title: [Purification and properties of Pichia guilliermondii yeast alkaline nucleotide pyrophosphatase hydrolyzing flavin adenine dinucleotide].
abstract: Alkaline nucleotide pyrophosphatase was isolated from the Pichia guilliermondii Wickerham ATCC 9058 cell-free extracts. The enzyme was 740-fold purified by saturation of ammonium sulphate, gel-chromatography on Sephadex G-150 and ion-exchange chromatography on DEAE-cellulose. Nucleotide pyrophosphatase is the most active at pH 8.3 and 49 degrees C. The enzyme catalyzes the hydrolysis of FAD, NAD+, NADH, NADPH, GTP. The Km value for FAD is 2.4 x 10(-4) M and for NAD+--5.7 x 10(-6) M. The hydrolysis of FAD was inhibited by NAD+, NADP+, ATP, AMP, GTP, PPi and Pi. The Ki for NAD+, AMP and Na4P2O7 was 1.7 x 10(-4) M, 1.1 x 10(-4) M and 5 x 10(-5) M, respectively. Metal chelating compounds, 8-oxyquinoline, o-phenanthroline and EDTA, inhibited completely the enzyme activity. The EDTA effect was irreversible. The molecular weight of the enzyme determined by gel-filtration on Sephadex G-150 and thin-layer gel-filtration chromatography was 78000 dalton. Protein-bound FAD of glucose oxidase is not hydrolyzed by the alkaline nucleotide pyrophosphatase. The enzyme is stable at 2 degrees C in 0.01 M tris-HCl-buffer (pH 7.5).
mesh_terms: D001203:Ascomycota / Q000201:enzymology*; D002384:Catalysis; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004789:Enzyme Activation; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008722:Methods; D009711:Nucleotides / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism; D010843:Pichia / Q000201:enzymology*; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D009711:Nucleotides; D005182:Flavin-Adenine Dinucleotide; D011755:Pyrophosphatases; C021899:nucleotide pyrophosphatase
vernacular_title: Ochistka i svoĭstva shchelochnoĭ nukleotidpirofosfatazy drozhzheĭ Pichia guilliermondii, gidrolizuiushcheĭ flavinadenindinukleotid.