Nucleotide pyrophosphatase from potato tubers

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Abstract
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 Mr of the enzyme, from gel filtration or sucrose density gradient centrifugation, is 343000 or 346000 respectively; and SDS electrophoresis indicates an Mr 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′)p2(5′)A– A(5′)p5(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 m7GMP from the 5′‐terminal m7G(5′)p3(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 Ki= 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.

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