Binding of H-Meromyosin with F-Actin at Low Ionic Strength

抄録

The reaction of the H-meromyosin-F-actin-ATP system was in-vestigated in the presence of various concentrations of ATP, F-actin and KCl in 1mM MgCl₂ and 10mM Tris-HCl at pH 7.5 and 25°C. The following results were obtained:
1. The binding ratio of H-meromyosin to F-actin was 3.3:1 by weight. When ATP was added to acto-H-meromyosin, the intensity of light scattered at high angles decreased to a level lower than the sum of those of H-meromyosin and F-actin. The weight average molecular weight of acto-H-meromyosin estimated from the angular distribution was slightly decreased but the z-average radius of gyration was increased markedly by the addition of ATP.
2. The ATP concentration necessary for obtaining half the maximum change in the intensity of light scattered at 90° by acto-H-meromyosin was increased by decrease in the ionic strength and by increase in the F-actin concentration. At a fixed ionic strength, the ATPase [EC 3. 6. 1. 3] activity of acto-H-meromyosin increased monotonously with ATP con-centration and the so called “substrate inhibition” was not observed. The activity in the presence of a sufficient amount of ATP was increased by increase in the F-actin concentration and by decrease in the ionic strength. The ATP concentration necessary for attaining half the maximum activity was increased by decrease in the ionic strength and by increase in the F-actin concentration, and was higher than that for inducing half the maximum change in the intensity of light-scattering. The effect of treatment of H-meromyosin with PCMB and β-mercapto-ethanol on the ATPase activity of acto-H-meromyosin was very similar to that observed when decreasing the ionic strength.
3. The ATPase activity of acto-H-meromyosin was unaffected by the addition of EGTA, but inhibited by the addition of EDTA. The inhibition was restored by the further addition of MgCl₂. About 40-80 per cent of ADP of F-actin were exchanged with nucleotide in the medium, when H-meromyosin and ATP were added to F-actin.
4. From these results the following reaction scheme is proposed for the reaction between H-meromyosin, F-actin and ATP:
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where M and A are the binding units of H-meromyosin and F-actin, and S is MgATP^--; k₁ and k₂ are the rate constants of ATP splitting by the myosin type and the actomyosin type ATPase, respectively. AMS^* is a complex of acto-H-meromyosin with ATP, which has a weight average molecular weight almost equal to that of AM but a radius of gyration much larger than AM. Furthermore, the polymerization state of F-actin in AMS^* is different from F-actin itself.