Purification and properties of cyclic nucleotide-independent phosvitin kinase from pig testis.
Cyclic nucleotide-independent and Ca2+-independent phosvitin kinase was purified from pig testis to apparent homogeneity by DEAE-cellulose, Sephadex G-200 chromatography, followed by subsequent DEAE-cellulose chromatography and phosvitin-Sepharose 4B affinity chromatography. The purified enzyme was homogeneous by analytical polyacrylamide gel electrophoresis, and it consisted of two polypeptides with molecular weights of 92000 (alpha) and 84000 (beta), which were present in the ratio of 1:2. Molecular weight of the native enzyme was estimated at 240000 by gel chromatography on Sepharose 6B, suggesting that the enzyme consists of alpha beta 2. The enzyme had maximal activity with phosvitin as the substrate. Casein was less active than phosvitin. Histone, protamine and myosin light-chains were practically ineffective. The enzyme possessed no ability to autophosphorylate. The apparent Km values were 7.4 microM for phosvitin, 65 microM for ATP and 0.6 mM for Mg2+. Vmax was 2.16 mumol/min per mg. The enzyme was inhibited by ammonium sulfate and heparin, and it was not affected by the addition of cyclic nucleotides and Ca2+ with calmodulin or phospholipid.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent; D002846:Chromatography, Affinity; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D009712:Nucleotides, Cyclic / Q000494:pharmacology; D009994:Osmolar Concentration; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine; D013737:Testis / Q000201:enzymology*
D002413:Cations, Divalent; D009712:Nucleotides, Cyclic; D011494:Protein Kinases; D002118:Calcium