Vallano M L
Department of Pharmacology, SUNY/Health Science Center, Syracuse 13210.
J Neurosci Methods. 1989 Oct;30(1):1-9. doi: 10.1016/0165-0270(89)90067-8.
Three distinct isozymes of a type II calcium/calmodulin-dependent protein kinase (CaM kinase II) from rat forebrain cytosol were separated using S-Sepharose cation-exchange resin. About 90% of the applied kinase activity was recovered in three protein peaks. Each isozymic form of the kinase was purified 200-300 fold by chromatography on S-Sepharose, calmodulin-affinity and gel filtration resins. All 3 forms of CaM kinase II had apparent molecular masses of 650-700 kDa, but contained variable proportions of 50 kDa and 58-60 kDa subunits. The molar ratios of the 50 kDa/58-60 kDa kinase subunits in each holoenzyme were determined by protein staining and [125I]calmodulin-binding studies and were approximately: 6/1, 3/1 and 1/1. The isozyme containing a 3/1 ratio of subunits corresponds to the predominant form of CaM kinase II in forebrain representing 70-80% of the total activity in cytosol, whereas the other forms each represent 5-10% of the total cytosolic activity. The substrate specificities and time courses of substrate phosphorylation for the isozymes were comparable. These studies provide a basis to examine regional, subcellular, and developmental differences in the isozymic forms of CaM kinase II which may subserve different neuronal functions.
利用S-Sepharose阳离子交换树脂分离了来自大鼠前脑胞质溶胶的三种不同的II型钙/钙调蛋白依赖性蛋白激酶（CaM激酶II）同工酶。大约90%的外加激酶活性在三个蛋白峰中得以回收。通过在S-Sepharose、钙调蛋白亲和和凝胶过滤树脂上进行层析，激酶的每种同工酶形式都被纯化了200 - 300倍。CaM激酶II的所有三种形式的表观分子量为650 - 700 kDa，但含有不同比例的50 kDa和58 - 60 kDa亚基。通过蛋白质染色和[125I]钙调蛋白结合研究确定了每种全酶中50 kDa/58 - 60 kDa激酶亚基的摩尔比，分别约为：6/1、3/1和1/1。亚基比例为3/1的同工酶对应于前脑中CaM激酶II的主要形式，占胞质溶胶总活性的70 - 80%，而其他形式各占胞质溶胶总活性的5 - 10%。这些同工酶的底物特异性和底物磷酸化的时间进程具有可比性。这些研究为研究CaM激酶II同工酶形式在区域、亚细胞和发育方面的差异提供了基础，这些差异可能具有不同的神经元功能。
