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 double (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 phosphatidylserine 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.
Koide
H
H
Department of Biochemistry, Kobe University School of Medicine, Japan.
Ogita
K
K
Kikkawa
U
U
Nishizuka
Y
Y
D000470:Alkaloids / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology; D001921:Brain / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D002227:Carbazoles / Q000494:pharmacology; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004789:Enzyme Activation / Q000187:drug effects; D015870:Gene Expression; D026121:Indole Alkaloids; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010743:Phospholipids / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011919:Rats, Inbred Strains; D011994:Recombinant Proteins / Q000302:isolation & purification; D019311:Staurosporine; D013379:Substrate Specificity
D000470:Alkaloids; D002227:Carbazoles; D026121:Indole Alkaloids; D010455:Peptides; D010743:Phospholipids; D012333:RNA, Messenger; D011994:Recombinant Proteins; D016718:Arachidonic Acid; C049985:staurosporine aglycone; D011493:Protein Kinase C; D019311:Staurosporine; D002118:Calcium