Kinetic study of the pre-steady state formation and the decay of the heavy meromyosin-adenosine triphosphate complex

Kinetic study of the pre-steady state formation and the decay of the heavy meromyosin-adenosine triphosphate complex

Biochimiea et Biophysica Acta, 328 (1973) 481-49 ° © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - P r i n t e d in T h ...

546KB Sizes 2 Downloads 59 Views

Biochimiea et Biophysica Acta, 328 (1973) 481-49 ° © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - P r i n t e d in T h e N e t h e r l a n d s

BBA

36572

K I N E T I C STUDY OF T H E P R E - S T E A D Y STATE FORMATION AND T H E DECAY OF T H E H E A V Y M E R O M Y O S I N - A D E N O S I N E T R I P H O S P H A T E COMPLEX

L O U I S H. S C H L I S E L F E L D

AND G E O R G E J. K A L D O R

Department of Contractile Proteins, Institute for Muscle Disease, Inc., 515 East 7rst St., New York, N.Y., and Department of Physiology, Medical College of Pennsylvania, Philadelphia, Pa. (U.S.A .) (Received M a y 28th, 1973)

SUMMARY

In the presence of Mg 2+ heavy meromyosin reacts with radioactive ATP to form a complex that can be prepared by precipitation with (NH4)2SO 4. This complex appears to be heavy m e r o m y o s i n . A D P . P i . The pre-steady state formation of this complex is a second order reaction with an average rate constant of 3.1' lO 6 M -1" s -1 in o.o2 M Tris-HC1 buffer and o.Io M NaC1 at p H 7.4 and room temperature. Analysis of the data suggests i complex site per molecule of heavy meromyosin, although this protein has 2 ATP binding sites per molecule (Schliselfeld, L. H. and B~rdmy, M. (1968) , Biochemistry 7, 32o6-3213). The decay of this complex is a first order reaction. The rate constant for m a x i m u m velocity of ATP hydrolysis is greater than the complex decay rate constant. Therefore both ATP binding sites must catalyze the hydrolysis of ATP. In 0.02 M Tris-HC1 buffer and o.oi M NaC1 at p H 7.4 and 25 °C the rate constants are o.047 s 1 for the complex site and 0.049 s -~ for the second ATP binding site. Increasing the NaC1 concentration to 1.5o M decreases both constants but to different extents, 0.0054 s -~ for the complex site and O.Ol7 s -1 for the second ATP binding site.

INTRODUCTION

A complex of myosin (EC 3.6.1.3) and ATP has been prepared by precipitation with (NH4)2SO4 of the protein from a solution of Mg 2+ and ATP (ref. I). This procedure gave identical complexes of ATP with heavy meromyosin and subfragment I (EC 3.6.1.3, ATP phosphohydrolase) l& The formation of the m y o s i n - A T P complex required Mg 2+ and native myosin. Concentrations of p-chloromercuribenzoate that inhibited the ATPase activity also inhibited the complex formation. The Km values for complex formation with myosin and actomyosin were similar to their Km values for ATP hydrolysis. When the complex of myosin prepared with [?-s~P]ATP was dissolved in IO M urea or washed in a high ionic strength buffer (pH 4.5) to denature the protein, all of the radioactivity was found in Pi. This showed that the complex

482

L.H.

S C H L I S E L F E L D , G.

j.

KALDOR

actually consisted of myosin and hydrolyzed ATP (myosin.ADP.Pi), where the ADP and Pi were bound by non-covalent means. This m y o s i n . A D P ' P i complex decayed as a first order reaction with a rate constant similar to the rate constant calculated from the ATPase activity. These findings led to the conclusion that this complex was an intermediate in the hydrolysis of ATP. The rate limiting step is the decay of the complex. Recently several laboratories have reported on the pre-steady state hydrolysis of ATP by myosin, heavy meromyosin, and Subfragment I (refs 3-9). This report will show that the pre-steady state formation of heavy meromyosin. ADP. Pi complex is a second order reaction with a rate constant similar to the value reported for A T P hydrolysis. MATERIALS

