Purification and characterization of protein kinase C from Pleurotus ostreatus

Abstract

Protein kinase C (PKC) from Pleurotus ostreatus mycelium (Basidiomycotina) was purified by affinity chromatography using activated agarose (Affigel 15) coupled with the protein kinase inhibitor N-(2-aminoethyl)-5-isoquinolinesulphonamide hydrochloride. Using buffer containing 2·0 M-arginine. PKC activity eluted as a single peak corresponding to a protein with apparent molecular mass of approximately 68000 Da as estimated by SDS-PAGE. The enzyme was not stimulated by Ca2+ alone, and activation by phosphatidylserine (PS) was significant only at 10−6 M-Ca2+. However, the activity was enhanced by the addition of 1,2-dioctanoyl-sn-glycerol in the presence of PS and Ca2+. Additionally, although sensitivity was lower than for mammalian PKC, the enzyme was activated by a tumour-promoting phorbol ester, phorbol-12-myristate 13-acetate (PMA). The saturation concentration of PMA was 50 g ml−1, which was 5 × 103 times the value for mammalian PKC.