THIOL GROUPS IN DEOXYRIBONUCLEIC ACID NUCLEOTIDYLTRANSFERASE

Abstract

The inhibitory effects of iodoacetate, iodoacetamide, p-hydroxy-mercuribenzoate and sarkomycin were studied in a partially purified preparation of deoxyribonucleic acid nucleotidyltransferase from Land-schutz ascites-tumour cells. All of these agents inhibited the activity of the enzyme, and it was shown that the inhibition exerted by the last three compounds obeyed non-competitive kinetics. Inclusion of glutathione or 2-mercaptoethanol in the enzyme assays did not prevent the inhibition by iodoacetate or iodoacetamide, but did prevent inhibition by p-hydroxymercuribenzoate. Inhibition by sarkomycin could be partially prevented by glutathione or 2-mercaptoethanol. The enzyme fraction also catalysed incorporation in the presence of only one triphosphate (thymidine 5[image]-triphosphate), and the limited incorporation observed in these circumstances was more resistant to the inhibitory action of iodoacetamide and p-hydroxymercuribenzoate than was the standard nucleotidyltransferase reaction (four triphosphates present). Levels of inhibition imposed on the standard reaction were achieved in the limited incorporation reaction with 2. 5-fold higher concentrations of the two inhibitors. The addition of certain bivalent cations to the standard system resulted in severe inhibition of the reaction: Zn2+ ions (10 [mu]M) gave 50% inhibition; ethylenediaminetetra-acetate (0-4m[image]) in the reaction mixture gave essentially complete protection against this inhibitory effect of Zn2+ ions. Deoxyribonucleic acid-nucleotidyl-transferase fractions prepared in the presence of a thiol and ethylenediaminetetra-acetate could be stored without loss of activity for 2 months at 0[degree] or for 1 year at -70[degree].