The following compounds were purchased: DEAE-cellulose (Whatman DE-52) from Reeve Angel; A T P from P-L Biochemicals; ESJ4CIATP from Schwartz BioResearch; Cleland's reagent from Calbiochem; twice crystallized salt-free trypsin and crystalline soybean trypsin inhibitor from Worthington Biochemical Corp. ; and 32Pi from New England Nuclear. The Ey-~2PIATP was synthesized b y the ~2Pi ATP exchange procedurO°, n. Rabbit skeletal muscle myosin was prepared by the procedure of B~r~iny and Oppenheimer 12. H e a v y meromyosin was prepared by the digestion of about 1.2 g myosin with trypsin 13, and its subsequent chromatography x4. Trypsin was added to a solution of myosin (myosin to trypsin weight ratio of IOO:I) in o.5o M K C l a n d o.o5 M KPO42- at room temperature and pH 6.2. After IO min 2 mg soybean trypsin inhibitor per mg trypsin was added to stop the digestion. The digest was dialysed at 5 °C overnight against 4 l of o.oi M KC1 and one change of this dialysate. The precipitate was collected by centrifuging at 27 ooo × g for IO rain, and it was washed once with 2o-4o ml of o.oi M KC1. The two supernatants were combined and sufficient o.4o M Tris-HC1 buffer (pH 8.o) was added to give o.o5 M Tris-HC1 buffer. The protein was placed on a 2.4 cm × 4 ° cm column of DEAE-cellulose equilibrated at 5 °C in o.o5 M Tris-HC1 buffer (pH 7.9). The column was washed with 250 ml of the o.o5 M Tris-HC1 buffer. Then a linear gradient of i.o 1 of o.o5o M Tris-HC1 buffer (pH 7.9) and I.O 1 of a solution containing o.o5 ° M Tris-HC1 buffer and o.5o M KC1 (pH 7-9) was started. As described by Lowey et al. '4 this procedure yields two ATPase activity peaks. The first peak is small and consists of Subfragment I. The second peak contains most of the ATPase activity and 48°,; of the protein placed on the column. The fractions for this peak were combined and solid (NH4)~SO a was added slowly with mixing at 5 °C to give 65 }g satn. I h or more after adding the (NH4)2S()4, the protein precipitate was collected by centrifugation at 27 ooo ,'< g for 15 rain. The precipitate was dissolved in 2o-3o ml of o.o5o M Tris-HC1 buffer (pH 7.9) and dialyzed overnight against 4 1 of o.oi M KC1. The dialyzed protein solution was then centrifuged at lO5 ooo × g for 2 11 to remove a fine precipitate. Then Cleland's reagent was added to o.oo5 M. This procedure yielded 250-35o mg of purified heavy meromyosin.

HEAVY MEROMYOSIN-ATPCOMPLEX

483

METHODS

Pre-steady state apparatus and procedure The interaction of [y-32pIATP with heavy meromyosin was followed in a Durrum multi-mixing system, Model D-I33. This apparatus is similar to the apparatus described by Lymn and Taylor 4. It contains three motor driven syringes. Syringe I contains the protein-buffer solution, and Syringe 2 contains an equal volume of EV-32PIATP buffer solution. Syringe 3 (with a volume equal to the sum of the volumes in Syringes I and 2) contains cold neutral saturated (NH4)2SO 4 solution. The motor caused the contents of Syringes I and 2 to mix and flow through a narrow tube of known length at a pre-determined speed. Upon leaving this tube the reaction solution was mixed with the (NH4)~SO 4 solution from Syringe 3 to stop the reaction. The first o.5-1.o ml of this mixture was discarded into a waste container, the remaining mixture was collected in a 5o-ml polycarbonate centrifuge tube. The reactions were performed at room temperature (20-25 °C) in a buffer consisting of 0.020 M Tris-HC1 buffer, o.oolo M MgSOa, and o.Ioo M NaC1 at pH 7.4. Immediately after collecting the stopped reaction mixture, cold neutral saturated (NH4)2SO * solution was added to raise the saturation level from 50 to 670/0 . After 30-60 rain at o °C the protein precipitate was centrifuged down. Each precipitate was then washed twice with 15 ml per tube of neutral 67% saturated (NH4)2SOa, 0.67 mM ATP, and 2. 7 mM KPO42-. Next each precipitate was washed once with 15 ml per tube of cold neutral 67% satd (NH4)2SO 4 solution. All centrifugations were carried out at 12 ooo × g for IO rain and the supernatants were discarded. Each precipitate was dissolved in 5.0 ml of o.io M NaC1; o.5o-ml samples were counted for radioactivity and the solution was assayed for protein.

