High molecular weight deoxyribonucleic acid polymerase from crown gall tumor cells of periwinkle (Vinca rosea).
A high molecular weight (6 S) plant DNA polymerase from axenic Vinca rosea tissue culture cells has been purified 2200-fold and characterized. The enzyme has a molecular weight of 105 000 (+/-5000). Sodium dodecyl sulfate-acrylamide gel electrophoresis of the purified enzyme yields polypeptide subunits having molecular weights of 70 000 and 34 000. The purified enzyme has a pH optimum of 7.5; a cation requirement optimum of 6 mM Mg2+ or 0.5 mM Mn2+; an apparent requirement for Zn2+; a Km of 1 muM for dTTP; and a 3.5-fold stimulation by 50 mM KCl. The enzyme is sensitive to N-ethylmaleimide (1 mM), heparin (0.1 muM), ethanol (5%), pyrophosphate (0.05 muM), and o-phenanthroline (0.1 mM) but is insensitive to rifamycin. Denatured DNA is found to be the best natural template, and only negligible activity can be demonstrated with the ribopolymer templates poly(dT)n-poly(rA)n and p(dT)10-poly(rA)n. In addition to the polymerization reaction, the enzyme catalyzes a pyrophosphate exchange reaction. Antibody to calf thymus 6-8S DNA polymerase does not inhibit DNA polymerase from Vinca rosea, suggesting no antigenic relationships between the mammalian and plant enzymes.
D001667:Binding, Competitive; D002414:Cations, Monovalent; D004254:DNA Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D010941:Plant Tumors; D010944:Plants / Q000201:enzymology*; D013698:Templates, Genetic; D015032:Zinc / Q000494:pharmacology
D002414:Cations, Monovalent; D008345:Manganese; D004254:DNA Nucleotidyltransferases; D008274:Magnesium; D015032:Zinc