Protein kinase activities in ripening mango, Mangifera indica L., fruit tissue. I: Purification and characterization of a calcium-stimulated casein kinase-I.
Frylinck L., Dubery I. A.
Author Affiliation: Department of Biochemistry, R.A.U. University, PO Box 524, Auckland Park 2006, Johannesburg, South Africa.
Biochimica et Biophysica Acta, Protein Structure and Molecular Enzymology 1382 : 65-79
Abstract : 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 sulfate fractionation and sequential anion exchange-, affinity-, hydrophobic interaction- and gel filtration chromatography. PK-I was purified about 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 Mr of 30.9 kDa and pI of 5.1. PK-I 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 KmATP and Km casein of PK-I were 9 µM and 0.26 mg ml-1, respectively. The KiADP of PK-I was 9µM.