SIMPLE, SPECIFIC RADIOMETRIC ASSAY FOR 5'-NUCLEOTIDASE

A radiometric assay for [Crotalus atrox venom] 5''-nucleotidase (EC 3.1.3.5) was developed, which is applicable for all 5''-nucleotide substrates. Various column materials and eluants were evaluated for their suitability in the separation of purine and pyrimidine bases and nucleosides produced in the reaction. Neutral alumina columns were the best. The unadsorbed nucleosides and their bases could be quantitatively eluted with 0.1 M Tris-HCl, pH 7.4; subsequent elution of the 5''-nucleotide was then accomplished with 0.2 M sodium phosphate, pH 7.4. Differential measurement of 5''-nucleotidase could be accomplished in the presence of acid or alkaline phosphatases by inclusion of concanavalin A into the reaction mixture. It completely inhibited 5''-nucleotidase without effecting the phosphatases. The applicability of this assay was demonstrated by studying the properties of 5''-nucleotidase present in a purified plasma membrane preparation from a rat tumor which was enriched with 5''-nucleotidase and alkaline phosphatase.