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 fraction ation and sequential anion exchange-, affinity-, hydrophobic interacti on-and gel filtration chromatography. PK-I was purified ca. 40-fold wi th an overall yield of < 1%. The final specific activity in the presen ce of 0.1 mM Ca2+ was 55 nmol min(-1) mg(-1). Analysis of the most hig hly purified preparations revealed a monomeric enzyme with an M-r of 3 0.9 kDa and pI of 5.1. PK-I efficiently phosphorylated casein and phos vitin, but did not phosphorylate histone II-S, histone III-S, protamin e sulphate or bovine serum albumin. PK-I activity was stimulated by mi cromolar concentrations of Ca2+ and was dependent on millimolar Mg2+ c oncentrations, which could not be substituted with Mn2+. PK-I activity was stimulated by, but was not dependent on Ca2+. Calmodulin and calm odulin inhibitors did not affect PK-I activity, but heparin and cAMP a cted as inhibitors, The pH and temperature optima of the enzyme under standard reaction conditions were 6.5 and 35 degrees 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 th e Mg2+-ATP complex binding first to the enzyme followed by binding of the protein substrate. The K(m)ATP and K(m)casein of PK-I were 9 mu M and 0.26 mg ml(-1), respectively. The K(i)ADP of PK-I was 9 mu M. (C) 1998 Elsevier Science B.V.