Rapid purification and characterization of protein kinase C from bovine retinal rod outer segments

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Abstract
A rapid FPLC procedure for the purification of protein kinase C from bovine rod outer segments is described. The enzyme is essentially homogenous 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 α 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.