Expression of the alpha, beta II, and gamma protein kinase C isozymes in the baculovirus-insect cell expression system. Purification and characterization of the individual isoforms.
Detailed in vitro comparisons of the biochemical characteristics of three protein kinase C isozymes were performed. As an alternative to earlier uncertain separation methods and expression schemes, highly purified and genetically distinct protein kinase C enzymes were produced using the baculovirus expression system. The baculovirus expression system yielded approximately 200-300 micrograms of the purified isozyme from 3 x 10(8) (100 ml of culture medium) baculovirus-infected insect cells. Biochemical characterization of the expressed isozymes indicated that the three isozymes had virtually indistinguishable Ca2+, Mg2+, and ATP dependencies. However, in certain critical functional characteristics such as phosphatidylserine dependencies, phospholipid and substrate preferences, and arachidonic acid activation, the gamma isozyme exhibited distinctive properties when compared with both the alpha and beta II subtypes. In addition, the activity of the beta II subtype was more dependent upon diacylglycerol or phorbol esters for activation than either the alpha or gamma isoforms. The alpha isozyme, unlike the beta II and gamma forms, was totally dependent on Ca2+ for activation in the presence of free arachidonic acid. These studies provide definitive characterizations of the pure isoforms; many of the findings were consistent with earlier enzymatic observations using hydroxyapatite-purified isoforms. Thus, the distinctive biochemical properties of the protein kinase C isozymes are consistent with the hypothesis that each isoform may have distinct roles in signal transduction processes.
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
D J
DJ
Burns
J
J
Bloomenthal
M H
MH
Lee
R M
RM
Bell
D000818:Animals; D001921:Brain / Q000201:enzymology; D002417:Cattle; D002460:Cell Line; D004789:Enzyme Activation; D005822:Genetic Vectors; D007304:Insect Viruses / Q000235:genetics*; D007313:Insecta; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008823:Micelles; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D010718:Phosphatidylserines / Q000494:pharmacology; D011485:Protein Binding; D011493:Protein Kinase C / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013379:Substrate Specificity
D007527:Isoenzymes; D008823:Micelles; D010718:Phosphatidylserines; D011994:Recombinant Proteins; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C