Okagaki T, Higashi-Fujime S, Ishikawa R, Takano-Ohmuro H, Kohama K
Department of Molecular Biology, Faculty of Science, Nagoya University, Aichi.
J Biochem. 1991 Jun;109(6):858-66. doi: 10.1093/oxfordjournals.jbchem.a123471.
ATP-dependent movement of actin filaments on smooth muscle myosin was investigated by using the in vitro motility assay method in which myosin was fixed on the surface of a coverslip in a phosphorylated or an unphosphorylated state. Actin filaments slid on gizzard myosin phosphorylated with myosin light chain kinase (MLCK) at a rate of 0.35 micron/s, but did not slide at all on unphosphorylated myosin. The movement of actin filaments on phosphorylated myosin was stopped by perfusion of phosphatase. Subsequent perfusion with a solution containing MLCK, calmodulin, and Ca2+ enabled actin filaments to move again. The sliding velocities on monophosphorylated and diphosphorylated myosin by MLCK were not different. Actin filaments did not move on myosin phosphorylated with protein kinase C (PKC). The sliding velocity on myosin phosphorylated with both MLCK and PKC was identical to that on myosin phosphorylated only with MLCK. Gizzard tropomyosin enhanced the sliding velocity to 0.76 micron/s. Gizzard caldesmon decreased the sliding velocity with increase in its concentration. At a 5-fold molar ratio of caldesmon to actin, the movement stopped completely. This inhibitory effect of caldesmon was relieved upon addition of excess calmodulin and Ca2+.
通过体外运动分析方法研究了肌动蛋白丝在平滑肌肌球蛋白上依赖ATP的运动，该方法是将肌球蛋白以磷酸化或未磷酸化状态固定在盖玻片表面。肌动蛋白丝在经肌球蛋白轻链激酶（MLCK）磷酸化的砂囊肌球蛋白上以0.35微米/秒的速度滑动，但在未磷酸化的肌球蛋白上根本不滑动。磷酸酶灌注可使肌动蛋白丝在磷酸化肌球蛋白上的运动停止。随后用含有MLCK、钙调蛋白和Ca2+的溶液灌注可使肌动蛋白丝再次运动。MLCK对单磷酸化和双磷酸化肌球蛋白的滑动速度没有差异。肌动蛋白丝在蛋白激酶C（PKC）磷酸化的肌球蛋白上不运动。同时用MLCK和PKC磷酸化的肌球蛋白的滑动速度与仅用MLCK磷酸化的肌球蛋白的滑动速度相同。砂囊原肌球蛋白将滑动速度提高到0.76微米/秒。砂囊钙调蛋白随着其浓度增加而降低滑动速度。当钙调蛋白与肌动蛋白的摩尔比为5倍时，运动完全停止。加入过量的钙调蛋白和Ca2+后，钙调蛋白的这种抑制作用得到缓解。
