Miglietta L A, Nelson D L
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison 53706.
J Biol Chem. 1988 Nov 5;263(31):16096-105.
An unusual monomeric cGMP-dependent protein kinase, enriched in cilia, was isolated from Paramecium cilia and whole cells. Cilia and whole cell extracts had relatively high ratios of cGMP-dependent to cAMP-dependent protein kinase activity (1:2). The calculated molecular weight of the native enzyme was 88,000. The enzyme was identified on sodium dodecyl sulfate-polyacrylamide gels as a 77,000 molecular weight band based on copurification of this protein with enzyme activity, 8-N3-[32P]cAMP labeling, and autophosphorylation. Based on the size of the native enzyme, it was concluded that the kinase is a monomer with cGMP-binding and catalytic activities on the same polypeptide. Dimer-sized cGMP-dependent protein kinase, like that of the well characterized mammalian enzyme, was never seen, despite stringent efforts to control proteolysis. The structure of the Paramecium cGMP-dependent protein kinase supports a model in which the dimeric vertebrate form of the enzyme evolved from an early monomeric form. The catalytic properties of the Paramecium enzyme differed in several respects from those of the mammalian enzyme: it could use GTP or ATP as the phosphoryl donor, it did not phosphorylate Kemptide effectively, and it had poor histone kinase activity with high Mg2+ concentrations. Quercertin, 5'-guanylyl imidodiphosphate, indomethacin, and the isoquinolinesulfonamide drug H7 inhibited Paramecium cGMP-dependent protein kinase activity. The enzyme had fast and slow binding sites (with kd values of 5-10 x 10(-3)s-1 and 0.44 x 10(-3)s-1) and showed an order of preference for cyclic nucleotides and cyclic nucleotide analogs similar to that of the mammalian enzyme.
从草履虫纤毛和全细胞中分离出一种不同寻常的单体环鸟苷酸依赖性蛋白激酶，该激酶在纤毛中含量丰富。纤毛提取物和全细胞提取物中环鸟苷酸依赖性蛋白激酶活性与环腺苷酸依赖性蛋白激酶活性的比例相对较高（1:2）。天然酶的计算分子量为88,000。基于该蛋白与酶活性的共纯化、8-N3-[32P]环腺苷酸标记以及自身磷酸化，在十二烷基硫酸钠-聚丙烯酰胺凝胶上鉴定该酶为分子量77,000的条带。根据天然酶的大小，得出该激酶是一种在同一多肽上具有环鸟苷酸结合和催化活性的单体。尽管严格控制了蛋白水解，但从未见过二聚体大小的环鸟苷酸依赖性蛋白激酶，就像已充分表征的哺乳动物酶那样。草履虫环鸟苷酸依赖性蛋白激酶的结构支持一种模型，即该酶的二聚体脊椎动物形式是从早期的单体形式进化而来的。草履虫酶的催化特性在几个方面与哺乳动物酶不同：它可以使用鸟苷三磷酸或三磷酸腺苷作为磷酰供体，它不能有效地磷酸化肯普肽，并且在高镁离子浓度下组蛋白激酶活性较差。槲皮素、5'-鸟苷酰亚氨基二磷酸、吲哚美辛和异喹啉磺酰胺药物H7抑制草履虫环鸟苷酸依赖性蛋白激酶活性。该酶具有快速结合位点和慢速结合位点（解离常数分别为5 - 10×10(-3)s-1和0.44×10(-3)s-1），并且对环核苷酸和环核苷酸类似物的偏好顺序与哺乳动物酶相似。
