The Pre-steady State of the Myosin-Adenosine Triphosphate System

Transcription

1 /. Biochem., 71, (1972) The Pre-steady State of the Myosin-Adenosine Triphosphate System XL Formation and Decomposition of the Reactive Myosin-Phosphate- Complex* Akio INOUE, Kazuko SHIBATA-SEKIYA and Yuji TONOMURA The Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka Received for publication, July 7, 1971 The amount and rate of formation of the reactive myosin-phosphate- complex, A DP Ep, was estimated by measuring the size and rate of the initial burst of Piliberation after stopping the reaction by trichloroacetic acid. Measurements on myosin-atpase [EC ] were made in 0.5 M KCl and 2.5 mm MgCU at ph7.8 and 0 C, unless otherwise stated. The following results were obtained. 1) At ATP concentrations below 0.3/iM, the values of K u and V /m for formation of Ep were 0.3/iM and 0.70mole Pi/min-4.8xl0 s g myosin, respectively, while at ATP concentrations above 0.3/^M, the values of K u and V /m were much larger. 2) The values of KM and V m of actomyosin-atpase were measured in 50 mm KCl and 2 mm MgCU at ph7.0 and 25 C. At ATP concentrations below lofim, they were 2.9/iM and 2.5 X 10 2 moles/min-4.8xl0 s g myosin, respectively, and were equal to those for Ep formation of myosin under the same conditions. At ATP concentrations above 10/iM, the rate of the actomyosin-atpase reaction was larger than that expected from the K u and V m values obtained at low ATP concentrations. 3) At ATP concentrations above 0.5^M, the values of K M and V a of myosin-atpase in the steady state were 1/iM and 0.44 mole Pi/min-4.8x10^ myosin, respectively, while at ATP concentrations below 0.3//M, the rate of the ATPase reaction was almost independent of the ATP concentration and had a V. value of about 1/4 of that obtained at high ATP concentrations. 4) The results of Lymn and Taylor on the dependence of the burst size on the ATP concentration was reexamined in 0.5 M KCl and 10 mm MgCl* at ph8.0 and 20 C. Both the rate in the steady state and the size of the initial burst of Piliberation were found to be independent of the ATP concentration at concentrations of 5 to 200/iM. * This investigation was supported by grants from the Muscular Dystrophy Associations of America, Inc., and the Ministry of Education of Japan. Abbreviations; TCA: trichloroacetic acid, NTP: /nnitrothiophenol or p-nitrothiophenyl, EDTA: ethylenediaminetetraacetic acid, PCMB: />-chloromercuribenzoate. Vol. 71, No. 1,

2 116 A. INOUE, K. SHIBATA-SEKIYA and Y. TONOMURA The initial rapid liberation of inorganic phosphate, which is conventionally called as the initial burst of myosin-atpase [EC ], was first observed by us (/, 2), and later confirmed by Sartorelli et al. (3) and Lymn and Taylor (4). We (2, 5, 6) attributed this initial burst to formation of the reactive myosin-phosphate- complex, Ep. We obtained the following results on the formation A DP of Ep : (i) a stoichiometric amount of H + is rapidly absorbed by myosin, and then it is liberated just before formation of E (7,8), (ii) NTP binds to myosin in the presence of MgATP and the NTP-myosin thus formed shows no initial burst of Pi-liberation (9 11) and (iii) during the initial phase of the reaction a P-exchange reaction occurs between the intermediate and the terminal phosphate of ATP (12). From these results, we proposed the following reaction mechanism (8): (1) (2).».E: (3) (4) '-P In this scheme, the reactive myosin-phosphate- complex, Ep., consists of two.... kinds of intermediates, E-" and E:' ~ P -.p The former can exchange P with ATP and is called phosphoryl myosin, while the latter cannot exchange P with ATP. We also concluded that step 2 is accelerated by ATP from the dependences on the ATP concentration of the rates of rapid HMiberation, the change in the UV-spectrum and the initial burst of Pi-liberation (5).