Studies on the regulation of protein kinase C by degradation of protein kinase C-activator

当在培养的人或小鼠成纤维细胞中加入蛋白激酶C （C-激酶）激活剂TPA时，细胞构象发生改变，细胞的egf结合活性降低。然而，一段时间后，即使细胞中仍有大量的结合TPA，这种作用也会消失。同时，细胞失去了对TPA的反应性。通过降解将TPA从这些细胞中去除带来了反应性的恢复。对这些现象进行了分析，结果表明，c -激酶的活化之后是酶的失活，而活化剂的去除在c -激酶的再活化中具有矛盾的积极作用。另一方面，对于TPA刺激的多形核白细胞产生的超氧化物，没有观察到去除TPA的这种积极作用。这些结果似乎表明，c -激酶在激活后是不可逆的失活，TPA抑制了酶的供应。或者，失活可能是可逆的，酶通过去除TPA获得原始的TPA活化状态。用纯化的c -激酶进行体外实验，分析其恢复机制。然而，他们没有成功，因为污染的钙蛋白酶活性不能从酶制剂中去除。在动物血清中发现了一种新的酯酶，这种酯酶可能参与体内TPA的降解，从而在TPA的重复施用过程中激活c激酶。从小鼠血清中纯化该酶，并对其酶学、化学和生物学特性进行了研究。纯化后的酶用于上述研究。此外，还发现了一种特异性的酯酶抑制剂，该抑制剂通过与酯酶共存来调节血清酶活性。它具有脂质性质，并对其进行了部分纯化和表征。

When TPA, an activator of protein kinase C (C-kinase) was added to cultured human or mouse fibroblasts, the cell conformation changed and EGF-bindingactivity of the cells decreased. However, such TPA effect disapeared after a while even when the cells still had large amount of bound TPA. At the sametime, the cells lost their reactivity to TPA. Removal of TPA from these cells by degradation brought bout recovery of the reactivity. Analyses of these phenomena were carried out and the results indicated that activation of C-kinase was followed by inactivation of the enzyme and that removal of the activator had a paradoxically positive role in the reactivation of C-kinase. On the otherhand, such positive effect of removal of TPA was not observed with regard tosuper oxide generation by TPA-stimulated polymorphonuclear leukocytes. These results seem to show that C-kinase is irreversibly inactivated after the activation and the supply of the enzyme is inhibited by TPA. Alternatively, it may be that inactivation is reversible and the enzyme acquires the original TPA-activatable status by removal of TPA. In vitro experiments using purified C-kinase was performed to analyze the mechanism of the recovery. They were notsuccessful, however, because contaminating calpain activity could not be removed from the enzyme preparation. A novel esterase which may take part in degradation of TPA in vivo, and therefore in activation of C-kinase during repeated administration of TPA, was demonstrated in sera from animals. The enzyme wasprufied from mouse serum and its enzymological, chemical and biological characteristics were clarified. The purified enzyme was utilized in the studies mentioned above. Also, an inhibitor specific for this esterase and which regulates the serum enzyme activity by co-existing with the esterase in the sera was found. It was of lipid nature and its partial purification and characterization was carried out.