, This study has been aided in part by a grant-in-aid for Fundamental Scientific Research of the Ministry of Education given to the Research Group on the "Chemistry of Muscular Contraction". Re/erences p. 2x.
VOL. 2 6
(1957)
INITIAL P H A S E
2I
OF A C T O M Y O S I N - A T P A S E SUMMARY
i. B y m e a n s of a n a p p a r a t u s c o n s t r u c t e d for t h e m e a s u r e m e n t of r a p i d reactions, A T P a s e a c t i v i t y in t h e first few s e c o n d s (the initial phase) of t h e r e a c t i o n h a s b e e n q u a n t i t a t i v e l y investigated. 2. I n t h e initial s t a g e t h e r a t e of P-liberation w a s a b o u t 5 t i m e s as h i g h as t h e r a t e in t h e stationary state. 3. U n d e r o u r e x p e r i m e n t a l conditions, t h e m a x i m u m a m o u n t o b s e r v e d in t h e .initial, a d ditional b u r s t of P-liberation w a s e v e n e q u i v a l e n t to a b o u t ~/3 of t h e t e r m i n a l P of a d d e d A T P . Therefore, t h i s p h e n o m e n o n c a n n o t be a t t r i b u t e d to a n i m p u r i t y of t h e A T P . 4. F r o m a n e x p e r i m e n t u s i n g ssP-labelled A T P as s u b s t r a t e , it h a s b e e n confirmed t h a t t h e initial b u r s t of P does n o t originate in P b o u n d p r e v i o u s l y to t h e protein. 5 - T h e Michaelis c o n s t a n t of t h e initial A T P a s e is 1.3" I o - ~ M . T h e a c t i v a t i o n e n e r g y is H . o kcal, w h i c h is smaller t h a n t h a t of t h e s t a t i o n a r y A T P a s e , i3. 7 kcal. No difference h a s been o b s e r v e d b e t w e e n t h e p H - d e p e n d e n c y of t h e initial a n d t h e s t a t i o n a r y s t a t e s . 6. E D T A p r e v e n t s t h e t r a n s i t i o n of A T P a s e a c t i v i t y . D N P a n d P C M B e n h a n c e b o t h t h e initial a n d t h e s t a t i o n a r y A T P a s e .
REFERENCES 1 A. W E B E R AND W . HASSELBACH, Biochim. Biophys. Acta, x5 (1954) 237W . KIELLEY AND H. M. KALCKAR, cited in H. M. KALCKAR, Science, H 9 (1954) 479. s M. F. MORALES, J. BOTTS, J. J. B L u ~ AND T. L. HILL, Physiol. Revs., 35 (1955) 475. Y. TONOMURA, S. W A T A N A B E AND K. YAGI, J. Biochem. (Tokyo), 4 ° (I953) 27. s W . E. COHN AND C. E. CARTER, J. Am. Chem. Sot., 72 (x95o) 4273. s R. HEMS AND W . BARTLEY, Biochem, J., 55 (I953) 434. 7 p. A. L~PAGE, Biochemical Preparations, Vol. I, J o h n W H e y a n d Sons, Inc., N e w York, x949. s j . B. MARTIN AND D. M. DorY, Anal. Chem., 2x (I949) 965. 9 W . F. H. M. MOr~MAERTS, J. Gen. Physiol., 39 (I956) 82i. l0 F. MORITA AND Y. TONOMURA, in p r e p a r a t i o n . 11 S. BERNHARD, cited in M. F. MORALES et al., Physiol. Revs., 35 (I955) 475. is F. BUCHTHAL, A. DEUTSCH, G. G. KNAPPEIS AND A. MUNCH-PETERSEN, Acta Physiol. Scan&, 24 (I95 I) 368. xs D. E. KOSHLAND, Jr., Z. BUDENSTEIN AND A. KOWALSKY, J. Biol. Chem., 2II (I954) 279. 1~ L. 0UELL~T, K. J. LAIDLER AND M. F. MORAU~S, Arch. Biochem. Biophys., 39 (I952) 37. 