Isolation and Characterization of the ε Subspecies of Protein Kinase C from Rat Brain

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 89; no. 4; pp. 1149 - 1153
• Main Authors: Koide, Hiroshi, Ogita, Kouji, Kikkawa, Ushio, Nishizuka, Yasutomi
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 15-02-1992; National Acad Sciences
• Subjects: Alkaloids - pharmacology; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Antibodies; Arachidonic Acid - pharmacology; Biochemistry; Biological and medical sciences; brain; Brain - enzymology; Calcium - pharmacology; Carbazoles - pharmacology; Cells, Cultured; Cercopithecus aethiops; Chromatography; COS cells; Diglycerides; Enzyme Activation - drug effects; Enzymes; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Gene Expression; Homogenization; Indole Alkaloids; Kinetics; Male; Molecular Sequence Data; Peptides - chemistry; Phosphatases; Phospholipids; Phospholipids - pharmacology; phosphorylation; Protein Kinase C - antagonists & inhibitors; Protein Kinase C - isolation & purification; Rats; Rats, Inbred Strains; Recombinant Proteins - isolation & purification; Research facilities; RNA, Messenger - genetics; Staurosporine; Substrate Specificity; Transferases; Molecular form; Protein kinase C; Rat; Enzyme; Rodentia; Activation; Arachidonic acid; Vertebrata; Mammalia; Enzymatic activity; Substrate specificity; Isolation; Kinetics; Brain (vertebrata)
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.89.4.1149

The ε subspecies of protein kinase C (ε 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 ε 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 ε PKC. When treated with potato acid phosphatase, both doublet proteins disappeared with the concomitant appearance of a single protein at 90 kDa, suggesting that ε 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 α-, βI-, βII-, and γPKC, εPKC was independent of Ca2+but absolutely required phosphatidylserine and diacylglycerol for its activation; a tumor-promoting phorbol ester could replace diacylglycerol. εPKC showed enzymological properties similar to those of δPKC, except that εPKC but not δPKC was greatly activated by free arachidonic acid. Immunoblot analysis revealed that, in marked contrast to δPKC, εPKC is expressed predominantly in the brain tissue and only in trace amounts in heart, lung, spleen, thymus, and testis.