Rapid purification and characterization of protein kinase C from bovine retinal rod outer segments.
A rapid FPLC procedure for the purification of protein kinase C from bovine rod outer segments is described. The enzyme is essentially homogeneous after purification and exhibits a molecular mass of approximately 85 kDa, as determined by SDS/PAGE. From its chromatographic behaviour on hydroxyapatite, and from Western-blotting experiments using isoenzyme-specific antibodies, we were able to identify the bovine rod outer segment protein kinase C as being of the alpha or type-III form. The purified protein kinase C has a specific activity of 1066 nmol 32P.min-1.mg protein-1, and shows a 30-fold activation upon the addition of the effectors Ca2+, PtdSer and 1,2-diacylglycerol. Arachidonic acid and linoleic acid were also found to enhance significantly the activity of the purified enzyme.
Max-Planck-Institut für Biophysik, Abteilung für Molekulare Membranbiologie, Frankfurt am Main, Federal Republic of Germany.
G
G
Wolbring
N J
NJ
Cook
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005227:Fatty Acids / Q000494:pharmacology; D007527:Isoenzymes / Q000302:isolation & purification; D011493:Protein Kinase C / Q000187:drug effects / Q000302:isolation & purification*; D012374:Rod Cell Outer Segment / Q000201:enzymology*
D005227:Fatty Acids; D007527:Isoenzymes; D011493:Protein Kinase C; D002118:Calcium