Hathaway D R, Adelstein R S
Proc Natl Acad Sci U S A. 1979 Apr;76(4):1653-7. doi: 10.1073/pnas.76.4.1653.
In an actomyosin fraction isolated from human platelets, phosphorylation of the 20,000-dalton light chain of myosin is stimulated by calcium and the calcium-binding protein calmodulin. The enzyme catalyzing this phosphorylation has been isolated by using calmodulin-affinity chromatography. Platelet myosin light chain kinase activity was monitored throughout the isolation procedures by using the 20,000-dalton smooth muscle myosin light chain purified from turkey gizzards as substrate. The partially purified myosin kinase requires both calcium and calmodulin for activity and has a specific activity of 3.1 mumol of phosphate transferred to the 20,000-dalton light chain per mg of kinase per min under optimal assay conditions. Km values determined for ATP and myosin light chains are 121 microM and 18 microM, respectively. Of several substrates surveyed as phosphate acceptors (alpha-casein, histone II-A, phosphorylase b, protamine, histone V-S, and phosvitin), only the 20,000-dalton myosin light chain is phosphorylated at a significant rate. These results suggest that platelet myosin light chain kinase is a calcium-dependent enzyme and that the requirement for calcium is mediated by the calcium-binding protein calmodulin.
在从人血小板中分离出的肌动球蛋白组分中，肌球蛋白20,000道尔顿轻链的磷酸化受钙和钙结合蛋白钙调蛋白的刺激。通过使用钙调蛋白亲和层析已分离出催化这种磷酸化的酶。在整个分离过程中，以从火鸡砂囊中纯化的20,000道尔顿平滑肌肌球蛋白轻链作为底物来监测血小板肌球蛋白轻链激酶活性。部分纯化的肌球蛋白激酶的活性需要钙和钙调蛋白两者，并且在最佳测定条件下，其比活性为每毫克激酶每分钟有3.1微摩尔磷酸转移至20,000道尔顿轻链。测定的ATP和肌球蛋白轻链的Km值分别为121微摩尔和18微摩尔。在所检测的作为磷酸受体的几种底物（α-酪蛋白、组蛋白II-A、磷酸化酶b、鱼精蛋白、组蛋白V-S和卵黄高磷蛋白）中，只有20,000道尔顿的肌球蛋白轻链能以显著速率被磷酸化。这些结果表明血小板肌球蛋白轻链激酶是一种钙依赖性酶，并且对钙的需求是由钙结合蛋白钙调蛋白介导的。
