Protein kinase activities in ripening mango, Mangifera indica L., fruit tissue I: Purification and characterization of a calcium-stimulated casein kinase-I

A Ca²⁺-stimulated protein kinase (PK-I), active with dephosphorylated casein as exogenous substrate, was purified from ripening mango fruit, The purification procedure involved 30-70% ammonium sulphate fractionation and sequential anton exchange-. affinity-. hydrophobic interaction- and gel filtration chromatography. PK-I was purified ca. 40-fold with an overall yield of < 1%. The final specific activity in the presence of 0.1 mM Ca²⁺ was 55 nmol min^-1 mg^-1. Analysis of the most highly purified preparations revealed a monomeric enzyme with an M(r) of 30.9 kDa and pI of 5.1. PK-1 efficiently phosphorylated casein and phosvitin, but did not phosphorylate histone II-S, histone III-S, protamine sulphate or bovine serum albumin. PK- I activity was stimulated by micromolar concentrations of Ca²⁺ and was dependent on millimolar Mg²⁺ concentrations, which could not be substituted with Mn²⁺. PK-I activity was stimulated by, but was not dependent on Ca²⁺. Calmodulin and calmodulin inhibitors did not affect PK-I activity, but heparin and cAMP acted as inhibitors. The pH and temperature optima of the enzyme under standard reaction conditions were 6.5 and 35°C, respectively. The kinetic reaction mechanism of PK-I was studied by using casein as substrate. Initial velocity and product inhibition studies with ADP as product inhibitor best fit an ordered bi-bi kinetic mechanism with the Mg²⁺-ATP complex binding first to the enzyme followed by binding of the protein substrate. The K^mATP and K^mcasein of PK-I were 9 μM and 0.26 mg ml^-1, respectively. The K(i)ADP of PK-I was 9 μM.