Bartelt D C, Moroney S, Wolff D J
Department of Pharmacology, UMDNJ-Robert Wood Johnson Medical School, Piscataway 08854.
Biochem J. 1987 Nov 1;247(3):747-56. doi: 10.1042/bj2470747.
A substrate-specific calmodulin-dependent myosin light-chain kinase (MLCK) was purified 45,000-fold to near homogeneity from bovine brain in 12% yield. Bovine brain MLCK phosphorylates a serine residue in the isolated turkey gizzard myosin light chain (MLC), with a specific activity of 1.8 mumol/min per mg of enzyme. The regulatory MLC present in intact gizzard myosin is also phosphorylated by the enzyme. The Mr-19,000 rabbit skeletal-muscle MLC is a substrate; however, the rate of its phosphorylation is at best 30% of that obtained with turkey gizzard MLC. Phosphorylation of all other protein substrates tested is less than 1% of that observed with gizzard MLC as substrate. SDS/polyacrylamide-gel electrophoresis of purified MLCK reveals the presence of a major protein band with an apparent Mr of 152000, which is capable of binding 125I-calmodulin in a Ca2+-dependent manner. Phosphorylation of MLCK by the catalytic subunit of cyclic-AMP-dependent protein kinase results in the incorporation of phosphate into the Mr-152,000 protein band and a marked decrease in the affinity of MLCK for calmodulin. The presence of Ca2+ and calmodulin inhibits the phosphorylation of the enzyme. Bovine brain MLCK appears similar to MLCKs isolated from platelets and various forms of muscle.
一种底物特异性钙调蛋白依赖性肌球蛋白轻链激酶（MLCK）从牛脑中纯化出来，纯化倍数达45000倍，产率为12%，纯度接近均一。牛脑MLCK能使分离出的火鸡砂囊肌球蛋白轻链（MLC）中的一个丝氨酸残基磷酸化，每毫克酶的比活性为1.8 μmol/分钟。完整砂囊肌球蛋白中的调节性MLC也能被该酶磷酸化。Mr为19000的兔骨骼肌MLC是一种底物；然而，其磷酸化速率最高仅为火鸡砂囊MLC磷酸化速率的30%。所测试的所有其他蛋白质底物的磷酸化程度均低于以砂囊MLC为底物时观察到的1%。纯化的MLCK经十二烷基硫酸钠/聚丙烯酰胺凝胶电泳显示存在一条主要蛋白带，其表观Mr为152000，该蛋白带能够以Ca2+依赖的方式结合125I-钙调蛋白。环磷酸腺苷依赖性蛋白激酶的催化亚基对MLCK的磷酸化导致磷酸掺入Mr为152000的蛋白带，并使MLCK对钙调蛋白的亲和力显著降低。Ca2+和钙调蛋白的存在会抑制该酶的磷酸化。牛脑MLCK似乎与从血小板和各种肌肉形式中分离出的MLCK相似。
