The Pre-Steady State of the Myosin-Adenosine Triphosphate System
IX. Effect of F-Actin on the Myosin-ATP System
1969 Volume 65 Issue 4 Pages 567-579
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1969 Volume 65 Issue 4 Pages 567-579
1. Myosin was treated with NTP by the method described in the preceding paper (2). As mentioned in the preceding paper (2), the rate of ATP-hydrolysis of myosin [ATP : phosphohydrolase, EC 3. 6. 1. 3] in the steady state was only slightly altered by treatment with NTP, but no initial burst of Pi-liberation could be observed with NTP-myosin. The actomyosin type of ATPase activity was inhibited by treatment of myosin with NTP. At low ionic strength and in the presence of MgCl2, no superprecipitation or clearing response could be observed with actomyosin reconstituted from NTP-myosin and F-actin.
2. At high ionic strength and low Mg++ concentration the amount of initial rapid P liberation was more than I mole per 4×105g of myosin; i.e., an extra-burst of Pi-liberation was observed. As mentioned in the preceding paper (2) the initial extra-liberation of hydrogen ion was accompanied by an extra-burst of P liberation. The dependencies of the amount of initial extra-burst of hydrogen ion-liberation on the ATP concentration were measured in the presence of various Mg++ concentrations. They were in good agreement with those of the amount of decrease in light-scattering intensity of reconstituted actomyosin induced by ATP. The rate of initial rapid hy-drogen ion-liberation was essentially equal to that of decrease in light-scattering.
3. The dependency of actomyosin ATPase on Mg++ concentration was studied. The ATPase activity increased with the concentration of Mg++ until the concentration of added MgCl2 reached 5 pss. At higher concentrations the activity decreased gradually with increasing Mg++ concentration. The rate of superprecipitation of actomyosin in the presence of 10μM MgCl2 was much faster than that in the presence of 1mM MgCl2.
4. When F-actin was added to the myosin-ATP system at various times (10-120 see) after mixing myosin with ATP, the initial rapid ATP-splitting, seen when ATP was added to actomyosin, was not detected, but only the steady state actomyosin ATPase was observed.
5. Based on the results given in this and the preceding paper (2), the following reaction mechanism is proposed for the function of F-actin in the myosin (E)-ATP (S) system:
F-actin inhibits the conversion of phosphoryl myosin, _??_to the weakly reactive ADP .P myosin-phosphate-ADP complex, _??_When the myosin component of actomyosin ADP is converted to the myosin-phosphate-ADP complex by ATP, reconstituted actomyosin dissociates to myosin and F-actin at high ionic strength. But at low ionic strength the presence of F-actin accelerates the decomposition of the myosin-phosphate-ADP complex to myosin, ADP and Pi.
