Son M, Gundersen R E, Nelson D L
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison 53706.
J Biol Chem. 1993 Mar 15;268(8):5940-8.
The ciliated protozoan Paramecium tetraurelia contains two protein kinase activities (CaPK-1 and CaPK-2) that are dependent on Ca2+ (Gundersen, R. E., and Nelson, D. L. (1987) J. Biol. Chem. 262, 4602-4609). We purified Ca(2+)-dependent protein kinase-1 (CaPK-1) 1,000-fold from the EGTA-extracted soluble fractions of Paramecium. The purified enzyme was a single polypeptide of 52 kDa on SDS-polyacrylamide gel electrophoresis with a native molecular mass of 60,000, suggesting that the active enzyme is a monomer. The purified kinase used casein as the best substrate in vitro, and its activity was absolutely dependent on Ca2+. The physical, catalytic and regulatory properties were clearly distinct from those of casein kinase, protein kinase C, and Ca2+/calmodulin-dependent protein kinases. CaPK-1 was half maximally activated by submicromolar (0.2 microM) free Ca2+, and the purified kinase bound Ca2+ in a blot overlay assay. CaPK-1 and the previously characterized CaPK-2 were biochemically and immunologically different enzymes sharing a similar activation mechanism. CaPK-1 and CaPK-2 appear to be members of a new family of Ca(2+)-dependent protein kinases. A protein immunologically related to the CaPKs was also detected in rat brain.
纤毛原生动物四膜虫含有两种依赖于Ca2+的蛋白激酶活性（CaPK-1和CaPK-2）（冈德森，R.E.，和纳尔逊，D.L.（1987年）《生物化学杂志》262，4602 - 4609）。我们从四膜虫经乙二醇双四乙酸（EGTA）提取的可溶性组分中，将依赖于Ca2+的蛋白激酶-1（CaPK-1）纯化了1000倍。纯化后的酶在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS - PAGE）上是一条52 kDa的单条多肽链，天然分子量为60,000，这表明活性酶是单体。纯化后的激酶在体外以酪蛋白作为最佳底物，其活性绝对依赖于Ca2+。其物理、催化和调节特性与酪蛋白激酶、蛋白激酶C以及依赖于Ca2+/钙调蛋白的蛋白激酶明显不同。CaPK-1在亚微摩尔浓度（0.2 microM）的游离Ca2+作用下被激活至最大活性的一半，并且纯化后的激酶在印迹覆盖分析中能结合Ca2+。CaPK-1和先前已鉴定的CaPK-2是生化和免疫特性不同但具有相似激活机制的酶。CaPK-1和CaPK-2似乎是一个新的依赖于Ca2+的蛋白激酶家族的成员。在大鼠脑中也检测到了一种与CaPKs免疫相关的蛋白。