* After the addition of a stoichiometric amount of ATP to myosin, the rate constants of liberation of (11) and of restoration of the change in the UV-spectrum (5) were almost equal to that of ATPase in the steady state, while the rate of slow liberation of H + (8, 10) and the recovery process of the initial * For details of our experiments on the reaction mechanism of myosin-atpase and its physiological meanings, see our monograph (13). burst of Pi-liberation (12) were much slower than the rate of the ATPase reaction in the steady state. - Therefore, we concluded that.. decomposition of E:' occurs in two steps: ' P h h E:' >E. +»E ' P (5) "P (6) + + Pi + H +, where k t is almost equal to the rate constant (V m ) of the ATPase reaction in the steady state and Ar 8 is only a fraction of this value. Furthermore, the initial burst of Pi-liberation was completely suppressed by />-nitrothiophenylation of myosin, while the ATPase reaction in the steady state was unaffected by this modification (11). These results strongly suggest that Ep is not an intermediate in the main path of ATP-decomposition in the steady state. From the ph-dependence of the ATPase activity and the effect of chemical modification of myosin, we proposed the following scheme for the ATPase reaction in the steady state (13-15): E. P "P 'P ++Pi+H + Recently, Taylor and his collaborators found that the rate of Ep formation is proportional to the ATP concentration (10) from 5 to loo^m (4) and that the rates of liberation of Pi and from Ep P are almost equal to the rate of the ATPase reaction in the steady state (16). Therefore, they proposed the following scheme:?=±es Ep DP E++P l +H +. Furthermore, they concluded that the myosin molecule has two hydrolytic sites which interact with each other (4, 16), since their results indicated that the burst size of Pi-liberation increases and approaches two moles per mole of myosin at an ATP concentration of about 30 /im (4). J. Biochem.

3 PRE-STEADY STATE OF MYOSIN-ATPase. XI 117 To clarify the cause of the differences between our results and conclusions and those of Taylor's group, we measured the rates of Ep DP formation and the ATPase reaction in the steady state over a wide range of ATP concentrations. From our results we concluded : (i) The intrinsic value of the K M of Ep formation was very low, but the apparent values of K u and V /m increased at ATP concentrations above 0:3 i*m. (ii) The rate of the ATPase reaction in the steady state could be expressed as the sum of the rates of two kinds of ATPase reactions: the one with lower values of K u and V m was a hydrolytic reaction via Ep^P and the other with higher values of K u and V m was a simple hydrolysis of ATP. (iii) The rate of the actomyosin-atpase re- action was equal to the rate of Ep formation of myosin at least at low ATP concentrations, (iv) In contrast to the results obtained by Lymn and Taylor (4), the amount of the initial burst of Pi-liberation and the rate of the ATPase reaction in the steady state were constant and were independent of the ATP concentration, at ATP concentrations above 5^M. EXPERIMENTAL Myosin was prepared from rabbit skeletal muscle by the method of Perry (77). It was further purified by phosphocellulose column chromatography using the method of Harris and Suelter {18). Fresh preparations of myosin were used throughout. The molecular weight of myosin was taken as 4.8 xlo 5 (79). F-Actin was prepared from acetone powder of rabbit skeletal muscle by the procedures of Mommaerts (20) with slight modifications (21). The crystalline sodium salt of ATP was purchased from Sigma Chemical Company. 