Higashi K, Fukunaga K, Matsui K, Maeyama M, Miyamoto E
Biochim Biophys Acta. 1983 Sep 28;747(3):232-40. doi: 10.1016/0167-4838(83)90102-4.
Myosin light-chain kinase was purified from porcine myometrium to apparent homogeneity at about 262-fold with an Mr of 130 000 as determined by SDS-polyacrylamide gel electrophoresis and a sedimentation coefficient of 4.5 S. The approximate content of the soluble myosin light-chain kinase was estimated to be about 0.85 microM. The purified enzyme exhibited strict substrate specificity only for 20-kDa myosin light chain and Ka values of 0.6 nM and 0.3 microM for calmodulin and Ca2+, respectively. The enzyme was phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase, which resulted in a decrease in the affinity for calmodulin of 4-7-fold without effect on the Vmax. The maximal amount of phosphate incorporated into the enzyme was 0.5-0.8 and 1.0-1.4 mol per mol of the enzyme in the presence and absence of Ca2+ and calmodulin, respectively. In the presence of a subsaturating concentration of calmodulin, the enzyme showed a lower sensitivity for Ca2+ by phosphorylation.
肌球蛋白轻链激酶从猪子宫肌层中纯化出来，达到了明显的均一性，纯化倍数约为262倍，经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定其分子量为130000，沉降系数为4.5 S。可溶性肌球蛋白轻链激酶的大致含量估计约为0.85微摩尔。纯化后的酶仅对20 kDa的肌球蛋白轻链表现出严格的底物特异性，对钙调蛋白和钙离子的解离常数分别为0.6纳摩尔和0.3微摩尔。该酶被环磷酸腺苷依赖性蛋白激酶的催化亚基磷酸化，这导致对钙调蛋白的亲和力降低4至7倍，而对最大反应速度无影响。在有和没有钙离子及钙调蛋白的情况下，每摩尔酶掺入的最大磷酸量分别为0.5至0.8摩尔和1.0至1.4摩尔。在钙调蛋白亚饱和浓度存在的情况下，该酶经磷酸化后对钙离子的敏感性降低。
