adenosine-kinase and Hemolysis

An assay suitable for initial velocity studies was developed to measure adenosine kinase (ATP: adenosine 5'-phosphotransferase, EC 2.7.1.20) activity in crude lystaes of human erthrocytes. The pH optimum for the reaction was dependent on the ratio of Mg2+/ATP used in the assay. With a Mg2+/ATP ratio of 5.0, the pH optimum was 5.1 and only 10% of the maximal activity was retained at pH 7.3. When a Mg2+/ATP ratio of 0.50 was used, the pH optimum was 6.2, but 70% of the maximal activity was retained at pH 7.3 For assays performed at pH 7.3, the optimal Mg2+/ATP ratio was 0.50 for ATP concentrations between 0.50 and 2.0 mmol/l. Mg2+ was required for reaction, presumably to form the physiological substrate MgATP2-. However, excess free (uncomplexed) Mg2+ was a strong inhibitor of the enzyme at pH 7.3. At pH 7.3, the Km values for adenosine and MgATP2- were 0.66 mu mol/l and 82 mu mol/l respectively.

Topics: Adenosine; Adenosine Kinase; Adenosine Triphosphate; Adult; Cations, Divalent; Chromatography, Thin Layer; Coformycin; Erythrocytes; Hemolysis; Humans; Hydrogen-Ion Concentration; Kinetics; Magnesium; Methods; Phosphotransferases; Reference Values