82 P-Labelled ATP was synthesized enzymatically by the method of Glynn and Chappel {22). The reaction was started by mixing 1 ml of M P-ATP solution with 1 ml of myosin solution and stopped by adding 2 ml of 10% TCA containing 1 mm ATP and 100 (im Pi as- carriers. The "Pi liberated was measured by the standard procedure used in this laboratory (72). In calculating the concentration of free ATP in the reaction mixture, corrections were made for hydrolysis and binding of ATP with myosin. The simple mixing apparatus devised by Kanazawa et al. (23) was used to follow the rapid initial reaction. When the reaction time was very long, a correction was made for non-enzymatic hydrolysis of ATP. Protein was estimated by the biuret reaction calibrated by nitrogen determination. RESULTS Dependence on the ATP Concentration of the Rate of Formation of the Reactive Myosin- Phosphate- Complex-The rate of Ep Wr formation, v f, has previously been measured by us (6) and later by Lymn and Taylor (4). Previously we measured it in the presence of 2mg/ml of myosin at ATP concentrations above 1.45 fim, while Lymn and Taylor measured it in the presence of 2mg/ml myosin at ATP concentrations above 5 ^M. Therefore, we measured the v f in a range of ATP concentrations from 0.06 to 5/*M in the presence of Fig. 1. Lineweaver-Burk plot of the rate of the initial burst of Pi-liberation, v/, in 0.5 M KC mg/ml myosin, 0.5 M KC1, 2.5 mm MgCl 2 and 25 mm Tris-maleate buffer at ph7.8 and 0 C. O,, x, A, different preparations of myosin, see text. 15 "Vol.. 71, No. 1, 1972

4 118 A. INOUE, K. SHIBATA-SEKIYA and Y. TONOMURA 0.03 mg/ml myosin in 0.5 M KC1, 2.5 mm MgCl 2 and 25 mm Tris-maleate buffer at ph 7.8 and 0 C. Figure 1 shows a double reciprocal plot of v f against the ATP concentration, [S]. In this experiment four myosin preparations were used, and the values of v f relative to that at 0.26 ftm ATP were plotted. The values of v f of these preparations at 0.26 pm ATP were 0.28, 0.35, 0.34 and 0.31 mole/min-4.8xl0 8 g myosin, and the average value of 0.32 min" 1 was used in the figure. The figure shows that a plot of V/- 1 versus [S]" 1 gave two straight lines bending at about 0.3 ftm ATP. At ATP concentrations below 0.3 ftm, the maximum value of v f, V fm, was 0.70 mole/min-4.8xl0 5 g myosin and the value of KM was 0.3 ftm. At ATP concentrations above 0.3 ftm, the values of K u and V fm were too large to measure accurately with our simple mixing apparatus, and Vf increased in proportion to ATP concentration. The apparent second order rate constant was about 1.2 xh^m" 1 min" 1. As shown in Fig. 2, a plot of v f ~ l versus [S]" 1 in 1.5 M KC1 gave two straight lines bending at 1 ftm of ATP. In the low ATP Fig. 2. Lineweaver-Burk plot of the rate of the initial burst of Pi-liberation, v t, in 1.5 M KC mg/ml myosin,' 1.5 M KC1, 2.5 mm MgCl 2 and 25 mm Tris-maleate buffer at ph7.8 and 0 C. O,, different preparations of myosin. concentration range, the values of K u and Vf m were 1A<M and 0.43 mole/min-4.8xl0! g myosin, while at high concentrations of ATP Vf increased in proportion to the ATP concentration. The value of v f after mixing 2.52 mg/ml (5.25 ^M) myosin with 5 ftm ATP was estimated as 5.9 moles/min-4.8xl0! g myosin, which was almost equal to the value of 6.0 moles/min- 4.8xl0 5 g myosin obtained in the presence of 0.03 mg/ml ( ftm) myosin (Fig. 1). To examine the order of the reaction of initial Pi-liberation, we plotted log(so x) and log(e x)/(so x) against time, where So, x and e are the initial concentration of ATP, the concentration of TCA-Pi liberated at time t and the molar concentration of active site, respectively. However, we could not decide the order of the reaction from this type of experiment, as shown in Fig. 3. Dependence of the Rate of the Actomyosin- ATPase Reaction on the ATP Concentration Myosin was pretreated by PCMB and /S-mercaptoethanol to strengthen the binding with F-actin (24), and the rates of the initial burst of Pi-liberation of 0.1 mg/ml myosin were measured in 50 mm KC1, 2 mm MgCl 2 and t 6 REACTION TIME (we) -Q005 -QOOi OQ002 Fig. 3. Time-course of the initial burst of Pi-liberation mg/ml myosin, 5/«M ATP, 0.5 M KC1, 2.5 mm MgCl 2 and 25 mm Tris-maleate buffer at ph7.8 and 0 C. The plot of log(so x) against t shows a first order reaction and the plot of log(e x)/(so x) against t shows a second order reaction. So, x and e are the initial concentration of ATP, the concentration of TCA-Pi liberated at time t and the molar concentration of active site. /. Biochem.

