Protein kinase activities in ripening mango, Mangifera indica L., fruit tissue. I: Purification and characterization of a calcium-stimulated casein kinase-I.
A Ca(2+)-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 anion 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-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 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 the Mg(2+)-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 microM and 0.26 mg ml-1, respectively. The KiADP of PK-I was 9 microM.
Frylinck
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Department of Biochemistry, R.A.U.-University, Johannesburg, South Africa.
Dubery
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IA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D047388:Casein Kinases; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D000242:Cyclic AMP / Q000494:pharmacology; D005638:Fruit / Q000201:enzymology* / Q000502:physiology; D007700:Kinetics; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics
D019000:Phosphotyrosine; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases; D047388:Casein Kinases; D002118:Calcium