Abstract
It has previously been shown that deoxy-ATP can induce contraction of myofibrils and the enzymatic activity of actomyosin type, and that deoxy-ATP is scarcely incorporated into G-actin. In this report, the superprecipitation of actomyosin by deoxy-ATP was shown to be sensitive to EGTA. The time-course of P1-liberation from the myosindeoxy-ATP system showed an initial burst of about I mole per 4×105g. of myosin which originated from the decomposition of a reactive myosinphosphate complex caused by trichloroacetic acid. Therefore, to clarify the role of ADP bound to F-actin in contraction of muscle models, we investigated the superprecipitation and the enzymatic activity of reconstituted actomyosin made with myosin and ADP free F-actin, which was prepared by the method of Kasai et al. or of Mommaerts, using deoxy-ATP as the substrate.
When Kasai's F-actin free from ADP was used, the deoxy-ATP induced superprecipitation of actomyosin was scarcely affected, but the enhancement of deoxy-ATPase activity of myosin by F-actin was decreased remarkably by the decrease in the ADP content of F-actin. When F-actin completely free from ADP was used, only 0.1 mole of deoxy-ATP per mole of myosin was hydrolyzed by the actomyosin type deoxy-ATPase, before the superprecipitation of actomyosin was completed. From these results, we concluded that the enzymatic activity of actomyosin type is unnecessary for the superprecipitation. Measurements of the light-scattering of the myosin-F-actin system showed that Kasai's F-actin free from ADP scarcely binds to myosin.
Not only the superprecipitation induced by deoxy-ATP but also the deoxy-ATPase activity of actomyosin reconstituted from myosin and Mommaerts' F-actin free from ADP were almost the same as those of the control actomyosin. Furthermore, this type of ADP free F-actin could bind to myosin to almost the same extent as the control F-actin. Therefore, it was concluded that the disappearance of actomyosin type deoxy-ATPase activity of actomyosin made with myosin and Kasai's F-actin free from ADP was not directly due to the removal of ADP but to a conformational change in actin occurring during the removal of nucleotide from G-actin by the method of Kasai et al.
When ATP was used as the substrate, the ATPase activity of myosin was considerably enhanced even by Kasai's F-actin free from ADP, and no significant difference in the enzymatic activities of the control actomyosin. and actomyosin free from ADP was observed, if the ratio of F-actin to myosin was higher than 1.5:8. This result suggests that ATP or ADP was incorporated into Kasai's F-actin during the superprecipitation of actomyosin.