5 PRE-STEADY STATE OF MYOSIN-ATPase. XI 119 Q&S0 Fig. 4. Lineweaver-Burk plots of the rate of the actomyosin-atpase reaction, CAM, and the rate of the initial burst of Pi-liberation of myosin-atpase,»/. 50 mm KC1, 2 mm MgCl 2 and 20 mm Tris-maleate buffer at ph7.0 and C. O, A, actomyosin- ATPase, mg/ml myosin pretreated with PCMB and /9-mercaptoethanol, 0.015mg/ml F-actin;, A, initial burst of Pi-liberation, 0.1 mg/ml myosin pretreated with PCMB and /J-mercaptoethanol. mm Tris-maleate buffer at ph 7.0 and C. They were compared with those of the ATPase reaction of actomyosin in the presence of mg/ml of myosin and mg/ml of F- actin under the same conditions. As shown in Fig. 4, at ATP concentrations below 10 pu the rate of the actomyosin-atpase reaction was equal to that of the initial burst of Pr liberation of myosin: the values of K u and V m were 2.9 pm and 2.5x10* moles/min-4.8x 10 s g myosin, respectively. At higher ATP concentrations, both the rate of the initial burst of the myosin-atpase reaction and the rate of the actomyosin-atpase reaction increased more than expected from the K m and V m values in the low ATP concentration range, as already reported by Kominz (25) on actomyosin-atpase. These results strongly support our conclusion (26, 27) that Ep DP is a reaction intermediate in ATP hydrolysis catalyzed by actomyosin. Kinetics of the Change in A TPase Activity after Adding EDT A to the Myosin-ATP System in the Presence of Magnesium Jon-Reactions were started by adding 6.6 [tu ATP to a solution containing 0.1 mg/ml myosin, 0.5 M KC1, lmm MgClt and 20 mm Tris-maleate REACTION TIME(min) Fig. 5. Time-course of Pi-liberation after adding EDTA to the myosin-atp system in the presence of Mg mg/ml myosin, 6.6 /IM ATP, 0.5 M KC1, 1 mm MgCl 2 and 20 mm Tris-maleate buffer at ph 7.0 and 0 C. x, Pi-liberation in the presence of 1 mm Mg 2+ ; O,,A,A,D, Pi-liberation after adding 25 mm EDTA to the system at the time indicated by the arrow, j ;, Pi-liberation when ATP was added to myosin in the presence of 1 mm MgCl, and 25 mm EDTA. buffer at ph 7.0 and 0 C, and at intervals 25 mm EDTA was added. As shown in Fig. 5, the initial burst of Pi-liberation occurred within 1 min after adding ATP. When EDTA was added to the system after the initial burst of Pi-liberation, the rate of the ATPase reaction gradually increased from that in the presence of Mg 2+ to that observed when 6.6 pm ATP was added to a solution containing both 1 mm MgCl 2 and 25 mm EDTA. (The kinetics of Pi-liberation from 6 min after EDTA-addition are not shown in the figure.) The induction period of this transition of ATPase was found to be 210 sec. When EDTA was added during the initial burst of Pi-liberation, the transition of ATPase was seen but the induction period was shorter than that when EDTA was added in the steady state. It is interesting to note that when F-actin is added to the myosin-atp Vol. 71, No. 1, 1972

6 120 A. INOUE, K. SHIBATA-SEKIYA and Y. TONOMURA system, the rate of the ATPase reaction increases to the high activity of the actomyosin type, without showing any lag phase (26). Dependence on the ATP Concentration of the Rate of Myosin-ATPase in the Steady State-The dependence on the ATP concentration of myosin-atpase has been measured by many workers. It has been measured at ATP concentration above 0.5 fim, except in the work reported by Lymn and Taylor (4, 16). Fig. 6. Lineweaver-Burk plot of the rate of the ATPase reaction in the steady state, v, in 0.5 M KC mg/ml myosin, 0.5 M KC1, 2.5 mm MgClj and 50 mm Tris-maleate buffer at ph7.8 and 0 C. O,, X, A, different preparations of myosin; the solid line indicates by: 0 33 v (mole/min-4.8xl0 5 g myosin)= Therefore, we measured the rate of ATPase in the steady state over a wide range of ATP