Purification of modulator-deficient myosin light-chain kinase by modulator protein-Sepharose affinity chromatography.
Purification of modulator-deficient myosin light-chain kinase by modulator protein-Sepharose affinity chromatography.
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通过调节蛋白-琼脂糖亲和层析纯化调节剂缺陷型肌球蛋白轻链激酶。
DOI：
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发表时间：
1978
期刊：
影响因子：
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通讯作者：
K. Yagi
中科院分区：
文献类型：
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作者：
M. Yazawa;K. Yagi
用调节蛋白-Sepharose 4 B亲和层析法从兔骨骼肌中纯化了缺乏调节蛋白的肌球蛋白轻链激酶。纯化的蛋白质在十二烷基硫酸钠中的聚丙烯酰胺凝胶电泳上显示单一条带（MW 80，000），并且如通过凝胶过滤所确定的，其以天然状态作为单体存在。缺乏调节剂的肌球蛋白轻链激酶（MW 80，000）、调节剂蛋白（MW 16，500）和Ca 2+是激酶活性所必需的。在过量调节蛋白与10 mM MgCl 2存在下，激酶的半最大活性为pCa 5.1，其中在肌钙蛋白-原肌球蛋白系统存在下观察到肌动球蛋白-ATP酶的完全活性。假设肌球蛋白轻链激酶与两种底物ATP和g2轻链之间快速平衡，则ATP和g2轻链的Km值分别评估为0.28 mM和0.024 mM。Vm/e为5.7 s-1。
Modulator-deficient myosin light-chain kinase from rabbit skeletal muscle was purified by modulator protein-Sepharose 4B affinity chromatography. The purified protein showed a single band (MW 80,000) on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, and it exists as a monomer in the native state as determined by gel filtration. The modulator-deficient myosin light-chain kinase (MW 80,000), modulator protein (MW 16,500) and Ca2+ were essential for the kinase activity. The half-maximal activity of the kinase in the presence of excess modulator protein with 10 mM MgCl2 was at pCa 5.1, where full activity of actomyosin-ATPase is observed in the presence of the troponin--tropomyosin system. Assuming a rapid equilibrium between myosin light-chain kinase and two substrates, ATP and g2 light-chain, Km values for ATP and g2 light chain were evaluated as 0.28 mM and 0.024 mM, respectively. Vm/e was 5.7 s-1.