Nucleotide pyrophosphatase from potato tubers

Abstract

Purification of potato tuber nucleotide pyrophosphatase was modified to furnish a rapid and reproducible procedure yielding a preparation purified 1800-fold and homogeneous in sodium dodecyl sulfate[SDS]/polyacrylamide gel electrophoresis. The MW of the enzyme, from gel filtration or sucrose density gradient centrifugation, is 343,000 or 346,000, respectively; SDS electrophoresis indicates MW for the subunit of 74,000. Analytical isoelectrofocusing reveals a broad isoelectric range of pH 8.3-8.7. The enzyme is a glycoprotein. The purified enzyme exhibits the previously reported activities vs. pyrophosphate linkages located at either the 5''-OH or 3''-OH of nucleosides, and phosphodiester linkages in: aryl esters of nucleoside 3''- and 5''-phosphates, p-nitrophenylphosphate and orthophosphate; and nucleoside cyclic 2'',3''-phosphates. The relative rates of activity towards these substrates and the corresponding V values differ significantly. The enzyme exhibits additional novel activities, including ability to cleave dinucleoside polyphosphates such as A(5'')p2(5'')A-A(5'')p5(5'')A, and aryl phosphonates. There is no activity towards nucleoside cyclic 3'',5''-phosphates. The present preparation is also devoid of endonucleolytic activity, so that it specifically cleaves m7GMP from the 5''-terminal m7G(5'')p3(5'')Gm of intact reovirus mRNA. NAD+ was the most effective inhibitor of enzyme activity vs. thymidine 5''-p-nitrophenylphosphate, with a Ki = 0.1 mM. Kinetic analyses demonstrated competitive inhibition between these 2 substrates. Both 2'',3''-cAMP and thymidine 3''-p-nitrophenylphosphate inhibit hydrolysis of NAD+ noncompetitively and vice versa.