concentrations from 0.1 to 5 ftu in the presence of mg/ml myosin, 0.5 M KC1, 2.5 mm MgCU and 50 mm Tris-maleate buffer at ph 7.8 and 0 C. Figure 6 shows results on four myosin preparations. The rates of each preparation are plotted relative to that at 2?M ATP. The rates of the preparations at 2 fim ATP were 0.36, 0.36, 0.35 and 0.33 mole/min 4.8xl0 5 g myosin, and the average value of 0.35 mole/min 4.8 xl0 5 g myosin was used as the rate at 2 fim ATP. At ATP concentrations above 1 /*M, the values of K u and V m were 1 pm and 0.44 mole/min-4.8x 10 s g myosin, respectively. At ATP concentrations below 0.3 ^M the rate deviated from the straight line obtained at higher ATP concentrations and became almost independent of the ATP concentration. The values of v over all the ATP concentrations used were given by: v (moles/min- 4.8 xlo 5 g myosin) 0.33 = A plot of v~ l versus [S]" 1 in 1.5 M KC1 gave a straight line (Fig. 7). The values of K M and V m were 0.038?M and 0.11 mole/min-4.8xlo 5 g myosin, respectively. Dependence of the Burst Size and the Rate of the ATPase Reaction on the ATP Concentration To examine whether Lymn and Taylor's conclusion (4) on the number of active sites of myosin is correct, the size of the Fig. 7. Lineweaver-Burk plot of the rate of the ATPase reaction in the steady state, v, in 1.5 M KC mg/ml myosin, 1.5 M KC1, 2.5 mm MgCl 2 and 50 mm Tris-maleate buffer at ph 7.8 and 0 C. /. Biochem.

7 PRE-STEADY STATE OF MYOSIN-ATPase. XI 121 Fig. 8. Dependences on the ATP concentration of the amount of the initial burst of Pi-liberation and the rate of the ATPase reaction in the steady state, lmg/ml myosin, 0.5 M KC1, 10 mm MgCl 2 and 0.1M Tris-HCl buffer at ph8.0 and 20 C. O, rate of the ATPase reaction in the steady state;, amount of the initial burst. initial burst of Pi-liberation and the rate of the ATPase reaction in the steady state were measured in a range of ATP concentrations from 5 to 200 (tm under the conditions used by Lymn and Taylor, i.e. in 1 mg/ml myosin, 0.5 M KC1, 10 mm MgCl 2 and 0.1 M Tris-HCl buffer at ph 8.0 and 20 C. As shown in Fig. 8, the size of the initial burst and the rate of the ATPase reaction in the steady state were independent of the ATP concentration. They were 1.1 mole/4.8xl0! g myosin and 1.1 mole/ min-4.8xl0 5 g myosin, respectively. DISCUSSION We proposed the following mechanism for formation of the reactive myosin-phosphate- complex, E, (1) (2) (3). E (4) - -P on the basis of the findings that the amount of the initial burst of TCA-labile Pi-liberation is one mole per mole of myosin (2), that one mole of H + per mole of myosin is rapidly absorbed by myosin and is liberated again slightly prior to the initial burst of Pi-liberation (7, 8), and that the rate of change in the UV-spectrum is equal to that of rapid liberation of H + (8). We concluded that step 2 is accelerated by ATP itself, since the values of Ti/2, the time for half the final change of rapid HMiberation, the change in the UVspectrum and the initial burst of Pi-liberation were almost independent of the ATP concentration when the latter was lower than the stoichiometric value, but decreased inversely with the ATP concentration when the latter was higher than the stoichiometric one (6). On the other hand, Lymn and Taylor (4) recently proposed a simpler scheme for the formation of Ep DP, from the finding that the rate of the initial burst of Pi-liberation is almost proportional to the ATP concentration at concentrations from 5 to 100 fim: E + S: :ES- r The main differences between these two reaction mechanisms are the presence of phos-.. phoryl myosin, E-', as an intermediate and ~P the dependence of the rate of E formation on the ATP concentration. We deduced that phosphoryl myosin was formed from the findings that NTP