Identification of two distinct populations of protein kinase C in rat brain membranes.
The regulatory enzyme protein kinase C (PKC) is proposed to be activated on its translocation from the cytosol to the membrane. However, a portion of the native activity is always associated with the membrane fraction. Using a noninvasive procedure to extract this endogenous activity from rat brain membranes, it has been possible to characterize the activity in a partially purified reconstituted system bearing resemblance to the in vivo system. Two subpopulations of membrane-associated PKC were identified and characterized at the level of activation, inhibition, and isozyme immunologic characteristics and chromatographic properties. One peak had properties similar to those of cytosolic PKC, whereas the second population, extracted as protein-lipid complexes, had considerable constitutive activity that could be stimulated further on addition of PKC activators. This latter activity was relatively resistant to staurosporine inhibition and phorbol ester treatment, but it phosphorylated the exogenous PKC substrates, histone 1 and the epidermal growth factor receptor peptide KTRLRR. The constitutive activity was totally dependent on its endogenous associated lipids coextracted by the solubilization procedure. The ratio between these two populations was ontogenetically regulated and modulated by phorbol ester treatment, suggesting that different PKC populations may serve unique functions in the rat brain regulated by the lipid environment. Analyses of the phospholipids extracted in these protein-lipid complexes showed differences in the major classes correlating to age. However, apart from a markedly lower cholesterol content in these complexes, no direct relationship between a specific lipid composition and the amount of constitutive PKC activity was evident.
Orr
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N
Department of Membrane Research, Weizmann Institute of Science, Rehovot, Israel.
Yavin
E
E
Lester
D S
DS
D000818:Animals; D000911:Antibodies, Monoclonal; D001921:Brain / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D007527:Isoenzymes; D008297:Male; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D051381:Rats; D011919:Rats, Inbred Strains; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate