Superprecipitation and Enzymatic Activity of Actomyosin as Affected by the Mode of Polymerization and Nucleotide of Actin

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G-actin was polymerized by adding 30mM KCl at pH 7.5 and 16-20°C for 20-30 hours at various concentrations of protein (0.04-1.2mg. per ml.) in the presence of 0.1mM ATP and 1mM dehydroacetate. The binding of polymerized actin with myosin or H-meromyosin, the extent and rate of superprecipitation and the activity of ATPase [EC 3. 6. 1. 3] of actomyosin reconstituted from myosin and actin were measured, and the following results were obtained : When G-actin was polymerized at extremely low protein concentrations, myosin and H-meromyosin scarcely bound with the type of polymerized actin obtained (F'-actin-ADP). The increase intensity of light-scattering due to the binding of myosin or H-meromyosin to actin occurred in parallel with the actin concentration used for polymerization and reached a constant value when the concentration used for polymerization was higher than 0.6mg. per ml. The light-scattering intensity from 30° to 135° of the system composed of F'-actin-ADP (0.04mg. per ml.) and myosin (0.12mg. per ml.) was equal to the sum of those myosin and F'-actin-ADP, and was unchanged on the addition of 1mM MgPP₁.
The time dependence and the final extent of superprecipitation (induced by ATP) of the mixture of myosin and polymerized actin were scarcely affected by the actin concentration for polymerization. On the other hand, the enhancement of ATPase activity of myosin by actin depended on the actin concentration for polymerization; the ATPase activity of the F'-actin-ADP-myosin system was only less than 20 per cent of that of the usual actomyosin. These results suggest that the actomyosin type of enzymatic activity is unnecessary for the occurrence of superprecipitation of actomyosin.
Under the condition in which the superprecipitation of actomyosin took place, no formation of C¹⁴-ITP or C¹⁴-ATP was detected in the F-actin-C¹⁴-IDP-myosin-ATP system or the F-actin-C¹⁴-ADP-myosin-ATP system. This result suggests that the nucleotide bound to F-actin exchanges with nucleotides in the medium during superprecipitation of actomyosin as a result of loosening of actin-nucleotide linkage, and that the ADP or IDP bound to F-actin is not the acceptor of the phosphate group from the reactive myosin-phosphate complex.