Decay of heavy meromyosin. A DP. Pi complex The decay of the heavy m e r o m y o s i n . A D P . P i complex was followed as described previously 2. Heavy meromyosin was incubated with radioactive ATP at 25 °C for 15-3o s to form the complex. Then a 2oo-fold excess of non-radioactive ATP was added to prevent the binding of anymore radioactive ATP. The complex remaining as a function of time was precipitated by the very rapid addition of 2 vol. of neutral saturated (NH4)2SO 4 solution. After 15 min at o °C the mixtures were centrifuged at 12 ooo × g for IO min to remove the supernatant. Each precipitate was then washed twice with 15 ml of neutral 67% satd (NH4)2SO, solution containing 0.67 mM ATP and 2.7 mM KPO42-. Then each precipitate was washed once with 15 ml of neutral 67 % satd (NH4)2SO 4 solution. Each wash solution was removed after centrifugation at 12 ooo × g for IO min. The washed precipitates were then each dissolved in o.Io M NaC1. These solutions were counted for radioactivity and assayed for protein.

Specific activity determination A 7.7o-ml solution of ATP was pre-incubated at 25 °C for 5 min. Then o.3o ml of heavy meromyosin solution, 15.1 mg/ml, was added to give a final reaction mixture of 0.o2o M Tris-HC1 buffer, I.OO mM MgSO4, o.Ioo M or 1.5o M NaC1, 0.566 mg/ml heavy meromyosin, and i.o mM ATP at pH 7.4. After 2O-lOO rain at 25 °C the

484

L. H . S C H L I S E L F E L D ,

G. J . K A L D O R

reactions were stopped by the addition of i.o ml 5.o M HC10 a. The resulting precipitates were centrifuged down in a clinical centrifuge, and the supernatants were poured into clean tubes. Zero-times samples were prepared by adding the 5.0 M HC104 prior to the addition of heavy meromyosin, and this was clarified like the reaction solutions. Then 0.5o ml 5}/o ammonium molybdate and o.5o ml I-anfino-2naphthol sulfonic acid reagent of Fiske and SubaRow ~5 was added. After io rain at room temperature the solutions were read in the Klett Summerson photoelectric colorimeter.

Other methods of analysis Protein was determined by precipitation in 5°/~ trichloroacetic acid, and assaying the precipitate for biuret protein ~6 as described by B~r~ny and B~r~ny 17. Radioactivity was determined by scintillation counting in I5 ml of the dioxanebased fluid as described by Kemp and Krebs is. All counting was performed for a sufficient time so that twice the standard error of the total counts was equal to or less than 3.3% of the total counts. The molecular weight for heavy meromyosin employed is 350 ooo daltons. RESULTS

Fig. IA shows the time course of the incorporation of radioactive ATP into the protein. At constant heavy meromyosin concentration r, the nmoles of [)/-32p]ATP per mg heavy meromyosin, increases with time until it reaches a plateau. Fig. IB shows that at constant heavy meromyosin concentration and constant reaction time the value of r increases with [ATP] until it reaches the same plateau. If the pre-steady state formation of the complex is a second order re?.ction then it should obey Eqn I: --log(/2 -- C)/E

--

(.4

--

E)kt/2.3o

-

log(// -- C)/~4

(l)

E is the initial heavy meromyosin reactive site concentration, A is the initial ATP concentration, C is the concentration of complex at reaction time l, and k is the second order rate constant. When the reaction time is short and A is large so that the fraction of substrate consumed is very small, then the term --log(A--C)/A will be small enough to ignore. At constant heavy meromyosin concentration r is proportional to C. Substituting r for C and rmax for E in Eqn i yields Eqn 2: --log(rmax

r)/rma×

--

(A

--

E)kt/2.3

o =

h't/2.3o

The pseudo-first order rate constant k' is given by Eqn 3 : k' = kA -- kE

(3)

Fig. 2 shows first order plots for the data in Fig. IA and of a second time study. Both reactions follow first order kinetics. However, both studies intersect tile time axis at --13.2 ms instead of at the origin. The dead time for the apparatus used, which is 4 ms, is too short to explain tile negative time value. This is interpreted to mean that I3.2 ms are required to raise the (NH4)2SO 4 concentration to a level sufficient

HEAVY M E R O M Y O S I N - A T P COMPLEX

485

.J i A

a8

o.6 0.4

0.2

20 ' i o ; o ' 80 IO0 ' REACTION TIME (ms)

120 '

:~ O.8

Eo6 -~0.4

,. 0.2 i. 50

I, I I tO0 15,0 20.0 [ATP] (~M)

I 25,0

Fig. I. Pre-steady state formation of heavy meromyosin-ATP complex. A. Formation of complex with increasing reaction time. The reaction time is the time spent in the narrow tube before addition of neutral saturated (NH4)2SO , solution. Heavy meromyosin concn, 3.3/zM; initial ATP concn, 5.2/zM. B. Formation of complex at a constant reaction time of 9.2 ms before addition of (NH4),SO 4 solution with increasing initial ATP concentration. Heavy meromyosin concn, 3.3/~M.

o5l 0.4

-20

Q

20

40 60 80 tO0 REACTION TIME (ms)

120

Fig. 2, First order plot of pre-steady state complex formation. The reaction time employed is the value calculated for the reaction before addition of (NH4),SO 4 solution 0, replot of points in Fig. IA; heavy meromyosin concn, 3.3/~M and initial ATP concn, 5.2/~M; (2), heavy meromyosin concn, 7.o/,M and initial ATP concn, lO. 4 IbM.

t o s t o p t h e r e a c t i o n . T h e r e f o r e w h e n c a l c u l a t i n g k' t h e t r u e r e a c t i o n t i m e is e q u a l to t h e s u m o f t h e a p p a r e n t r e a c t i o n t i m e plus 13.2 ms. Fig. 3 p r e s e n t s p l o t s of k' versus i n i t i a l [ A T P ] w i t h t h r e e d i f f e r e n t p r e p a r a t i o n s of h e a v y m e r o m y o s i n . W h i l e e a c h s t u d y has s o m e r a n d o m s c a t t e r of p o i n t s , satisf a c t o r y s t r a i g h t lines a r e o b t a i n e d . F r o m E q n 3 it is seen t h a t t h e slope of e a c h line

486

L. H, SCHLISELFELD, G. J. KALDOR

/

80

S

x

60

A

T

-~ 4 0

20

I

10

20

30

40

50

ZATP] ("M) Fig. 3. Effect of i n i t i a l A T P c o n c e n t r a t i o n on t h e p r e - s t e a d y s t a t e first orde r r a t e c o n s t a n t s . All r e a c t i o n s c o n t a i n e d o.o2 M T r i s - H C l buffer, o.ooi M MgSO4, a n d o. i o o M NaC1 a t p H 7.4. The r e a c t i o n s were c a r r i e d o u t a t r o o m t e m p e r a t u r e w h i c h v a r i e d from 20-25 °C. H e a v y m e r o m y o s i n c o n c e n t r a t i o n is as follows: 3.3/~M (0), 7.81~M ( × ) a n d 7.7/~M ( ~ ) .

represents the second order rate constant and the abscissa intercept represents the reactive site concentration. Table I summarizes these results plus the results of a fourth study not shown in Fig. 3. The second order rate constant varied from o.96.IO6 ~z o.36.1o 8 to 6 . 7 2 . 1 o 6 ± o.29.1o 6 M-l.s -1 with an arithmetic average of 3.o 9. Io 6 M -1.s -~. This 7-fold variation in the rate constants may be due to the differences in the room temperature for each study and to experimental error. Lynm and Taylor 5 have reported that the pre-steady state hydrolysis of ATP catalyzed by TABLE I

SUMMARYOFPARAMETERS OFPRE-STEADYSTATEREACTION Heavy meromyosin concn (aM)

A T P conch range* (t*M)

k :k S.E. ( × , 0 -6) ** ( M -1 s -1)

j E l l [ p r o t e i n ] ratio (mole~mole)

3-3 7 .8 7.7 5.6

5.2-I5.4 lO-O-25.o io.o 25.0 lO. 4 3I.O

6.72 2.38 2.30 0.96

0.94 0.86 t.oo 1.o3

~_ i ± ±

0.29 0.46 0.25 0.36

A v e r a g e 3.09

0.96

* At A T P c o n c e n t r a t i o n s of 5 °/~M or g r e a t e r t h e m a x i m u m c o m p l e x v a l u e w a s a l w a y s obtained. ** Second ord er r a t e c o n s t a n t -4 S.E.

