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

蛋白激酶C激活剂降解调节蛋白激酶C的研究

基本信息

批准号：
61440031
负责人：
EGAWA KOHJI
金额：
$ 8.32万
依托单位：
THE UNIVERSITY OF TOKYO
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (A)
财政年份：
1986
资助国家：
日本
起止时间：
1986 至 1987
项目状态：
已结题

项目摘要

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.

将蛋白激酶C（C-激酶）激活剂TPA加入培养的人或小鼠成纤维细胞中，细胞构象发生变化，细胞与EGF结合活性降低。然而，这种TPA效应在一段时间后消失，即使细胞仍然具有大量结合的TPA。同时细胞对TPA失去反应性。通过降解从这些细胞中去除TPA带来了反应性的恢复。对这些现象进行了分析，结果表明，C-激酶的激活，随后由失活的酶和去除的激活剂有一个矛盾的积极作用，在C-激酶的重新激活。另一方面，在TPA刺激的多形核白细胞产生超氧化物方面，没有观察到去除TPA的这种积极作用。这些结果似乎表明，C-激酶在活化后不可逆地失活，并且酶的供应被TPA抑制。或者，失活可能是可逆的，并且酶通过去除TPA获得原始TPA可活化状态。使用纯化的C-激酶进行体外实验以分析恢复的机制。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。一种新的酯酶，它可能参与TPA在体内的降解，因此在TPA的重复给药过程中的C-激酶的激活，从动物血清中被证明。从小鼠血清中分离纯化了该酶，并对其酶学、化学和生物学特性进行了研究。纯化的酶用于上述研究。此外，还发现了一种对该酯酶有特异性的抑制剂，它通过与血清中的酯酶共存来调节血清酶的活性，它是脂类的，并对其进行了部分纯化和鉴定。