Calcium-stimulated guanosine--inosine nucleosidase from yellow lupin (Lupinus luteus).
Guanosine-inosine-preferring nucleoside N-ribohydrolase has been purified to homogeneity from yellow lupin (Lupinus luteus) seeds by ammonium sulfate fractionation, ion-exchange chromatography and gel filtration. The enzyme functions as a monomeric, 80kDa polypeptide, most effectively between pH 4.7 and 5.5. Of various mono- and divalent cations tested, Ca(2+) appeared to stimulate enzyme activity. The nucleosidase was activated 6-fold by 2mM exogenous CaCl(2) or Ca(NO(3))(2), with K(a)=0.5mM (estimated for CaCl(2)). The K(m) values estimated for guanosine and inosine were 2.7+/-0.3 microM. Guanosine was hydrolyzed 12% faster than inosine while adenosine and xanthosine were poor substrates. 2'-Deoxyguanosine, 2'-deoxyinosine, 2'-methylguanosine, pyrimidine nucleosides and 5'-GMP were not hydrolyzed. However, the enzyme efficiently liberated the corresponding bases from synthetic nucleosides, such as 1-methylguanosine, 7-methylguanosine, 1-N(2)-ethenoguanosine and 1-N(2)-isopropenoguanosine, but hydrolyzed poorly the ribosides of 6-methylaminopurine and 2,6-diaminopurine. MnCl(2) or ZnCl(2) inhibited the hydrolysis of guanosine with I(50) approximately 60 microM. Whereas 2'-deoxyguanosine, 2'-methylguanosine, adenosine, as well as guanine were competitive inhibitors of this reaction (K(i) values were 1.5, 3.6, 21 and 9.7 microM, respectively), hypoxanthine was a weaker inhibitor (K(i)=64 microM). Adenine, ribose, 2-deoxyribose, 5'-GMP and pyrimidine nucleosides did not inhibit the enzyme. The guanosine-inosine hydrolase activity occurred in all parts of lupin seedlings and in cotyledons it increased up to 5-fold during seed germination, reaching maximum in the third/fourth day. The lupin nucleosidase has been compared with other nucleosidases.
Szuwart
Maciej
M
Department of Biochemistry and Biotechnology, Agricultural University, 35 Wołyńska Street, 60-637 Poznań, Poland.
Starzyńska
Elzbieta
E
Pietrowska-Borek
Małgorzata
M
Guranowski
Andrzej
A
D002118:Calcium / Q000737:chemistry / Q000494:pharmacology*; D002412:Cations / Q000737:chemistry; D002850:Chromatography, Gel; D003116:Color; D006151:Guanosine / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D031297:Lupinus / Q000201:enzymology* / Q000254:growth & development; D015394:Molecular Structure; D008970:Molecular Weight; D009699:N-Glycosyl Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D010936:Plant Extracts; D021122:Protein Subunits / Q000378:metabolism; D036226:Seedlings / Q000201:enzymology / Q000254:growth & development; D012639:Seeds / Q000201:enzymology; D013379:Substrate Specificity
D002412:Cations; D010936:Plant Extracts; D021122:Protein Subunits; D006151:Guanosine; D009699:N-Glycosyl Hydrolases; C043277:inosine nucleosidase; D002118:Calcium