Purification and characterization of Novikoff ascites tumor protein kinase.
Purification and characterization of Novikoff ascites tumor protein kinase.
复制标题
Novikoff 腹水肿瘤蛋白激酶的纯化和表征。
作者：
M. Dahmus;J. Natzle
一种蛋白激酶，设计为KII，已经从Novikoff腹水肿瘤细胞中纯化了5000倍。该纯化程序还允许纯化第二种主要蛋白激酶，命名为KI，以及RNA聚合酶I和II。纯化的KII具有7.6 S的沉降常数和39 A的斯托克斯半径，表明分子量为约122000。在十二烷基硫酸钠存在下的酶的聚丙烯酰胺凝胶电泳表明，该酶是由分子量为44 000，40 000，和26 000的亚基存在的摩尔比为1：1：2。在[γ-32 P]ATP存在下单独孵育酶导致26 000-道尔顿亚基磷酸化。蛋白激酶II能使卵黄蛋白、酪蛋白和非组蛋白染色体蛋白质磷酸化，但不能使碱性蛋白质如组蛋白或鱼精蛋白磷酸化到可感知的程度。在4 mM Mg 2+存在下测定ATP的Km值为3.6 μ M，GTP的Km值为6.5 μ M。该酶既不受环腺苷酸或环鸟苷酸的刺激，也不受兔肌蛋白激酶调节亚基的抑制。其活性在浓度低于0.2 M时受KCl刺激，在较高浓度时受抑制。
A protein kinase, designed KII, has been purified 5000-fold from Novikoff ascites tumor cells. The purification procedure also allows for the purification of a second major protein kinase, designated KI, as well as RNA polymerase I and II. Purified KII has a sedimentation constant of 7.6 S and a Stokes radius of 39 A, suggesting a molecular weight of about 122000. Polyacrylamide gel electrophoresis of the enzyme in the presence of sodium dodecyl sulfate suggests the enzyme is composed of subunits of molecular weights 44 000, 40 000, and 26 000 present in a molar ratio of 1:1:2. Incubation of the enzyme alone in the presence of [gamma-32P]ATP results in the phosphorylation of the 26 000-dalton subunit. Protein kinase II actively phosphorylates phosvitin, casein, and nonhistone chromosomal proteins but does not phosphorylate basic proteins such as histones or protamine to an appreciable extent. Km values of 3.6 micron for ATP and 6.5 micronM for GTP were determined in the presence of 4mM Mg2+. The enzyme is neither stimulated by cyclic adenosine 3',5'-monophosphate or cyclic guanosine 3', 5'-monophosphate nor inhibited by the regulatory subunit of rabbit muscle protein kinase. Its activity is stimulated by KCl at concentrations below 0.2 M and inhibited by higher concentrations.