Identification of isoenzymic forms of hepatic Ca2+-activated-phospholipid-dependent protein kinase in various animal models.
We have examined the protein kinase C that are present in mouse, rat, guinea pig and rabbit liver. Initial subcellular fractionation analysis indicated that the majority (75-85%) of the activity was associated with particulate fraction of the liver. The bound protein kinase C was dissociated by homogenization of livers in buffer containing EGTA, EDTA and various proteolytic inhibitors and the solubilized extract was used to resolve multiple forms of the enzyme. The fractionation procedure, sequentially utilized (NH4)2SO4 precipitation, ion exchange chromatography, gel permeation chromatography, and hydroxylapatite column chromatography. With hydroxylapatite, protein kinase C was resolved into three isoenzymic forms designated C-I, C-II and C-III. In each case, the predominant activity consisted of C-II and C-III and together they represented about 80-88% of the total activity. All three isoenzymes from each source demonstrated an absolute requirement for PS + Ca2+ (approximately 25-50 fold stimulation over basal activity); for maximal activity the isoenzymes also required the presence of divalent metal ion, Mg2+ (5-10 mM) and lysine rich histone (H1). Both diolein and TPA decreased the Ca2+ and PS requirement of the enzyme and directly stimulated enzyme activity in the presence of suboptimal concentrations of Ca2+ and PS. In conclusion, the present studies suggest that protein kinase C in mammalian liver exists in isoenzymic forms.
Department of Medicine, Stanford University School of Medicine, California.
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Azhar
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Butte
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Reaven
D000818:Animals; D002118:Calcium / Q000378:metabolism; D004075:Diglycerides / Q000378:metabolism; D006168:Guinea Pigs; D007527:Isoenzymes / Q000032:analysis*; D008099:Liver / Q000201:enzymology*; D008297:Male; D051379:Mice; D010743:Phospholipids / Q000378:metabolism; D011493:Protein Kinase C / Q000032:analysis*; D011817:Rabbits; D051381:Rats; D013045:Species Specificity; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism
D004075:Diglycerides; D007527:Isoenzymes; D010743:Phospholipids; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium