Protein kinase activities in ripening mango, Mangifera indica L., fruit tissue. III. Purification and characterisation of a calcium-regulated protein kinase.
Frylinck L., Dubery I. A.
Author Affiliation: Department of Biochemistry, R.A.U.-University, P.O. Box 524, Auckland Park 20006, South Africa.
Biochimica et Biophysica Acta, Protein Structure and Molecular Enzymology 1387 : 342-354
Abstract : A calcium-dependent protein kinase (PK-III), not requiring calmodulin for activity, was purified from extracts of ripening mango fruits. Purification was achieved by ammonium sulfate fractionation and sequential anion exchange-, hydrophobic interaction-, dye ligand affinity- and gel filtration chromatography. The purified enzyme was a monomer with a MW of 49?000, but was resolved by denaturing electrophoresis into 2 related protein bands of 49 and 45 kDa. Enzyme activity was activated > 30-fold by micromolar amounts of free calcium and was dependent upon millimolar Mg2+ or Mn2+ concentrations. Calmodulin (1 µM) had no effect on enzyme activity. The calmodulin antagonists, calmidazolium and chlorpromazine (0-100 µM), inhibited activity in a dose-dependent manner, suggesting a regulatory domain that was similar to calmodulin. PK-III phosphorylated histone III-S and to a lesser extent casein, but did not phosphorylate histone II-S, phosvitin or protamine sulfate. The enzyme phosphorylated substrate proteins on either serine or threonine but not tyrosine. PK-III displayed a broad pH optimum (pH 6.6-9.5), and the best reaction temperature with histone III-S as substrate was 35°C.