15 H. LXNEWEAVER AND D. BVRK, J. Am. Chem. Soc., 56 (I934) 658. 111T. NIHEI, in p r e p a r a t i o n . 17 E. T. F R m s s , Arch. Biochem. Biophys., 51 (I954) I7. is W . J. BOWEN AND T. D. KERWIN, J. Biol. Chem., 2 I I (I954) 237. x9 E. T. FRmSS, M. F. MORALES AND W . J. BOWEN, Arch. Biochem. Biophys., .53 (I954) 3 T M 20 y. TONOMURA, H. MATSUMIYA AND S. KITAGAWA, Biochim. Biophys. Acta, 24 (I957) 568. 21 G. D. GREVILLE AND D. M. NEEDHAM, Biochim. Biophys. Acta, I6 (I955) 284. ~ J. B. CHAPPELL AND S. V. PERRY, Biochim. Biophys. Acta, x6 (I955) 285. s s W. W . KmLLEY AND L. B. BRADLZY, J. Biol. Chem., 218 (I956) 653. Y. TONOMtrRA, F. MORXTA AND K . YAGL J. Phys. Chem., 6i (I957) 605. H . H. WSBER AND H. PORrZZHL, Advances in Protein Chem., 7 (I952) 16I. ]3. S. HARTL~Y AND B. A. KILBY, Biochem. J., 5 ° (I952) 6x4. s7 B. S. HARTLSY AND B. A. KXLBV, Biochem. J., 56 (x954) 288. 2s H. H. W s v s R AND H. PORTZ~HL, Progress in Biophys. and Biophys. Chem., 4 (1954) 60. z W.
Received
May 8th, 1957
SINGLE ELECTROPLAX PREPARATION I
I5
xo. The depolarization of the cell produced by the quaternary derivatives is due to an action upon the synaptic region. i i. Blocking and depolarizing action by quaternary compounds cannot be dissociated. Experiments with curare show t h a t they act exclusively upon the synaptic junction. x2. New evidence is offered t h a t the tertiary and quaternary compounds tested compete for the same receptor. REFERENCES x D. NACHMANSOHN, Harvey Lectures, (I953/I954). 2 D. NACX~MANSOH~,in JOHN F. FULTON, A Textbook o] Physiology, I7th Ed., W. B. Saunders Co., Philadelphia, I955, P. I92. s D. NACHMANSOHN, in Ergebnisse der Physiologie, Vol. 48, Springer Verlag, Heidelberg, I955, P. 575. 4 M. ALTAMIRANO, C. W. COAXES, H. GRUNDFEST AND D. NACHMANSOHN, J. Gen. Physiol., 37 (I953) 9 I5 M. ALTAMIRANO, W. L. SCHLEYER, C. W. COATES AND D. NACHMANSOHN, Biochim. Biophys. ACta, I6 (I955) 268. 6 W. L. SCHLEYER, Biochim. Biophys. Acta, I6 (I955) 396. M. ALTAMmANO, J. Cellular Comp. Physiol., 46 (x955) 249. 8 M. ALTAMmANO, Biochim. Biophys. Acta, 2o (x956) 323. 8 E. SCHOFFENmLS, Arch. intern, physiol, et biochem., 63 (I955) 36t. t o E. SCHOFFENmLS, Arch. intern, physiol, et biochem., 63 (I955) 513. n E. SCHOFFENmLS, Arch. intern, physiol, et biochem., 64 (x956) 57 I. 11 E. SCHOFFENmLS AND M. ALTAMmANO, Federation Prec., ~5 (I956) 536. Is T. H. BULLOCK, D. NACHMA~SOHN AND M. A. ROTHENBERG, J. Neurophysiol., 9 (I946) 9x4 M. A. ROTHENBERG. D. B. SeRXNSON AND D. NACHMANSOHN, J. Neurophysiol., II (I948) I I I . x5 D. LUDWIG AND M. C. BARSA, Biol. Bull., t i e (x956) 77" x8 G. E. PALADE, in O. H. GAEBLER, Enzymes: Units o[ Biological Structure and Function, Academic Press, Inc., New York, i956. l~ I. 13. WrLSON AND M. ALTAMmANO,in S. R. KOREY, Neurochemistry, Hoeber-Harper, New York, I956, p. x55. xs I. B. WILSON AND E. CABIn, J. Am. Chem. Soc., 78 (I956) 202.