487

HEAVY M E R O M Y O S l N - A T P COMPLEX

heavy meromyosin in the presence of Mg 2+ at 2o °C in o.o5 M KC1 buffered at p H 8.o is 2.4" lO 6 M -1" s -1. This is in reasonable agreement with the rate constant obtained for complex formation. Finally, only one of the 2 ATP binding sites per protein molecule 1,6,19-22 appears to be involved in the complex formation since EEl/[heavy meromyosinl ranges from o.86 to 1.o 3 with an arithmetic average of o.96. Since the error involved in making the necessary extrapolations to determine the abscissa intercept is unknown, the determination of I complex site per molecule must be taken with caution.

Decay of heavy meromyosin.ADP.P, complex I t was previously shown that the addition of an excess of non-radioactive ATP to a pre-incubated solution of myosin and radioactive ATP caused a first order loss of radioactivity in the complex 2. Fig. 4 presents similar studies with heavy meromyosin. Both CS-14CIATP and [~-s2pIATP yields complexes which have first order decay kinetics. Table I I summarizes the ATPase specific activities, the complex decay constants calculated from the slopes in Fig. 4, and the differences between each ATPase rate constant and the decay constant determined under identical conditions. The ATP concentrations employed to determine the ATPase activities and the complex decay constants are over I5oo-fold greater than the Km values reported

OD -0.2

-0.8

'°'f \\ - 1.0

e~

I

L

I

t

L

i

I

DECAY TIME (3)

Fig. 4. Decay of the complex of h e a v y m e r o m y o s i n a n d radioactive ATP. (D, i n c u b a t e d 5.3 mg h e a v y m e r o m y o s i n with o.o52/,mole [y-3*PIATP in o . I o M NaC1 at 25 °C for 15 s. T h e n added i o . o / , m o l e s non-radioactive A T P to block the binding of a n y m o r e [y-32PlATP. This solution contained 5.o ml of o.o2o M Tris-HC1 buffer, o . o o l o M MgSO4, o . i o M NaC1, i.o6 mg h e a v y meromyosin]ml, and 2.o mM A T P at p H 7.4. At v a r y i n g time intervals rapidly added io ml of n e u t r a l s a t u r a t e d (NH4)2SO ~ solution. Zero time samples were p r e p a r e d b y adding the (NH,)2SO 4 solution before adding the I o / , m o l e s non-radioactive ATP. The resulting precipitates were collected, washed, a n d assayed as described u n d e r Methods. 0 , i n c u b a t e d 6.95 mg h e a v y m e r o m y o s i n w i t h o.o52/,mole [8-1*C]ATP for 3 ° s in o . I o M NaC1 at 25 °C. T h e n added IO.O /,moles of non-radioactive ATP. This gave 5.oo ml containing o.o2o M Tris-HC1 buffer, o . o o l o M MgSOa, o . I o M NaC1, 1.39 m g h e a v y m e r o m y o s i n / m l , and 2.o mM A T P at p H 7-4. Followed the disappearance of radioactivity in the 67% satd (NH4)2SO 4 precipitated proteins as described above. [], i n c u b a t e d 3.78 m g h e a v y m e r o m y o s i n w i t h o.o2o 4 / , m o l e r_y-3*P~ATP for 15 s in 1.5o M NaC1 at 25 °C. T h e n added 5,o/,moles non-radioactive ATP. E a c h reaction consisted of 5.oo ml of o.o2o M Tris-HC1 buffer, o . o o l o M MgSO 4, 1.5o M NaC1, o.756 m g h e a v y m e r o m y o s i n / m l , and I.O mM A T P at p H 7.4. Followed the disappearance of radioactivity f r o m the 67% satd (NH4)2SO * precipitated protein as described above.

488

L. H. S C H L I S E L F E L D ,

TABLE Ii COMPARISON OF KINETIC CONSTANTS FOR COMPLEX DECAY AND A T P

NaCl conch (M )

Spec. act. *

o. I o o. I o 1.5°

I6. 5 :> 0. 3 16. 5 ± 0. 3 3.7 :t_ 0 . 0 4 * *" "*" * ** tt*

Radioactive'* subslrate

[8-*aC]ATP IT-a2P]ATP IT-a2p!ATP

G. J . K A L D O R

HYDROLYSIS

Rate conslants (s -1) .A ",l'Pase*"

