Purification and kinetic properties of polynucleotide kinase from rat testes.
Polynucleotide kinase (EC 2.7.1.78) has been purified from rat testes, and an approximately 2000-fold purification was obtained. The purified enzyme had an Mr of 38000 +/- 3800. The enzyme phosphorylated micrococcal nuclease-treated calf thymus DNA and (dT)10 while 5'-HO-tRNA was a very poor substrate. A certain degree of specificity towards purine-containing 5'-HO-nucleotides was observed. The polynucleotide kinase had an absolute requirement for a divalent cation. Both Mg2+ and Mn2+ could be used, but 10 mM MgCl2 gave optimal activity. The monovalent cations Na+, K+ and NH4+ all stimulated enzyme activity, and the optimal concentration was 0.1 M. The enzyme was inhibited by inorganic phosphate, pyrophosphate and sulphate. A 50% inhibition was obtained with 20, 0.3 and 2 mM, respectively. At 2 mM MgCl2, 1 mM spermine enhanced the enzyme activity 3-times. The apparent KATP was estimated to be 36 microM and KHO-DNA was found to be 2 microM.
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D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000838:Anions / Q000494:pharmacology; D002845:Chromatography / Q000379:methods; D004247:DNA / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D008297:Male; D010766:Phosphorylation; D010770:Phosphotransferases / Q000302:isolation & purification*; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D012492:Salts / Q000494:pharmacology; D013096:Spermine / Q000494:pharmacology; D013379:Substrate Specificity; D013737:Testis / Q000201:enzymology*
D000838:Anions; D012492:Salts; D013096:Spermine; D000255:Adenosine Triphosphate; D004247:DNA; D010770:Phosphotransferases; D011116:Polynucleotide 5'-Hydroxyl-Kinase