Received May 2oth, i957
THE
INITIAL
PHASE
OF
ACTOMYOSIN-ADENOSINETRIPHOSPHATASE* Y U J I TONOMURA A~D SHOTARO KITAGAWA
Research Institute/or Catalysis, and Chemistry Department, Faculty o/Science, University o/Hohhaido, Sapporo (Japan)
I t h a s b e e n o b s e r v e d b y A. WEBER AND HASSELBACH 1 a n d KIELLEY AND KALCKAR 2 t h a t t h e r a t e of l i b e r a t i o n of P f r o m t h e a c t o m y o s i n - A T P s y s t e m is, i n e a r l i e r s t a g e s , several times higher than the constant value ultimately attained at a stationary state. A c c o r d i n g t o t h e f o r m e r a u t h o r s , t h i s p h e n o m e n o n is n o t d u e t o t h e a c c u m u l a t i o n of e n d p r o d u c t s n o r t o a fall i n s u b s t r a t e c o n c e n t r a t i o n ; i t a p p e a r s e v e n w h e n f r e s h A T P is a d d e d a f t e r t h e o r i g i n a l s u b s t r a t e h a s b e e n u s e d u p . * The following abbreviations have been used: ATP, adenosine triphosphate; ATPase, adenosinetriphosphatase; ADP, adenosine diphosphate; P, inorganic orthophosphate; EDTA, ethylenedlaminetetra~cetic acid; DNP, z,4-dinitrophenol: PCMB, p-chloromercuribenzoate.
Relerences p. eL
16
Y. TONOMURA, S. KITAGAWA
VOL. 9.6
(x957)
It may be expected that more intensive investigation of this phenomenon will throw some light on the nature of actomyosin-ATPase and also on the process of dephosphorylation of ATP in living muscle. However, it is almost impossible to evaluate quantitative properties of this effect by the usual kinetic procedures, bemuse the phenomenon disappears in a rather short time. Furthermore, as was pointed out by MORALES et al. 3, the effect might also be explainable as a displacement of P left over by the previous reaction, or by the presence of a labile impurity in the ATP preparation. In view of these facts, attempts were made to construct an apparatus with which the rate of the ATPase reaction during an extra-short time could be measured, 82p_ labelled ATP being used to follow the course of the reaction. In this way it was confirmed that the initial, additional burst of P liberation is due to a higher catalytic activity of ATPase in an early stage of the reaction than at the stationary state. Several quantitative aspects of the initial phase of ATPase are also presented.
EXPERIMENTAL
Materials Myosin B ( n a t u r a l a c t o m y o s i n ) w a s p r e p a r e d f r o m r a b b i t skeletal m u s c l e according to s t a n d a r d practice in t h i s laboratory 4. T h e p r o t e i n solution w a s s t o r e d a t I ° C a n d n o t u s e d later t h a n t w o w e e k s a f t e r p r e p a r a t i o n . I n a l m o s t all e x p e r i m e n t s , a S i g m a p r e p a r a t i o n of " c r y s t a l l i n e " A T P ( d i s o d i u m salt) w a s used. I n s o m e e x p e r i m e n t s , t h e A T P u s e d w a s purified on a c o l u m n of D o w e x - I b y t h e u s u a l chloride cycle e l u t i o n 5. T h e d e p h o s p h o r y l a t i o n r a t e s were, however, n o t affected, w i t h i n e x p e r i m e n t a l a c c u r a c y , b y t h e m e t h o d of purification. A T P labelled w i t h 81p w a s p r e p a r e d according to t h e HEMS AND BARTLEY procedureS; A T P , K H s n P O 4 , Mg ++ a n d a - k e t o g l u t a r a t e were i n c u b a t e d w i t h pig h e a r t h o m o g e n a t e for 4 ° m i n . Labelled A T P t h u s p r o d u c e d w a s isolated b y t h e m e t h o d described b y I ~ PAGE7, a n d purified o n a c o l u m n of D o w e x - i b y m e a n s of t h e u s u a l chloride cycle elution. E D T A , D N P a n d P C M B were o b t a i n e d c o m m e r c i a l l y .
