牛脑Ca^(2+)/CaM依赖性蛋白激酶Ⅱ的纯化和鉴定

Purification and Characterizaiton of Bovine Brain Ca^(2+)/CaM Dpendent Protein Kinase Ⅱ

通过一系列层析法,首次从牛脑纯化得到胶凝电泳匀一的Ca^(2+)/CaM PKⅡ。凝胶过滤法测定全酶分子量为550kD,SDS-PAGE法测定亚基分子量为55kD,推测牛脑Ca^(2+)/CaM PK Ⅱ由十个相同的亚基组成。该酶活性绝对依赖于Ca^(2+)和CaM,以63kD PDE同工酶为底物,其AC_(50)分别为0.85μmol/L和0.18μmol/L;以酪蛋白为底物,其AC_(50)分别为0.22μmol/L和0.06μmol/L。牛脑Ca^(2+)/CaM PK Ⅱ旣能催化63kD PDE同工酶等多种蛋白或酶磷酸化,又能进行自身磷酸化。该酶催化63kD PDE同工酶最大磷酸参入量为1mol/mol亚基。磷酸化型63kD PDE同工酶的Ca^(2+)的AC_(50)高于非磷酸化型。

Ca2+/CaM PK II has first been purified from bovine brain to homog-enety on SDS-PAGE by using combination of several column chromatographies. The molecular weight of the holoenzyme of the kinase determined by gel filtration was 550 kD and the subunit molecular weight determined by SDS-PAGE was 55 kD. The kinase therefore consisted of ten identical subunits. The kinase activity showed an absolute dependence on Ca2+ and CaM:the AC50 were 0.85 μmol/L and 0.18 μmol-/L using 63 kD PDE isozyme as a substrate respectively; the AC50 were 0.22UUUUmol/L and 0.06μmol/L L.Using casein as a substrate respectively Bovine brain Ca2+/CaM PK Ⅱ was found to autophosphorlate in addition to phosphorylating 63 kD PDE isozyme and the other protein or enzymes.The phosphorylation of 63 kD pDE isozyme by the bovine brain Ca2+/CaM PK Ⅱ resulted in the maximal incorporation of 1 mol phosphate per mol subunit of 63 kD PDE isozyme. The AC50 for Ca2+ of the phosphory-lated form of the 63 kD PDE isozyme was higher than that of the nonphosphoryla-ted form of the 63 kD PDE isozyme.