5'-Nucleotidase activities in human erythrocytes. Identification of a purine 5'-nucleotidase stimulated by ATP and glycerate 2,3-bisphosphate.
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of the enzyme to DEAE-Trisacryl, as evidenced by the different elution profile obtained with fresh as compared with outdated blood.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.
Bontemps
F
F
Laboratory of Physiological Chemistry, Université Catholique de Louvain, Brussels, Belgium.
Van den Berghe
G
G
Hers
H G
HG
D019794:2,3-Diphosphoglycerate; D015720:5'-Nucleotidase; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D002852:Chromatography, Ion Exchange; D004163:Diphosphoglyceric Acids / Q000494:pharmacology*; D004790:Enzyme Induction / Q000187:drug effects; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D009708:Nucleotidases / Q000097:blood* / Q000302:isolation & purification; D013379:Substrate Specificity; D013997:Time Factors
D004163:Diphosphoglyceric Acids; D019794:2,3-Diphosphoglycerate; D000255:Adenosine Triphosphate; D009708:Nucleotidases; C029296:purine 5'-nucleotidase; D015720:5'-Nucleotidase