A l~paratus and p~'ocedures T h e a p p a r a t u s for t h e m e a s u r e m e n t of t h e r a p i d initial p h a s e of t h e A T P a s e r e a c t i o n (Fig. i) w a s c o n s t r u c t e d according to | h e i n s t r u c t i o n s of Dr. Y. OGURA a n d Mr. T. NAKAMURA of T o k y o U n i v e r s i t y . E i g h t e e n m l of t h e e n z y m e solution, c o n t a i n i n g a n a p p r o p r i a t e a m o u n t of m y o s i n B, o . 6 M KC1, t h e buffer solution a n d a n a c t i v a t o r , were p o u r e d into a conical flask (A). Stirring of t h e solution b y m e a n s of a m a g n e t i c stirrer (B) w a s c o m m e n c e d j u s t before t h e s t a r t of t h e reaction. A t t h e lower e n d s of t w o glass t u b e s (C a n d C'), i .2 c m in d i a m e t e r a n d 26 c m in l e n g t h , celluloid p l a t e s were a t t a c h e d b y silicone grease ( K G X - 5 7 o o * ) ; 2 m l of A T P solution w a s p u t into one t u b e a n d 2 m l of 3o % perchloric acid solution into t h e other. T h e glass t u b e s were fixed so t h a t their lower e n d s were s i t u a t e d j u s t n e a r t h e s u r f a c e of t h e e n z y m e solution. A glass rod (D or D'), o. 7 c m t h i c k a n d 28 c m long, e q u i p p e d w i t h a celluloid b r i m , w a s inserted into each glass t u b e a n d h u n g b y its b r i m on a d u r a l u m i n plate (E or E'). B y m e a n s of e l e c t r o m a g n e t s (F a n d F'), t h e s e plates could be m o v e d h o r i z o n t a l l y a t a k n o w n t i m e interval, t h u s c a u s i n g t h e glass r o d s (D a n d D') to fall, w h i c h resulted in t h e celluloid plates b e c o m i n g d e t a c h e d f r o m t h e glass t u b e s (C a n d C'). A t first A T P solution w a s r~leased into t h e e n z y m e solution b y t h e fall of rod D, t h u s s t a r t i n g t h e reaction; t h e n , after a k n o w n t i m e interval, t h e reaction w a s s t o p p e d b y allowing rod D ' to drop a n d release t h e perchloric acid solution. T e s t s w i t h d y e s h a v e s h o w n t h a t w i t h this a p p a r a t u s m i ~ i n g is n e a r l y c o m p l e t e o.o 5 sec after t h e fall of t h e glass rod. Fig. 2 s h o w s a c o m p l e t e w i r i n g - d i a g r a m of t h e circuit w h i c h r e g u l a t e s t h e t i m e i n t e r v a l b e t w e e n t h e a c t i o n s of t h e t w o e l e c t r o m a g n e t s . B y s w i t c h i n g B on, t h e e l e c t r o m a g n e t s a c t a t a k n o w n t i m e i n t e r v a l b y t h e cooperative f u n c t i o n of a c o n d e n s e r C 4 a n d a relay discharge t u b e 2D21, t h e t i m e interval being d e t e r m i n e d b y t h e c a p a c i t a n c e of C 4 a n d t h e r e s i s t a n c e of R s. T w o sets of four resistances were used, w h i c h fixed reaction t i m e s a t i .4, 2.8, 4.2 a n d 5.6 sec a n d 4.7, 9.4, i I . 7 a n d I4.2 sec respectively. W h e n t h e r e a c t i o n t i m e w a s m o r e t h a n 2o sec, t h e d e p h o s p h o r y l a t i o n r a t e w a s d e t e x m i n ~ i * K i n d l y supplied b y Mr. K. HAYASHX of S h i n - e t s u K a g a k u Co.
Re/erences p. 2I.
VOL. 26 (1957)
INITIAL PHASE OF ACTOMYOSIN-ATPASE
17
by a usual method. Throughout the present study experiments were carried out in 0.6 M KCi and 7 m M Ca ++, and at pH 6. 7 (tris-maleate buffer, o.o 3 Air) and 24-25 ° C, unless otherwise stated. .Inorganic phosphate was isolated from the ~ reaction mixture by the isobutanol extraction proE ~'~-~, ] cedure s and determined according to the MARTIN AND DOTY methods. The radioactivity was measured F" F, by a Riken Model z6 G-M counter. The content of protein was calculated by multiplying by a factor of 6 the nitrogen content determined by the microKjehldahl method.
T
_R,
R2
:o
IIM~ II
v,i C ,. E Fig. I. Apparatus for measuring rapid reactions. A, conical flask; B, agitator driven by a rotating magnet; C and C', glass tubes conraining substrate and perchloric acid solution respectively; D and D', glass rods; E and E', duralumin plates; F and F', electromagnets.
