PROTEIN-KINASE-C FROM RAINBOW-TROUT BRAIN - IDENTIFICATION AND CHARACTERIZATION OF 3 ISOZYMES

Free and membrane-associated fractions of protein kinase C (PKC) from rainbow trout (Onchorynchus mykiss) brain tissue were separated by hyd roxylapatite chromatography and characterized kinetically. In both res ting fish and in fish swum to exhaustion, approximately 40% of the tot al PKC activity was bound to membranes. Quantification of the three di stinct hydroxylapatite chromatography peaks (PKC types gamma, beta and a) in cytosolic and membrane fractions revealed different isozyme dis tributions. The cytosolic fraction contained 21% PKC type gamma, 52% P KC type beta and 27% PKC type alpha. The membrane-associated fraction contained 23% PKC type gamma, 28% PKC type beta and 49% PKC type alpha . Kinetic characterization of the three isozymes showed that PKC type gamma was almost completely activated by Ca2+ alone whereas PKC type b eta and PKC type alpha were 40% and 60% activated by Ca2+. Full activi ty for all enzymes was observed only in the presence of phosphatidylse rine and diacylglycerol. Differences in the kinetic constants for the three isozymes were also apparent. PKC type gamma had a much lower aff inity for Histone III-S when compared with PKC types beta and alpha (1 00 mu g/ml as compared with 1.7 and 5.7 mu g/ml). PKC type gamma also had a lower affinity for calcium (0.22 mu M) when compared with PKC ty pe beta (0.08 mu M) and PKC type alpha (0.05 mu M). PKC type alpha had a lower affinity for phosphatidylserine (8.6 mu g/ml) when compared w ith PKC type gamma (0.37 mu g/ml) and PKC type beta (0.89 mu g/ml).