Complex decay*

A T Pase complex deccty

0.096 ± 0.002 o . o 9 6 ~: o . 0 0 2 0 . 0 2 2 ~2 0 . 0 0 0 2

0.0432 -- o.ooi5'* o.o511 ± o.o37*t 0 . 0 0 5 4 - - 0.0003***

0.053 o.o45 O.Ol 7

T h e n m o l e s P i f o r m e d ' m i n - l " m g -1 in i . o m M A T P a t 25 °C ± S . E . Substrate used to prepare the radioactive complex. R a t e c o n s t a n t = s p e c . a c t . , 3 5 o o o o / 6 o - i o ~ :k S . E . R a t e c o n s t a n t s f o r c o m p l e x d e c a y a r e c a l c u l a t e d f r o m s l o p e s o f l i n e s in F i g . 4 7L S . E . D e c a y o f r a d i o a c t i v e c o m p l e x f o l l o w e d i n 2.0 m M A T P . Decay of radioactive complex followed in i.o mM ATP.

for myosin and heavy meromyosint, 4. Therefore the ATP concentrations are saturating for both studies summarized in Table II. The decay constants for heavy meromyosin. [14CIADP. Pi and heavy meromyosin-ADP. !a2PtPi complexes are identical with values of o.o43 and o.o51 s -t, respectively, for an average of o.o47 s -t in o.io M NaC1. This indicates that the complex decay concentration in o.Io M NaC1 is half of the ATPase rate constant. Since heavy meromyosin has only 2 ATP binding sites per molecule t,G,~9-22 the ATPase rate constant must be equal to the sum of the rate limiting constants at the two sites. The decay constant of the heavy meromyosin complex represents the rate constant for the complex site. The rate constant for the second site on the protein must therefore be equal to the ATPase rate constant minus the complex decay constant. In o.Io M NaC1 the rate constants for the second site with 18-14C?ATP and [),-a~PIATP are o.o53 and o.o45 s -t, respectively, with an average value of o.o49 s -1. Increasing the salt concentration to 1.5o M NaC1 decreases both rate constants but to different extents. The complex site has a rate constant, o.oo54 s -1, that is 32% of the rate constant for the second site, O.Ol7 s -1. DISCUSSION

Myosin and heavy meromyosin in the presence of Mg 2+ form a complex with radioactive ATP that can be isolated by precipitation with (NH4)~S04. Previous studies have shown the complex with myosin to be m y o s i n . A D P . P i (ref. I). The very rapid addition of (NH~)2SO4 to precipitate the complex was previously shown to cause a partial denaturation of the protein 'a. Denatured myosin does not retain its bound nucleotide. Correction for the myosin denatured by the rapid addition of (NH4)2SO 4 gave I complex site per molecule. The properties of this complex are those expected for an intermediate in the hydrolysis of ATP. Lymn and Taylor 4 and Trentham et al. 9 have proposed the formation of a myosin" ADP" Pi complex as an intermediate in the hydrolysis of ATP. The present studies demonstrate that the pre-steady state formation of the complex is a second order reaction with an average rate constant of 3.I" lOGMq " s '1 in 0.I0 M NaC1 at ,oom temperature. This value is similar to the rate constant of

HEAVY M E R O M Y O S I N - A T P COMPLEX

489

2. 4. lO 6 M -1. s -1 for the heavy meromyosin catalyzed hydrolysis of ATP (ref. 5). All of the plots of k' versus initial EATP] yield positive values for the abscissa intercept which represents the reactive complex site concentration at zero time. The ratio of EE]/Eheavy meromyosin] varied from 0.86 to 1.o3 moles per mole protein. This suggests that only one of the 2 ATP binding sites per molecule of heavy meromyosinl,6,19-22 is involved in the formation of this complex. This is supported by earlier analytical results which gave I complex site per molecule of myosin 1. In these studies the concentrations of ATP and of heavy meromyosin used are greater than the Km values reported for myosin and heavy meromyosin of 0.03 #M (ref. 4) and 0. 4 #M (ref. I). Therefore the ATP binding sites are completely filled with nucleotide when the steady state condition is reached. It is clear that more studies are necessary to distinguish conclusively between I and 2 complex sites per protein molecule. Extrapolation of the steady state hydrolysis of ATP catalyzed by myosin or heavy meromyosin to zero time yields a positive ordinate intercept. This is termed the initial burst value. Myosin has been reported to have initial burst values of I mole Pi formed per mole protein23, 24 and 1.8 moles Pi formed per mole protein 4. Inone et alY and L y m n and Taylor 5 have independently reported initial burst values for heavy meromyosin of I mole Pt formed per mole protein. The similarity of the initial bulst values and the complex site values suggests that these two properties belong to the same ATP binding site. One possible explanation for the similar initial burst values and complex site values would be that only one of the 2 ATP binding sites can hydrolyze ATP. It has been shown that the complex of myosin and ATP consists of m y o s i n . A D P . P i (ref. I)*. Therefore the formation and decay of this complex must involve the hydrolysis of ATP by at least one ATP binding site on the protein molecule. Since the ATPase rate constant is greater than the complex decay rate constant under identical conditions (see Table II) both ATP binding sites on the protein must hydrolyze ATP. A second possibility is that there is some difference in the catalytic mechanism at the 2 ATP binding sites. This is suggested b y the difference between the values of the rate constants for the 2 ATP binding sites in 1.50 M NaC1, although no such difference is found using 0.I0 M NaC1. Tokiwa and Tonomura 25 have suggested that the 2 ATP binding sites have a significant catalytic difference. The results of this study lend support to this hypothesis. ACKNOWLEDGMENTS