combines with a carboxyl group of a glutamic acid residue of myosin only in the presence of Mg 2 + and ATP (77, 28) and that this binding completely suppresses the initial burst of Pi-liberation but does not affect the ATPase reaction in the steady state (77), and that the P-exchange reaction occurs between the reactive myosin-phosphate- complex and the terminal phosphate of ATP during the initial phase of the reaction (72). However, this problem is not related to the present work, and will not be discussed here (see Ref. 13). As shown in Figs. 1 and 2, the Michaelis constant of Ep P formation was 0.3 //M at ATP concentrations below 0.3 ftu. Furthermore, at high ATP concentrations, the rate of Ep formation increased with the ATP concentration. These results are. consistent Vol. 71, No. 1, 1972

8 122 A. INOUE, K. SHIBATA-SEKIYA and Y. TONOMURA with our previous results (6*) on the rapid liberation of H +, the change in the UV-spectrum and the initial burst of Pi-liberation. To investigate the mechanism of increase in the rate of Ep formation at high ATP concentrations, we measured the rate of the initial burst of Pi-liberation after adding 5 ^M ATP to 5.25 ftm of myosin. The rate was almost equal to that obtained by adding 5 ftm ATP to (im myosin. Therefore, if we assume that ATP at high concentrations induces a conformational change in the myosin molecule, this change must not be accompanied by a decrease in the MgATP concentration as substrate of the ATPase reaction. If we adopt this assumption, the reaction mechanism of Ep formation is given by: (low ATP concentration) I E + S 5=^ES.. (high ATP concentration), E,. High concentrations of MgATP induce a change in myosin to a new conformational state, E*. This conformational change is ac- companied by acceleration of E-" forma- ~P tion from the ES complex. Since our experiments on the rapid absorption and liberation of H* were performed in the presence of several pm of ATP, the rapid change in H* might be attributed to the conformational change in myosin induced by ATP: E + S 7 I :ES % i^=ie*s+h + We have also reported acceleration of formation of the phosphorylated intermediate by ATP itself with the Na + -K + dependent ATPase (23) and the Ca* + -Mg l+ dependent ATPase of the sarcoplasmic reticulum (29). The rate of the actomyosin type of ATPase reaction was equal to the rate of the initial burst of P r liberation of myosin, as shown in Fig. 4. This supports our previous conclusion (27) that in the actomyosin-atpase reaction, ATP. is decomposed via the E-' and E: ~P --P complexes and decomposition of these complexes is accelerated by adding F-actin. On the other hand, we concluded previously that the main route of the ATPase reaction in the steady state is decomposition of ATP by simple hydrolysis, since (i) the rate of slow H + -liberation (8, 10) and the recoveryof the initial burst of Pi-liberation (12) after! addition of a stoichiometric amount of ATP to myosin is several times lower than the rate of the ATPase reaction in the steady state, and (ii) />-nitrothiophenylation of myosin suppresses the initial burst of Pi-liberation but does not affect the ATPase activity in the steady state (11). Furthermore, we concluded].. that the E:' complex is decomposed via " P two steps, since the rate of liberation of.. + S E ++P:+Ht ' P -.2 '*P from E:' is of the same order of magni- ' P tude of that of the ATPase reaction in the steady state and is much higher than those of slow HMiberation and the recovery of the initial burst (//). Thus, E + S- E:' + -P Taylor and his collaborators (16) recently obtained results suggesting that the rates of A DP liberation of Pi and from the Ep complex are in the same order of magnitude as that of ATPase reaction in the steady state. /. Biochem.

