Dinucleoside 5', 5"'-P1, P3-triphosphate hydrolase from yellow lupin (Lupinus luteus) seeds: purification to homogeneity and hydrolysis of mRNA 5'-cap analogs

Protein Expression and Purification
A GuranowskiE Darzynkiewicz

Abstract

Three separable forms of diadenosine 5',5"'-P1, P3-triphosphate (Ap3A)-degrading activity were revealed when proteins obtained from the meal of yellow lupin seeds by ammonium sulfate precipitation were chromatographed on a DEAE-Sephacel column. The major form, which eluted first at the lowest salt concentration (0.15 M KCl), was free of any activity converting the reaction products, ADP and AMP. Its further purification by gel filtration on Sephadex G-200 and by affinity elution from an AMP-agarose column yielded homogeneous protein as demonstrated on SDS-polyacrylamide gel electrophorograms. The enzyme is a single polypeptide chain of M(r) 41 kDa. Eleven guanosine-containing dinucleoside triphosphates, including analogs of the mRNA 5'-cap structure, have been tested as potential substrates of the lupin "Ap3A hydrolase." All have been hydrolyzed yielding mixtures of corresponding nucleoside mono- and diphosphates. Asymmetrical compounds gave four products; m7Gp3G, et7Gp3G, and bz7Gp3G were hydrolyzed randomly, whereas m7Gp3A, m7Gp3C, and m7Gp3U yielded at least 80% m7GMP plus corresponding NDP and 20% or less NMP plus m7GDP. Hydrolysis of the guanosine-containing hybrids, Ap3G, Cp3G, and Up3G, yielded at least 85% GMP plus corr...Continue Reading

Citations

Apr 29, 2008·The Journal of Biological Chemistry·Shin-Wu LiuMegerditch Kiledjian
Aug 29, 2002·The EMBO Journal·Hudan LiuMegerditch Kiledjian
Mar 20, 2003·Plant Physiology·Małgorzata Pietrowska-BorekAndrzej Guranowski
Aug 13, 2008·FEBS Letters·Andrzej GuranowskiWojciech J Stec
Dec 21, 2004·Biochemical and Biophysical Research Communications·Benjamin S Szwergold, Sundeep Lal
Feb 26, 2015·Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry·Narasinga Rao PalepuMohan Rao Kollipara
Jul 17, 2015·Bioscience Reports·Anna M Wojdyła-Mamoń, Andrzej Guranowski
Dec 13, 1997·Biochimica Et Biophysica Acta·Z WieczorekR Stolarski

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