Nucleotide pyrophosphatase from potato tubers. Purification and properties.
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-nitrophenylphosphate, with a Ki = 0.1 mM. Kinetic analyses demonstrated competitive inhibition between these two substrates. Both 2',3'-cAMP and thymidine 3'-p-nitrophenylphosphate inhibit hydrolysis of NAD+ noncompetitively and vice-versa.
D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D010944:Plants / Q000201:enzymology*; D011487:Protein Conformation; D011755:Pyrophosphatases / Q000302:isolation & purification* / Q000378:metabolism; D012314:RNA Cap Analogs / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D013379:Substrate Specificity
D012314:RNA Cap Analogs; D012333:RNA, Messenger; D009243:NAD; D011755:Pyrophosphatases; C021899:nucleotide pyrophosphatase