FISH-MUSCLE PURINE AND PYRIMIDINE NUCLEOSIDE PHOSPHORYLASES

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Abstract
Three purine nucleoside phosphorylase preparations (isoenzymes) were obtained by ammonium sulfate fractionation and DEAE-cellulose chromatography of aqueous extracts of lingcod muscle. Dialysis, adsorption on alumina Cγ, and elution with 0.4 M phosphate buffer yielded further purification. The most active enzyme preparation had about 120 times the activity of initial extracts. It utilized hypoxanthine, 6-mercaptopurine, guanine, 8-azaguanine, xanthine, adenine, 2,6-diaminopurine and 6-methylpurine in presence of ribose 1-phosphate or deoxyribose 1-phosphate. Several substituted purines were not utilized and did not inhibit the reaction between hypoxanthine and the pentose phosphates. The Kmwith inosine as substrate was 3.2 × 10−6 M. A pyrimidine nucleoside phosphorylase, distinct from the purine nucleoside phosphorylase, occurred in the DEAE-cellulose fraction comprising one of the purine nucleoside phosphorylases. Its activity was much lower than that of the purine nucleoside phosphorylase preparations. Uridine and thymidine were the best substrates. Deoxyuridine was a poor substrate, and neither cytidine nor deoxycytidine was utilized. The equilibrium with all preparations was about 80% in favor of nucleoside formation. The purified enzymes were all destroyed by freezing.