Strugovshchikova L P, Tesliar G E, Shavlovskiĭ G M
Ukr Biokhim Zh (1978). 1981 Jan-Feb;53(1):39-45.
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).
碱性核苷酸焦磷酸酶是从季也蒙毕赤酵母Wickerham ATCC 9058的无细胞提取物中分离得到的。通过硫酸铵饱和、Sephadex G - 150凝胶色谱和DEAE - 纤维素离子交换色谱对该酶进行了740倍的纯化。核苷酸焦磷酸酶在pH 8.3和49℃时活性最高。该酶催化FAD、NAD +、NADH、NADPH、GTP的水解。FAD的Km值为2.4×10(-4) M，NAD +的Km值为5.7×10(-6) M。FAD的水解受到NAD +、NADP +、ATP、AMP、GTP、PPi和Pi的抑制。NAD +、AMP和Na4P2O7的Ki分别为1.7×10(-4) M、1.1×10(-4) M和5×10(-5) M。金属螯合化合物8 - 羟基喹啉、邻菲罗啉和EDTA完全抑制了酶的活性。EDTA的作用是不可逆的。通过Sephadex G - 150凝胶过滤和薄层凝胶过滤色谱法测定的该酶分子量为78000道尔顿。葡萄糖氧化酶的蛋白结合FAD不被碱性核苷酸焦磷酸酶水解。该酶在0.01 M tris - HCl缓冲液（pH 7.5）中于2℃稳定。
