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

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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.