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The Journal of Biochemistry
Vol. 93 (1983) No. 1 P 205-223

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In an attempt to elucidate the Ca2+-regulated mechanism of motility in Physarum plasmodia, we improved the preparation method for myosin B and pure myosin. The obtained results are as follows:
1. We obtained two types of myosin B which are distinguishable from each other with respect to their sensitivity to Ca2+. The inactive type of myosin B had low superprecipitation activities both in the presence and in the absence of Ca2+. The active type showed very high superprecipitation activity in EGTA, and the activity was conspicuously inhibited by Ca2+. The active type was converted into the inactive type by treatment with potato acid phosphatase. Also the inactive type or the phosphatase-treated active type was converted into the active type upon reacting with ATP-γ-S.
2. In the reaction with ATP-γ-S, only the myosin HC of myosin B was phospho-rylated. The phosphorylation was independent of Ca2+ and calmodulin, and the extent was about 1 mol/mol HC.
3. The Ca2+ sensitivity in the superprecipitation of the active type was not decreased by adding an excess amount of F-actin. Besides, the actin-activated Mg2+-ATPase activity of purified phosphorylated myosin was not Ca2+-Sensitive. Therefore, presence of a Ca2+-dependent inhibitory factor(s) that could bind to myosin was suggested.
4. The Mg2+-ATPase activity of purified phosphorylated myosin was 7-8 times enhanced by F-actin, but that of dephosphorylated myosin was hardly activated at all.
5. In a gel filtration in 0.5M KCl, phosphorylated myosin was eluted behind dephosphorylated myosin. Electron microscopy applying the rotary-shadow method showed significant difference in flexibility in the tail between phosphorylated and dephosphorylated myosin molecules.
6. In 40mM KCl and 5-10mM MgCl2, phosphorylated myosin formed thick filaments, but dephosphorylated myosin did not, whether there was ATP or not.
The above results clearly show that the phosphorylation of myosin HC is indispensable to ATP-induced superprecipitation, the actin-activated Mg2+-ATPase activity, and the formation of thick filaments of myosin. A myosin-linked factor(s) that inhibits an actin-myosin interaction in a Ca2+-dependent manner may exist.

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