Deoxynucleotide-polymerizing Enzymes of Calf Thymus Gland

Abstract

Abstract The stoichiometry, equilibrium, and kinetic properties of a terminal deoxynucleotidyltransferase isolated from calf thymus gland are described. The reaction catalyzed may be written dXTP + d(pY)m (Mg++)/→ d(pY)m(pX)n + nPPi and the ratio of polymer nucleotide to monomer at equilibrium (pH 7.0, 35°) is about 99 when X and Y = adenine. Purine deoxyribonucleoside triphosphate polymerizations proceed readily in 8 mm Mg++ at low buffer strengths (40 mm). For deoxyadenosine triphosphate, under these conditions, Km = 1.1 x 10-4 m and Vmax varies from about 0.03 to 0.30 µmole min-1 mg-1 with various oligodeoxynucleotide initiators. At a constant chain length, Km values for oligodeoxynucleotide decrease in the order cytosine g thymine g adenine g guanine g hypoxanthine. Within a homologous series, a limiting value of Km and a maximum value for Vmax are reached at the penta- to septanucleotide. Pyrimidine deoxyribonucleoside triphosphates polymerize best in the presence of 1 mm Co++ at higher buffer strengths (200 mm). Syntheses of polydeoxyadenylate, polydeoxyinosinate, polydeoxycytidylate, and polydeoxythymidylate in greater than 80% yield are achieved under optimal conditions. Preparation of random copolymers is also described.