Purification of Bovine Brain Protein Kinase C Employing Metal Ion Dependent Properties

Abstract

Within the sequence of protein kinase C (PKC), a Ca²⁺ and phospholipid dependent enzyme (reviewed in Nishizuka 1989), no conserved Ca²⁺ binding site could be identified and the enzyme does not bind Ca²⁺ as an isolated enzyme at physiological Ca²⁺ concentrations (Bazzi & Nelsestuen 1990). The primary structure of PKC reveals within the phorbol ester binding domain two “zinc finger like” consensus sequences as well as a number of highly conserved histidine and cysteine residues (Parker et al. 1986). Ono et al. (1989) demonstrated the necessity of putative Zn²⁺ ligands within the CX₂CX₁₃CX₂C sequence for phorbol ester binding. Yet the functional consequences of metal binding to these structures or to other conserved potential ligands are poorly understood. Studies investigating the effects of divalent metal ions on PKC led to contradictory results, especially reporting activatory or inhibitory effects of zinc on the enzyme (Csermely et al. 1988, Speizer et al. 1989). Hence we developed a purification protocol which is suitable to characterize metal binding dependent properties of protein kinase C. Immobilized metal ion chromotography is suitable for preparative purification and subserves an analytical tool to evaluate surface accessible metal ion binding sites (Hemdan et al. 1989). The elucidation of metal binding properties of PKC may enhance our understanding of yet another mechanism regulating enzyme activation.