SYNTHESIS OF NUCLEOSIDE DIPHOSPHATES AND TRIPHOSPHATES BY A SALMON MILT ENZYME PREPARATION

Abstract

A cell-free enzyme preparation capable of forming nucleoside (or deoxynucleoside) di- and tri-phosphates from the corresponding nucleoside monophosphates in the presence of adenosine triphosphate (ATP) was prepared from immature salmon milts. The preparation was not free from phosphatase activities. The corresponding nucleoside (or deoxynucleoside) di- and tri-phosphates were readily formed from cytidine monophosphate, adenosine monophosphate, uridine monophosphate, and deoxyuridine monophosphate; guanosine monophosphate, deoxycytidine monophosphate, inosine monophosphate, and deoxyribosylthymine monophosphate were poorer substrates. The reaction required ATP. Guanosine triphosphate, uridine triphosphate, cytidine triphosphate, deoxyribosylthymine triphosphate, and inosine triphosphate could replace ATP, though they were in general less effective. With cytidine monophosphate as substrate, the formation of cytidine triphosphate was accelerated by Mg++, Ca++, Mn++, Co++, Ni++, and CN−, and was inhibited by Cu++, Ag+, Zn++, Hg++, F−, iodoacetate, p-hydroxymercuribenzoate, and ethylenediaminetetraacetate. Neither glutathione nor 2-mercaptoethanol in concentrations of between 1 × 10−3 and 1 × 10−1 M protected the enzyme. The optimum pH was 7.5–8.0. The enzyme was very unstable, losing two-thirds of its activity in 1 hour at 30 °C. Under optimum conditions with cytidine monophosphate as substrate, 8.5 μmoles of cytidine triphosphate were formed per hour per milligram of protein nitrogen at 25 °C.