9 PRE-STEADY STATE OF MYOSIN-ATPase. XI 123 TABLE I. Comparison of the rate of simple hydrolysis, K«2, with that of decomposition of ATP via KCl (M) MgCl 2 (tan) PH Temp. ( C) Method V. 1 : V m - 1 Reference H* liberation H* liberation H* liberation recovery of burst kinetics of steady state kinetics of steady state < 1:9 1:4 1 : 3 *(?) (8) (10) (8) (12) this paper this paper They proposed the following scheme:, ES- E++P,. E+S; Their finding that the rate of liberation of Pi from E was almost equal to that of does not agree with our reaction mechanism. The cause of this difference is not yet clear, but it is possible that E. in our reaction ' P mechanism is actually a new conformational state of myosin, E, which does not contain P and this conformational state of myosin returns to the original state very slowly: E"".. E' + + P-, -*-» E+ + P; +Ht According to our mechanism, hydrolysis of ATP catalyzed by myosin occurs via two different routes. If they have different Ku and V m values, they can be distinguished by measuring the dependence of the rate of the ATPase reaction in the steady state, v, on the ATP concentration, [S]. As shown in Fig. 6, the rate of the ATPase reaction in the steady state in 0.5 M KCl was given by v(mole/min 4.8 x 10 s g myosin) 0.33 = Since the formation of Ep is much faster than its decomposition, the K u of ATP hydrolysis via Ep DP and E. (or E ) must be P Vol. 71, No. 1, 1972 A DP lower than the K«of Ep r formation, 0.3 The value of V m of this route must be a small fraction of the whole for ATP hydrolysis, since this is not the main route of hydrolysis. Furthermore, the rate constant of Ep DP E. r ' P + (or E ++Pi) is much higher than that of E. -*E+Pi (or E -»E), as mentioned "P above. Therefore, the most stable intermediate in the ATP-hydrolysis via Ep r is E. (or r 'P E ). It must be added that the dissociation constant of the binding of ATP with myosin measured by the luciferin-luciferase method (30) and UV spectroscopy (37) is in the range of a few /am. This indicates that the Michaelis complex of ATP-hydrolysis in the range of high ATP concentrations (^l^m) contains ATP or, while that in the range of low ATP concentrations «1 (iu) contains no nucleotide. Therefore, it seems probable that the route of ATP hydrolysis with a V m of ATYP 0.11 min" 1 and Ki,<lf*M is the one via Ep * and E. (or E ) and that the route with a P V m of 0.33 min" 1 and Ku of 1 ^M is that involving simple hydrolysis of ATP via ES. * From the rate constant of decomposition of Ep given above and that of Ep formation given in Fig. 1, we calculated the Ku values of ATP hydrol- ysis via Ep at ATP concentrations above and below 0.3/IM as 0.09 and 0.05//M, respectively.

