Rapid purification of protein kinase C from rat brain

Abstract

We describe a rapid purification of protein kinase C from rat brain cytosol employing a specific substrate, protamine-coupled to agarose. Subsequential chromatography on DEAE-Sephacel, phenyl-Sepharose CL-4B, and protamine-agarose columns resulted in a 1500-fold purification of protein kinase C. SDS-PAGE analysis of the purified enzyme resolved a doublet protein of 77-80 kDa. This doublet was recognized by a polyclonal antiserum against protein kinase C. Proteolytic digestion of each protein band generated similar peptide fragments. The underlying principle of the protamine sulfate purification method was also clarified. Protamine can serve as a Ca2+/phospholipid-independent substrate. We demonstrate phosphorylation of protamine on the column; phoshorylated protamine did not bind the enzyme with the same affinity and this covalent modification was phosphorylated responsible for releasing the bound enzyme from the column after addition of Mg2+ and ATP. The C kinase inhibibitor, H7, inhibits protamine phosphorylation in a dose-dependent fashion but does not prevent binding of the enzyme to a protamine-agarose column. We therefore conclude that protamine interacts with the active center of the enzyme of the enabling it to be phosphorylated, upon which it loses its binding affinity for C kinase.