Ganguly C, Baines I C, Korn E D, Sellers J
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1992 Oct 15;267(29):20900-4.
The actin-activated Mg(2+)-ATPase activity of filamentous Acanthamoeba myosin II is inhibited by phosphorylation of 3 serine residues at the tip of the tail of each heavy chain. From previous studies, it had been concluded that the activity of each molecule in the filament was regulated by the global state of phosphorylation of the filament and was independent of its own phosphorylation state. The actin-activated Mg(2+)-ATPase activity of monomeric phosphorylated myosin II was not known because it polymerizes under the ionic conditions necessary for the expression of this activity. We have now found conditions to maintain myosin II monomeric and active during the enzyme assay. The actin-activated Mg(2+)-ATPase activities of monomeric dephosphorylated and phosphorylated myosin II were found to be the same as the activity of filamentous dephosphorylated myosin II. These results support the conclusion that phosphorylation regulates filamentous myosin II by affecting filament conformation. Consistent with their equivalent enzymatic activities, monomeric and filamentous dephosphorylated myosin II were equally active in an in vitro motility assay in which myosin adsorbed to a surface drives the movement of F-actin. In contrast to their very different enzymatic activities, however, filamentous and monomeric phosphorylated myosin II had similar activities in the in vitro motility assay; both were much less active than monomeric and filamentous dephosphorylated myosin II. One interpretation of these results is that the rate-limiting steps in the two assays are different and that, while the rate-limiting step for actin-activated Mg(2+)-ATPase activity is regulated only at the level of the filament, the rate-limiting step for motility can also be regulated at the level of the monomer.
丝状棘阿米巴肌球蛋白II的肌动蛋白激活的Mg(2+)-ATP酶活性受到每条重链尾部末端3个丝氨酸残基磷酸化的抑制。根据之前的研究得出结论，细丝中每个分子的活性受细丝磷酸化的整体状态调节，且与其自身的磷酸化状态无关。单体磷酸化肌球蛋白II的肌动蛋白激活的Mg(2+)-ATP酶活性尚不清楚，因为它在表达该活性所需的离子条件下会聚合。我们现在已经找到了在酶测定过程中维持肌球蛋白II单体且有活性的条件。发现单体去磷酸化和磷酸化肌球蛋白II的肌动蛋白激活的Mg(2+)-ATP酶活性与丝状去磷酸化肌球蛋白II的活性相同。这些结果支持了磷酸化通过影响细丝构象来调节丝状肌球蛋白II的结论。与它们相当的酶活性一致，单体和丝状去磷酸化肌球蛋白II在体外运动测定中同样活跃，在该测定中吸附在表面的肌球蛋白驱动F-肌动蛋白的运动。然而，与它们非常不同的酶活性形成对比的是，丝状和单体磷酸化肌球蛋白II在体外运动测定中具有相似的活性；两者都比单体和丝状去磷酸化肌球蛋白II的活性低得多。这些结果的一种解释是，两种测定中的限速步骤不同，虽然肌动蛋白激活的Mg(2+)-ATP酶活性的限速步骤仅在细丝水平受到调节，但运动的限速步骤也可以在单体水平受到调节。
