Bontemps F, Van den Berghe G, Hers H G
Laboratory of Physiological Chemistry, Université Catholique de Louvain, Brussels, Belgium.
Biochem J. 1988 Mar 15;250(3):687-96. doi: 10.1042/bj2500687.
A purine 5'-nucleotidase has been separated by DEAE-Trisacryl chromatography from other 5'-nucleotidase activities present in human haemolysates and purified approx. 30,000-fold by subsequent chromatography on Blue Sepharose. The enzyme has an Mr of around 250,000, displays hyperbolic substrate-saturation kinetics and hydrolyses preferentially IMP, GMP and their deoxy counterparts. It is much less active with AMP and dAMP. The purine 5'-nucleotidase is inhibited by Pi, and is strongly stimulated by ATP, dATP and GTP, and by glycerate 2,3-bisphosphate. Stimulators decrease Km and increase Vmax. Glycerate 2,3-bisphosphate is the most potent stimulator of the enzyme and, under physiological conditions, over-rides the influence of the other effectors. Glycerate 2,3-bisphosphate also influences the binding of the enzyme to DEAE-Trisacryl, as evidenced by the different elution profile obtained with fresh as compared with outdated blood. It is concluded that the glycerate 2,3-bisphosphate-stimulated purine 5'-nucleotidase is responsible for the dephosphorylation of IMP and GMP, but not of AMP, in human erythrocytes.
通过DEAE - Trisacryl色谱法已从人溶血产物中存在的其他5'-核苷酸酶活性中分离出一种嘌呤5'-核苷酸酶，并通过随后在蓝色琼脂糖凝胶上的色谱法将其纯化了约30000倍。该酶的相对分子质量约为250000，表现出双曲线型底物饱和动力学，优先水解肌苷酸（IMP）、鸟苷酸（GMP）及其脱氧对应物。它对腺苷酸（AMP）和脱氧腺苷酸（dAMP）的活性要低得多。嘌呤5'-核苷酸酶受到无机磷酸盐（Pi）的抑制，并受到三磷酸腺苷（ATP）、脱氧三磷酸腺苷（dATP）、三磷酸鸟苷（GTP）以及2,3-二磷酸甘油酸的强烈刺激。刺激剂降低米氏常数（Km）并增加最大反应速度（Vmax）。2,3-二磷酸甘油酸是该酶最有效的刺激剂，在生理条件下，它会抵消其他效应物的影响。2,3-二磷酸甘油酸还会影响该酶与DEAE - Trisacryl的结合，新鲜血液与过期血液洗脱曲线不同就证明了这一点。得出的结论是，2,3-二磷酸甘油酸刺激的嘌呤5'-核苷酸酶负责人类红细胞中IMP和GMP的去磷酸化，但不负责AMP的去磷酸化。