The authors wish to thank Dr Michael B/trAny for m a n y helpful discussions and suggestions. This work was supported by the Muscular Dystrophy Associations of America and Canada, and b y N.I.H. Grants NB o6517 and H D 06267. REFERENCES I Schliselfeld, L. H. and B~.r£ny, M. (1968) Biochemistry 7, 32°6-3213 2 Schliselfeld, L. H., Conover, T. E. and B~.r~my, M. (197o) Biochemistry 9, 1133-1139 3 Finlayson, B. and Taylor, E. W. (1969) Biochemistry 8, 8o2-81o * Recent studies with ethanol extraction of the complexes of heavy meromyosin with [7-82P]ATP and [8-14C]ATP yield [a~P]Pl and [14C]ADP; no radioactive ATP is found.

490 4 5 6 7 8 9 io Ii 12 13 14 15 16 17 18 19 20 21 22 23 24 25

L. H. SCHLISELFELD, G. J. KALDOR

Lymn, R. V~T. and Taylor, E. W. (I97 o) Biochemistry 9, 2975-2983 Lymn, 1~. XV. and Taylor, E. W. (1971) Biochemistry io, 4617-4624 Morita, F. (1969) Biochim. Biophys. ,4cta 172, 319-327 Inone, A., Shibata-Sekiya, K. and Tonomura, Y. (1972) J. Biochem. Tokyo 71, i i 5 - i 2 4 Pemrick, S. M. and W'alz, Ji., F. G. (1972) J. Biol. Chem. 247, 2959-2961 Trentham, D. iR., Bardsley, R. G., Eccleston, J. F. and Weeds, A. G. (1972) Biochem. J. 126, 635-644 Glynn, I. M. and Chappell, J. B. (1964) Biochem. J. 9 ° , 147-149 BArAny, M., Conover, T. E., Schliselfeld, L. H., Gaetjens, E. and Goffart, M. (1967) Eur. J. Biochem. 2, 156-164 BArAny, K. and Oppenheimer, H. (1967) Nature 213, 626-627 Lowey, S. and Cohen, C. (I962) J. Mol. Biol. 4, 293 308 Lowey, S., Slayter, H. S., Weeds, A. G., and Baker, H. (1969) J. Mol. Biol. 42, 1-29 Fiske, C. H. and SubbaRow, Y. (1925) J. Biol. Chem, 66, 375 4 °0 Gornall, A. G., Bardawill, C. J. and David, M. M. (1949) J. Biol. Chem. 177, 751-766 BArAny, M. and BArAny, K. (1959) Biochim, Biophys. dcta 35, 293-309 Kemp, R. G. and Krebs, E. G. (I967) Biochemistry 6, 423-434 I~:iely, B. and Martonosi, A. (1968) J. Biol. Chem. 243, 2273 2278 Lowey, S. and Luck, S. M. (1969) Biochemistry 8, 3195-3199 Eisenberg, E. and Moos, C. (197o) Biochemistry 9, 41o6-411o Murphy, A. J. and Morales, M. F. (197 o) Biochemistry 9, 1528-1532 Nanazawa, T. and Tonomura, Y. (1965) J. Biochem. Tokyo 57, 6o4-615 Sartorelli, L., Frolnm, H. J., Benson, R. W. and Boyer, P. D. (1966) Biochemistry 5, 2877-2884 Tokiwa, T. and Tonornura, Y. (1965) J. Biochem. Tokyo 57, 616-626