1~
Fig. 2. A circuit for regulating the time interval between the action of two electromagnets. R 1, 3 kQ; R s, i k.Q; R s, to be adjusted; R 4, i o o Q ; Cp 2o/~F; Cs, i o / , F , WV 350; Cs, io/~F, WV 15o; C4, io/~F; V1, 12 F; Vs, VR 135; V~, 2D2I; M t and Ms, electromagnets (R = i k.Q); T, transformer, 25o V, 6o mA.
RESULTS
Initial phase o/adenosinetriphosphatase WEBER AND HASSELBACH1 noted that the rate of P-liberation in the first 15 sec was twice as great as its rate at the stationary state. But, as shown in Fig. 3, the genuine initial rate was found to be about 4-6 times as high as the stationary one. The quantity of the initial, additional burst, i.e., the intercept obtained by extrapolating the linear P-liberation to zero time, was under our standard condition 1.3-1. 9 /~g P/mg protein. Since the weight unit for the ATP binding site is 2- 4. lO 5 4,9,1o the initial burst of P per one ATP binding site becomes 9-25 molecules. The initial burst was increased b y addition of D N P and PCMB, because, as will be described below, these substances enhanced both the initial and the stationary rate of Pliberation. For example, in the presence of 5 m M D N P and 7 m M Ca ++, the burst was about 6.5/~g/mg protein. In other words, when 0.8 m M ATP was added to a reaction medium containing 2.5 mg myosin B/ml and 7 m M Ca ++, the initial burst was found to be about 18% of the terminal P of ATP added, and when the same quantity of ATP was incubated with 1.2 mg myosin B/ml, 7 m M Ca ++ and 5 m M DNP, it came up to even 30% of the terminal P. This fact shows clearly that the initial phase of P-liberation is not due to the presence of a labile impurity in the A T P ' . In one experiment s'P-labelled ATP, of which the radioactivity was diluted with carrier ATP to I,OOO counts/min//~g terminal P, was incubated with myosin B, and the quantity of liberated P was determined b y both MARTIN AND DOTY'S method * BERNHARDla has recently noted that the acid-catalyzed hydrolysis of "c~'stalline" ATP also exhibits an initial, very rapid phase. But, in this case, the initial burst of P-liberation was only about 1-2 % of the terminal P of the added ATP. Re/erences p. 2z.
18
Y. TONOMURA, S. KITAGAWA
VOL. 2 6
(1957)
and by its radioactivity. As indicated in Fig. 4, the rates of P-liberation measured b y these two methods coincided with each other. Therefore, it can be concluded that the initial burst of P originates from the terminal P of ATP and not from P absorbed on the protein TM by the previous reaction, because it has been demonstrated by KOSHLAND, BUDENSTEIN AND KOWALSKY13 that the exchange of KH282PO4 with ATP is not catalyzed by myosin. (x)
(
s,ooo~ ~
°
o~
E o
,x /
3b
~b
,~ec
9b
50
Fig. 3- Initial phase of P-liberation. 0.8 m M ATP, o . 6 M KCI, pH 6.7, 24 ° C. O, 2.5 mg myosin B]ml, 7 m M Ca++; ×, o.6 mg myosin B/ml, io m M EDTA.
These lation point phase
100
~
150
Fig. 4- Rate of liberation of P, determined both by colorimetry (O) and radioactivity (×). o.96 mg myosin B/m], I.I m M 8*P-labelled ATP, o.6M KC1, 9 m M Ca++, p H 6. 7, 24 ° C.
two series of experiments show clearly that the change in the dephosphoryrate is due to a change in the catalytic activity of myosin itself. From a kinetic of view, the transition of ATPase activity from the initial to the stationary can be folanulated in terms of a monomolecular reaction: d [M]s d'----'~ = k [M]i
where [M~s and [M] ~ represent the concentration of myosin B in the stationary and the initial phase respectively*. The full line A in Fig. 3, which was calculated by putting 0.2 and 0.035 tzg P/mg protein/sec for the initial and the stationary activity respectively, and 0.o83 sec -1 for k, showed rather satisfactory agreement with the experimental values.
