Protein kinase activities in ripening mango, Mangifera indica L., fruit tissue. III. Purification and characterisation of a calcium-regulated protein kinase.
A calcium-dependent protein kinase (PK-III), not requiring calmodulin for activity, was purified from extracts of ripening mango fruit tissue. Purification was achieved by ammonium sulfate fractionation and sequential anion exchange-, hydrophobic interaction-, dye ligand affinity- and gel filtration chromatography; which allowed a recovery of 1-5% of the total available kinase activity. The final specific activity in the presence of 1 mM Ca2+ was consistently 9 nmol min-1mg-1. The purified enzyme was a monomer with a Mr of 49000, but was resolved by denaturing electrophoresis into two 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 microM) had no effect on PK-III activity but the calmodulin antagonists, calmidazolium and chlorpromazine, inhibited PK-III in a dose-dependent manner over a range of 0 to 100 microM. The results suggest a regulatory domain that is 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. Some endogenous substrates and the ability to autophosphorylate were revealed by autoradiographic studies. PK-III displayed a broad pH optimum (pH 6.6-9.5), and the optimum reaction temperature with histone III-S as substrate was 35 degreesC. The kinetic reaction mechanism of PK-III was studied by using casein as substrate. The KmATP and Kmcasein of PK-III were determined as 10 microM and 1.0 mg ml-1, respectively.
Frylinck
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Department of Biochemistry, R.A.U.-University, P.O. Box 524, Auckland Park 20006, South Africa.
Dubery
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D000255:Adenosine Triphosphate / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000737:chemistry; D002364:Caseins / Q000378:metabolism; D002746:Chlorpromazine / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005638:Fruit / Q000201:enzymology*; D006657:Histones / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D010766:Phosphorylation; D010940:Plant Proteins / Q000737:chemistry; D011494:Protein Kinases / Q000737:chemistry*; D012694:Serine / Q000378:metabolism; D013379:Substrate Specificity; D013912:Threonine / Q000378:metabolism
D002147:Calmodulin; D002364:Caseins; D004791:Enzyme Inhibitors; D006657:Histones; D007093:Imidazoles; D010940:Plant Proteins; D013912:Threonine; D012694:Serine; C031938:calmidazolium; D000255:Adenosine Triphosphate; D011494:Protein Kinases; C115256:protein kinase III; D002118:Calcium; D002746:Chlorpromazine