10 124 A. INOUE, K. SHIBATA-SEKIYA and Y. TONOMURA Table I summarizes our results on the ratio of the rates of the two routes of ATP hydrolysis under various conditions. Taylor and his co-workers (4, 16, 32) have tried to explain the complicated properties of myosin-atpase by assuming the presence of two ATPase sites on the myosin molecule, which interact with each other. However, as described in our previous paper (33), the amount and the rate of the initial burst of Pi-liberation per two moles of subfragment-1 were exactly the same as those per mole of myosin. Furthermore, as shown in Fig. 8, the burst size of Pi-liberation was constant, being about 1 mole/mole of myosin, and was independent of the ATP concentration even under the conditions used by Taylor et al. The authors are greatly indebted to Dr. Y. Hayashi for his valuable suggestions on the preparation of myosin. REFERENCES 1. Y. Tonomura and S. Kitagawa, Biochim. Biophys. Ada, 28, 15 (1957). 2. T. Kanazawa and Y. Tonomura, /. Biochem., 57, 604 (1965). 3. L. Sartorelli, H.J. Fromm, R.W. Benson and P.D. Boyer, Biochemistry, 5, 2877 (1966). 4. R.W. Lymn and E.W. Taylor, Biochemistry, 9, 2975 (1970). 5. S. Kitagawa and Y. Tonomura, Biochim. Biophys. Ada, 57, 416 (1962). 6. H. Onishi, H. Nakamura and Y. Tonomura, /. Biochem., 63, 739 (1968). L 7. T. Tokiwa and Y. Tonomura, /. Biochem., 57, 616 (1965). 8. Y. Tonomura, H. Nakamura, N. Kinoshita, H. Onishi and M. Shigekawa, /. Biochem., 66, 599 (1969). 9. Y. Tonomura, S. Kitagawa and J. Yoshimura, /. Biol. Chem., 237, 3660 (1962). 10. K. Imamura, T. Kanazawa, M. Tada and Y. Tonomura, /. Biochem., 57, 627 (1965). 11. N. Kinoshita, S. Kubo, H. Onishi and Y. Tonomura,./. Biochem., 65, 285 (1969). 12. H. Nakamura and Y. Tonomura, /. Biochem., 63, 279 (1968). 13. Y. Tonomura, " Muscle Proteins, Muscle Contraction and Cation Transport," Tokyo Univ. Press, Tokyo, (1972) in press. 14. T. Nihei and Y. Tonomura, /. Biochem., 46, 305 (1959). 15. N. Azuma and Y. Tonomura, Biochim. Biophys. Ada, 73, 499 (1963). 16. E.W. Taylor, R.W. Lymn and G. Moll, Biochemistry, 9, 2984 (1970). 17. S.V. Perry, "Method in Enzymology," ed. by S.P. Colowick and N.O. Kaplan, Academic Press Inc., New York, Vol. Ill, 582 (1955). 18. M. Harris and C.H. Suelter, Biochim. Biophys. Ada, 113, 393 (1967). 19. Y. Tonomura, P. Appel and M. Morales, Biochemistry, 5, 515 (1966). 20. W.F.H.M. Mommaerts, /. Biol. Chem., 198, 445 (1952). 21. W. Dravikowski and J. Gergely, /. Biol. Chem., 237, 3412 (1962). 22. I.M. Glynn and J.B. Chappel, Biochem. /.. 90, 147 (1964). 23. T. Kanazawa, M. Saito and Y. Tonomura, /. Biochem., 67, 693 (1970). 24. S. Kitagawa, J. Yoshimura and Y. Tonomura, /. Biol. Chem., 236, 902 (1961). 25. D.R. Kominz, Biochemistry, 9, 1792 (1970). 26. N. Kinoshita, T. Kanazawa, H. Onishi and Y. Tonomura, /. Biochem., 65, 567 (1969). 27. Y. Tonomura and T. Kanazawa, /. Biol. Chem., 240, 4110 (1965). 28. S. Kubo, N. Kinoshita and Y. Tonomura, /. Biochem., 60, 476 (1966). 29. T. Kanazawa, S. Yamada, T. Yamamoto and Y. Tonomura, /. Biochem., 70, 95 (1971). 30. L.B. Nanninga and W.F.H.M. Mommaerts, Proc. Natl. Acad. Sci. U.S., 46, 1155 (1960). 31. K. Sekiya and Y. Tonomura, /. Biochem., 61, 787 (1967). 32. B. Finlayson and E.W. Taylor, Biochemistry, 8, 802 (1969). 33. Y. Hayashi and Y. Tonomura, /. Biochem., 68, 665 (1970). J. Biochem.