Effect of Na + and K + on the Adenylate Kinase of Human Erythrocytes. ∗

The presence of a Na+, K+-activated adenosinetriphosphatase (ATPase) in eryth-rocyte membranes has been reported by several investigators (1-3). Evidence suggesting the participation of this enzyme in the process of monovalent cation selectivity of erythrocytes has been reviewed(4). The various enzyme preparations described thus far all release two equivalents of phosphate from each adenosinetriphosphate (ATP) molecule. A possibility which could account for the observed release of 2 phosphates is the presence of an adenylate kinase in the ATPase preparations. This investigation was undertaken to determine: (1) if adenylate kinase is present in the erythrocyte membrane preparations and (2) the effect of Na+ and K+ on whatever enzyme activity is responsible for release of the second phosphate of ATP. A present finding shows that contrary to a previous report(5), kinase activity can be detected in membranes prepared by several different methods and that this enzyme is also activated by monovalent cations. After the completion of this work Post and Sen(6) also reported the presence of adenylate kinase in erythrocyte membrane preparations used for the study of ATPase. They did not, however, study the effect of Na+ and K+ on the kinase activity.

Materials and methods. Sodium salts of ATP and adenosinediphosphate (ADP) were obtained from Sigma Chemical Co. (St. Louis), and converted to the tris salts by passage through columns of cation exchange resin Dowex SO in tris form. Adenosine-5′-monophosphate (AMP) and yeast hexokinase, either crystalline or Type IV, were also purchased from Sigma. All other chemicals used were of reagent grade quality.

Adenylate kinase of various preparations was measured, according to Colowick and Kalckar(7), by incubating aliquots with 5 μmoles of ADP, 100 K.M. units of hexokinase, 5 μmoles of Mg⁺⁺, 100 μmoles of tris-HC1 (pH 7.4), and 9 mg of glucose, at 37°.