Nucleotide pyrophosphatase from potato tubers

Purification of potato tuber nucleotide pyrophosphatase (EC 3.6.1.9) has been modified to furnish a rapid and reproducible procedure yielding a preparation purified 1800‐fold and homogeneous in sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The M r of the enzyme, from gel filtration or sucrose density gradient centrifugation, is 343000 or 346000 respectively; and SDS electrophoresis indicates an M r for the subunit of 74000. 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 versus pyrophosphate linkages located at either the 5′‐OH or 3′‐OH of nucleosides, and phosphodiester linkages in: (a) aryl esters of nucleoside 3′‐ and 5′‐phosphates, p ‐nitrophenylphosphate and orthophosphate, and (b) nucleoside cyclic 2′,3′‐phosphates. However, 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′)p 2 (5′)A– A(5′)p 5 (5′)A, and aryl phosphonates. Contrary to previous reports, there is no activity towards nucleoside cyclic 3′,5′‐phosphates. The present preparation is also devoid of endonucleolytic activity, so that it specifically cleaves m 7 GMP from the 5′‐terminal m 7 G(5′)p 3 (5′)Gm of intact reovirus mRNA. NAD + was found to be the most effective inhibitor of enzyme activity versus thymidine 5′‐ p ‐nitro‐phenylphosphate, with a K i = 0.1 mM. Kinetic analyses demonstrated competitive inhibition between these two substrates. Both 2′,3′‐cAMP and thymidine 3′‐β‐nitrophenylphosphate inhibit hydrolysis of NAD + non‐competitively and vice‐versa.