ISOLATION AND CHARACTERIZATION OF THE EPSILON-SUBSPECIES OF PROTEIN-KINASE-C FROM RAT-BRAIN

The epsilon-subspecies of protein kinase C (epsilon-PKC) was purified to near homogeneity from the soluble fraction of rat brain by successive chromatographies on DEAE-cellulose, threonine-Sepharose, phenyl-5PW, Mono Q, heparin-5PW, and hydroxyapatite columns. The enzyme from COS-7 cells that were transfected with an epsilon-PKC cDNA expression plasmid showed the same elution profile. The purified enzyme from the brain was a doublet (96 and 93 kDa) on SDS/PAGE. Both the doublet proteins were recognized by antibodies raised against several oligopeptides that were parts of the deduced amino acid sequence of the rat brain epsilon-PKC. When treated with potato acid phosphatase, both doublet proteins disappeared with the concomitant appearance of a single protein at 90 kDa, suggesting that epsilon-PKC exists in the tissue as phosphorylated forms. The physiological significance of this phosphorylation is unknown. The enzymes from the rat brain and COS-7 cells were indistinguishable from each other in their kinetic and catalytic properties. Unlike alpha-, beta-I-, beta-II-, and gamma-PKC, epsilon-PKC was independent of Ca2+ but absolutely required phosphatidyl-serine and diacylglycerol for its activation; a tumor-promoting phorbol ester could replace diacylglycerol. Epsilon-PKC showed enzymological properties similar to those of delta-PKC, except that epsilon-PKC but not delta-PKC was greatly activated by free arachidonic acid. Immunoblot analysis revealed that, in marked contrast to delta-PKC, epsilon-PKC is expressed predominantly in the brain tissue and only in trace amounts in heart, lung, spleen, thymus, and testis.