Dependence o/initial adenosinetriphosphatase activity on substrate concentration, pH and temperature As in the case of the stationary ATPase 4,14, the relation between the reaction velocity (v) of the initial ATPase and the concentration of ATP ([S]) was given by the Michaelis-Menten formula. In Fig. 5 the Michaelis constant in the initial phase was determined according to the LINEWEAVER-BURK procedure 15 and found to be 1.3" Io-4M, which was slightly lower than that (1. 9. Io-4M) in the stationary phase under the same conditions le. In Fig. 6 the activities of the initial and the stationary ATPase as a function of pH are compared. The two activity-pH curves are very similar to each other and * We shall refer to ATPase in these two states as "stationary" and "'initial" ATPase, respectively. Re/erences p. 2I.
VOL. 26 (1957)
INITIAL PHASE OF ACTOMYOSIN-ATPASE
19
both have two optima at pH 6.6 and 9.5. At pH 8.2 the dephosphorylation rate in glycine-KOH buffer was compared with the rate in tris-maleate buffer. The two rates were identical within the experimental error, The temperature coefficient of the rate was measured between 5 and 25 ° C and the activation energies of the initial and the stationary ATPase were calculated to be iI.O and I3.7 kcal, respectively. 1.0
E' (J,
°(~5 >*
/
/
t !
I
,
,
0
10
20
o
~o
6.b
7b
s~
9b p.~ab
V(s) (VM) x 10-3
Fig. 5. LINEWEAVER-BURKplot of reciprocal velocity of initial ATPase (I/V) against reciprocal substrate concentration (I/[S]). o.6M KC1, 7 mM Ca++, pH 6.7, 24° C.
Fig. 6. Rate of initial P-liberation, as a/unction of pH. Reaction time, 2.8 sec. 0.8 mM ATP, o.6M KCI, 7 mM Ca++, 24° C. x, Ixis-maleate buffer; 0, glycine--KOHbuffer. The dotted line indicates the activity-pH curve of the stationary state.
Effect o/modifiers It is well-known that the activity of (acto)myosin-ATPase is remarkably enhanced by EDTA 17-2° in the presence of high concentrations of KC1 and also by DNP2U 2 and PCMB ~3 in the presence of Ca ++. DNP and PCMB increase both the initial and the stationary rates to an almost identical degree. For example, in the presence of 7 m M Ca ++ and at pH 7.4, the initial and the stationary rates were increased 54 and 46% respectively b y the addition of 4 - I o - 5 M PCMB]g myosin B, and the transition of the rate was not changed substantially. On the other hand, EDTA inhibited completely the transition of the dephosphorylation rate, in the presence of io m M EDTA the initial rate agreeing perfectly with the stationary rate (line B in Fig. 3). FRIESS, MORALES AND BOWENz9 have concluded that the activating effect of EDTA is mediated by Mg++ intrinsically bound to the protein and KIELLEY AND BRADLEY23 have suggested that PCMB activates myosin-ATPase b y its combination with SH groups, which are involved in the binding of intrinsic Mg++ to the protein. Accordingly, from the above observations it may be suggested that intrinsic Mg++ is not a necessary factor for the transition of the dephosphorylation rate. On the contrary, the EDTA effect on the transition may preferably be interpreted as resulting from its chelation with "free" Mg++, with which myosin B preparations are contaminated (the concentration of Mg++ present as contamination in our myosin B was about 2.5 moles/Is 5 g protein*C). Re/erences p. 2x.
20
Y. TONOMURA, S. KITAGAWA
VOL. 2 6
(1957)
DISCUSSION
As already described, the estimation of the quantity of the initial, additional burst of P-liberation and the measurement of the rate of the dephosphorylation of 32p. labelled ATP have clearly shown that the initial, very rapid phase of the dephosphorylation is due to a higher ATPase activity in the early phase than in the stationary state. For the gradual decrease of the catalytic activity, any of the three following mechanisms might be possible. The first is a change of the protein induced by formation of the enzyme-substrate complex. If the transition originates in this complex formation, then the line I/V v e r s u s I/[S] of the initial phase must approach asymptotically to the line of the stationary one, as IS] becomes smaller than the Michaelis constant. Such a tendency could, however, not be recognized. The second possibility is the physical change of myosin B induced by ATP; this accompanies changes of viscosity, light scattering, etc., of the solution .5. This is also improbable, because the initial rapid phase can be observed also in myosin-ATPasO and, as stated above, in the presence of I0 m M EDTA and 0.8 m M ATP the transition is completely prevented, in spite of the fact that a nearly maximum change of scattering intensity occurs2°. Thirdly, if the reaction mechanism of ATPase consists of two steps, such as : M + ATP ~ M.ADP + P (I) and M.ADP --+ M + ADP (2) and the second step is much slower than the first one, I mole P per I mole of the ATPase site will be produced rapidly at the initial stage. Such a phenomenon has already been noted in the hydrolysis of p-nitrophenyl ethyl carbonate catalyzed by chymotrypsin 2s, 27. However, this mechanism cannot be of importance for the present case, since, as already mentioned, there are 9-25 times as many moles of P from the initial, additional burst as there are moles of ATP binding site, and, as discussed by WEBER AND HASSELBACH1, the transition of the rate of P-liberation is too slow to be interpreted by such a mechanism. Thus, several possible mechanisms for the transition of the rate of P-liberation have been excluded by the present experiments. It has been suggested that the transition of ATPase activity is induced by splitting ATP at the ATPase site several times and "free" Mg ++ may be a cofactor necessary to the transition. However, the intricate molecular mechanism still remains to be determined. It is highly probable that the remarkable, reversible and very rapid decrease of ATPase activity may be one of the mechanisms regulating the rate of the dephosphorylation of ATP in living muscle, and, as pointed out b y WEBER AND HASSELBACH 1, the difference between living muscle and muscle models m may be attributed to the difference in the states of their ATPase. Therefore, a more advanced study of the initial phase of ATPase is urgently needed. ACKNOWLEDGEMENTS
We should like to express our gratitude to Prof. J. HORIUTI (Research Inst. for Catalysis) and Prof. H. TAMIYA (Tokyo Univ.) for their support and encouragement and to Dr. Y. OGURA (Tokyo Univ.) and Prof. T. KWAN (Research Inst. for Catalysis) for their valuable advice, This study has been aided in part by a grant-in-aid for Fundamental Scientific Research of the Ministry of Education given to the Research Group on the "Chemistry of Muscular Contraction". Re/erences p. 2x.
VOL. 2 6
(1957)
INITIAL P H A S E
2I
OF A C T O M Y O S I N - A T P A S E SUMMARY
i. B y m e a n s of a n a p p a r a t u s c o n s t r u c t e d for t h e m e a s u r e m e n t of r a p i d reactions, A T P a s e a c t i v i t y in t h e first few s e c o n d s (the initial phase) of t h e r e a c t i o n h a s b e e n q u a n t i t a t i v e l y investigated. 2. I n t h e initial s t a g e t h e r a t e of P-liberation w a s a b o u t 5 t i m e s as h i g h as t h e r a t e in t h e stationary state. 3. U n d e r o u r e x p e r i m e n t a l conditions, t h e m a x i m u m a m o u n t o b s e r v e d in t h e .initial, a d ditional b u r s t of P-liberation w a s e v e n e q u i v a l e n t to a b o u t ~/3 of t h e t e r m i n a l P of a d d e d A T P . Therefore, t h i s p h e n o m e n o n c a n n o t be a t t r i b u t e d to a n i m p u r i t y of t h e A T P . 4. F r o m a n e x p e r i m e n t u s i n g ssP-labelled A T P as s u b s t r a t e , it h a s b e e n confirmed t h a t t h e initial b u r s t of P does n o t originate in P b o u n d p r e v i o u s l y to t h e protein. 5 - T h e Michaelis c o n s t a n t of t h e initial A T P a s e is 1.3" I o - ~ M . T h e a c t i v a t i o n e n e r g y is H . o kcal, w h i c h is smaller t h a n t h a t of t h e s t a t i o n a r y A T P a s e , i3. 7 kcal. No difference h a s been o b s e r v e d b e t w e e n t h e p H - d e p e n d e n c y of t h e initial a n d t h e s t a t i o n a r y s t a t e s . 6. E D T A p r e v e n t s t h e t r a n s i t i o n of A T P a s e a c t i v i t y . D N P a n d P C M B e n h a n c e b o t h t h e initial a n d t h e s t a t i o n a r y A T P a s e .
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Received
May 8th, 1957