Construction and Application of Protein Kinase C Subspecies-specific Express ion Systems and Developmental Research for Screening of Selective Effectors

昆虫蛋白Idnase C（PKC）是一个由多个亚种组成的大家族。目前已鉴定出PKC的8个亚型（α、β I、β II、γ、δ、β I、β II、β I、β II、β II、β III、β II、β II、β III、β III、为了发现亚种特异性激活剂或抑制剂，必须纯化每个亚种。迄今为止，已从大鼠脑中纯化出PKC的α、β（β I + β II）和γ亚型，并阐明了它们的酶学性质。然而，其他亚种还没有得到很好的研究。在这项研究中，从大鼠脑中分离出PKC的δ，β和ζ亚种，并与COS-7表达系统中获得的PKC进行比较。制备亚种-太平洋抗肽抗血清并用于鉴定每个PKC亚种。为了阐明PKC亚型在细胞周期调控中的作用，我们还从S.酿酒酵母和非洲爪蟾卵母细胞。酵母酶被分离为一个单一的实体，并发现是非常不同的哺乳动物的酶，因为它是不激活的肿瘤促进佛波醇酯，虽然它被激活的磷脂酰丝氨酸的存在下，由二酰基甘油。另一方面，爪蟾PKC被分离为两种细微不同的酶。这两种蛋白质的酶学性质与哺乳动物的α-PKC非常相似，如对钙的依赖性和被佛波醇酯激活。然而，它们在用肿瘤促进剂处理的卵母细胞中的反应不同。也就是说，一个PKC消失非常迅速佛波酯治疗后，而其他保持不变，长达4小时。

The mammarian protein Idnase C (PKC) exists as a large family of multiple subspecies. At present, 8 subspecies of PKC (alpha, betaI, betaII, gamma, delta, epsilon, zeta, and eta (L) ) have been identified. To find out subspecies-specific activators or inhibitors, it is essential to purify each subspecies. Up to now, alpha, beta (betaI +betaII) and gamma subspecies of PKC have been purified from rat brain and their enzymological properties have been clarified. However, other subspecies have not been studied well. In this study, delta, epsilon, and zeta subspecies of PKC were isolated from rat brain and characterized in comparison with those obtained from COS-7 expression systems. Subspecies-pacific anti-peptide antisera were prepared and used to identify each PKC subspecies. The isolated enzymes were different from each other in the mode of activation by various substances and in substrate specificity.In order to clarify the roles of PKC subspecies in the cell cycle control, we also isolated PKC subspecies from S. cerevisiae and-from Xenopus oocytes. The yeast enzyme was isolated as a single entity and found to be quite different from mammalian enzymes in that it was not activated by the tumor-promoting phorbol esters, although it was activated by diacylglycerol in the presence of phosphatidylserine. On the other hand, the Xenopus PKC was isolated as two subtly different enzymes. Both of them were very similar to the mammalian alpha-PKC in their enzymological properties such as dependency on calcium and activation by phorbol esters. However, they responded differently in oocytes treated widi the tumor promoter. Namely, one PKC disappeared very rapidly upon phorbol ester treatment, while the other remained unchanged up to 4 hr.