Isolation and Characterization of δ-Subspecies of Protein Kinase C from Rat Brain

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 89; no. 5; pp. 1592 - 1596
• Main Authors: Ogita, Kouji, Miyamoto, Shin-ichi, Yamaguchi, Keizo, Koide, Hiroshi, Fujisawa, Naoko, Kikkawa, Ushio, Sahara, Setsuko, Fukami, Yasuo, Nishizuka, Yasutomi
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 01-03-1992; National Acad Sciences
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Antibodies; Biochemistry; Biological and medical sciences; brain; Brain - enzymology; Cells, Cultured; Cercopithecus aethiops; Cloning, Molecular; COS cells; Elution; Enzymes; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Homogenization; In Vitro Techniques; Kinetics; Molecular Weight; Peptide Mapping; Phosphatases; Plasmids; Protein Kinase C - isolation & purification; Protein Kinase C - metabolism; Rats; Recombinant Proteins - metabolism; Research facilities; Substrate Specificity; Transferases; Vertebrata; Molecular form; Protein kinase C; Mammalia; Purification; Enzymatic activity; Rat; Enzyme; Rodentia; Brain (vertebrata)
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.89.5.1592

The δ-subspecies of protein kinase C (δ PKC) was purified to near homogeneity from the Triton X-100 extract of the rat brain particulate fraction by successive chromatographies on S-Sepharose fast flow, phenyl 5PW, heparin 5PW, hydroxyapatite, and Mono Q columns. The purified enzyme was a doublet with molecular masses of 78 and 76 kDa on SDS/PAGE. The doublet proteins were separated partially by Mono Q column chromatography; both were recognized by the antibodies raised against synthetic oligopeptides, parts of the deduced amino acid sequence of the rat δ PKC. Protein phosphatase 2A treatment suggested that the 78-kDa protein was a phosphorylated form of the 76-kDa protein. To confirm the structural and genetic identity of the doublet proteins, δ PKC was expressed in COS 7 cells by transfecting its cDNA-constructed plasmid and was purified for comparison. This recombinant enzyme was also a doublet. The enzymes isolated from the brain and COS 7 cells showed identical reactivities with δ PKC-specific antibodies, chromatographic behaviors, and V8 protease peptide mappings. In addition, these two enzyme preparations were indistinguishable from each other in their responses to phosphatidylserine, diacylglycerol, phorbol esters, free fatty acids, Ca2+, and enzyme inhibitors. Comparison was also made between the enzymologic properties of δ PKC and α PKC, which were distinctly different from each other.