Walsh M P, Vallet B, Autric F, Demaille J G
J Biol Chem. 1979 Dec 10;254(23):12136-44.
Myosin light chain kinase, which is located primarily in the soluble fraction of bovine myocardium, has been isolated and purified approximately 1200-fold with 16% yield by a three-step procedure. The approximate content of soluble myosin light chain kinase in heart is calculated to be 0.63 microM. The isolated kinase is active only as a ternary complex consisting of the kinase, calmodulin, and Ca2+; the apparent Kd for calmodulin is 1.3 nM. The enzyme also exhibits a requirement for Mg2+ ions. Myosin light chain kinase is a monomeric enzyme with Mr = 85,000. The enzyme exhibits a Km for ATP of 175 microM, and a K0.5 for the regulatory light chain of cardiac myosin of 21 microM. The optimum pH is 8.1. Kinase activity is specific for the regulatory light chain of myosin. The specific activity of the isolated enzyme (30 nmol 32P/min/mg of protein) is considerably less than and corresponding values reported for the skeletal and smooth muscle light chain kinases. This is probably due to proteolysis during extraction of the myocardium, a phenomenon which has, as yet, proven impossible to eliminate. In contrast to the smooth muscle enzyme (Adelstein, R.S., Conti, M.A., Hathaway, D.R., and Klee, C.B. (1978) J. Biol. Chem. 253, 8347-8350), the cardiac kinase is not phosphorylated by the catalytic subunit of cAMP-dependent protein kinase.
肌球蛋白轻链激酶主要存在于牛心肌的可溶性部分，通过三步法已将其分离纯化，纯化倍数约为1200倍，产率为16%。心脏中可溶性肌球蛋白轻链激酶的大致含量经计算为0.63微摩尔。分离出的激酶仅作为由激酶、钙调蛋白和Ca2+组成的三元复合物具有活性；钙调蛋白的表观解离常数（Kd）为1.3纳摩尔。该酶还表现出对Mg2+离子的需求。肌球蛋白轻链激酶是一种单体酶，相对分子质量（Mr）为85,000。该酶对ATP的米氏常数（Km）为175微摩尔，对心肌肌球蛋白调节轻链的半最大激活浓度（K0.5）为21微摩尔。最适pH为8.1。激酶活性对肌球蛋白的调节轻链具有特异性。分离出的酶的比活性（30纳摩尔32P/分钟/毫克蛋白质）远低于骨骼肌和平滑肌轻链激酶报道的相应值。这可能是由于心肌提取过程中的蛋白水解作用，这种现象至今仍无法消除。与平滑肌酶（阿德尔斯坦，R.S.，孔蒂，M.A.，海瑟薇，D.R.，和克利，C.B.（1978年）《生物化学杂志》253，8347 - 8350）不同，心脏激酶不会被环磷酸腺苷（cAMP）依赖性蛋白激酶的催化亚基磷酸化。
