Separation of Subfragment-1 of H-Meromyosin into Two Equimolar Fractions with and without Formation of the Reactive Enzyme-Phosphate-ADP Complex

Abstract

H-Meromyosin (HMM) was digested with insoluble papain [EC 3.4.22.2]. Neither the size of the initial burst of P, liberation (0.5 mole/mole of myosin head) nor the Mg²⁺-ATPase [EC 3.6.1.3] activity of HMM in the steady state was affected by this treatment. Acto-S-1 was obtained by mixing F-actin with HMM digested with insoluble papain (HMM-S-1).
The size of the initial burst of P₁ liberation of acto-S-1 was 0.35 mole/mole of S-1 at an ATP concentration of 0.5 mole/mole of S-1, and 0.5 mole/mole of S-1 at ATP concentrations above 1 mole/mole of S-1.
F-Actin (2mg/ml) was mixed with 3.4mg/ml (20μM) of HMM-S-1 in 50mM KCl, 2mM MgCl₂, and 10mm Tris-HCl at pH 7.8 and 0°. After adding 10μM ATP (0.5 mole/mole of S-1) with 4mg/ml of pyruvate kinase [EC 2.7.1.40] and 12mm PEP, the reaction mixture was centrifuged at 1.6×105×g and 0° for 40-60min. The amounts of S-1 bound -to F-actin in the precipitate and of S-1 dissociated from F-actin in the supernatant were measured by SDS-gel electrophoresis. S-1 was divided equally between the supernatant and the precipitate, but the sizes of the initial bursts of P₁ liberation of S-1 in the supernatant and the precipitate were about 0.7 and 0.3 mole/mole of S-1, respectively. When the separation procedures were repeated, the size of the initial burst of S-1 in the supernatant increased to 0.8 mole/mole of S-1, while that of S-1 in the precipitate decreased to 0.25 mole/mole of S-1.
Myosin was separated into two fractions, one bound to F-actin (precipitate) and the other dissociated from F-actin (supernatant), by centrifugation of actomyosin in the presence of various concentrations of ATP with an ATP-regenerating system at high ionic strength. At all ATP concentrations used, the myosin in the precipitate and supernatant gave the same sizes of initial burst (0.5 mole/mole of myosin head) as myosin before the separation.