Protein kinase activities in ripening mango (Mangifera indica) fruit tissue. II. Purification and characterization of a casein kinase I

A protein kinase (PK-II), phosphorylating casein, was purified from ripening mango, Mangifera indica L., fruit tissue. The purification procedure consisted of ammonium sulphate fractionation and sequential anion exchange-, dye-ligand, and gel filtration chromatography. The enzyme was purified over 500-fold to near homogeneity with a recovery of 4%. The purified enzyme had a specific activity of ca 1 μmol mg^-1 protein min^-1 with ATP as phosphoryl donor. SDS-PAGE results indicated a monomeric enzyme with molecular mass of 35 kDa. The protein kinase phosphorylated the acidic substrates casein and phosvitin, but had a very low activity with histones and protamine sulphate. The optimum pH and temperature for catalysis were determined to be 9.6 and 35°C, respectively. Mn²⁺ could not substitute for the Mg²⁺ needed for activity and Ca²⁺ had a slight stimulatory effect. Phospholipids, cAMP, calmodulin and the calmodulin inhibitor, calmidazolium, did not have any significant effect on activity, but the enzyme was inhibited by heparin and the specific inhibitor, CKI-7, (N-[2-aminoethyl]-5-chloroisoquinoline-8-sulphonamide). Autoradiographic studies revealed the ability of the protein kinase to autophosphorylate as well as the presence of endogenous protein substrates in the crude extract. Initial velocity studies with casein as substrate and product inhibition studies with ADP indicated a K(m) (ATP) and K(m) (casein) of 14 μM and 0.18 mg ml^-1, respectively, with a K(i) (ADP) of 3.2 μM. The enzyme can be classified as a casein kinase 1 type of protein kinase (EC 2.7.10).