Abstract
Abstract Human erythrocyte membranes were incubated with a low concentration of terminally labeled ATP with the aim of detecting a phosphorylated intermediate of adenosine triphosphatase activity. A large portion of material labeled briefly at 37° turned over and had properties consistent with participation in sodium ion-stimulated ATPase activity, including the following: (a) magnesium ions present, addition of sodium ions stimulated an increase in both 32P bound to trichloracetic acid-precipitated membrane material and the rate of ATP hydrolysis. (b) The Na+-activated bound 32P turned over and correlated quantitatively with the amount of bound 32P sensitive to hydroxylamine, in agreement with properties of phosphorylated intermediate of ATPase of other preparations. (c) Addition of potassium ions stimulated breakdown of the 32P bound in the presence of sodium ions. At 0°, a large portion of material labeled briefly was sensitive to hydroxylamine. Its turnover was apparent upon addition of excess ATP or ADP, but not guanosine triphosphate, deoxyguanosine diphosphate, AMP, or 3-phosphoglycerate. The addition of ethylenediamine tetraacetate allowed dephosphorylation of the bound 32P to proceed, and it was shown that the approximate rate of turnover was similar to the rate of ATP hydrolysis. Although the addition of sodium ions stimulated an increase in bound 32P and in ATPase activity at 2 µm ATP concentration, manifestations of altered sensitivity to sodium and potassium ions at 0° have been attributed to temperature-induced changes in conformation of catalytic sites. The relationship of membrane (14C)ADP-ATP exchange activity to ATPase activity was investigated. It was found that at least 30% of the exchange activity was associated with material which could be separated from ATP hydrolytic activity and which was not labeled with 32P after incubation with terminally labeled ATP. This activity did not appear to be related to other membrane activities tested, including nucleoside diphosphokinase, adenylate kinase, and phosphoglycerate kinase.
Highlights
MethodsErythrocyte membranes were prepared by osmotic lysis of fresh, 0.9% NaCl washed human erythrocytes, which was carried out by rapidly adding 10 volumes of distilled water to 1 volume of packed cells
ATPase activity, including the following: (a) magnesium ions present, addition of sodium ions stimulated an increase in both 32P
Bound to trichloracetic acid-precipitated membrane material and the rate of ATP hydrolysis. (b) The Na+-activated bound 32P turned over and correlated quantitatively with the amount of bound 32P sensitive to hydroxylamine, in agreement with properties of phosphorylated intermediate of ATPase of other preparations. (c) Addition of potassium ions stimulated breakdown of the 32P bound in the presence of sodium ions
Summary
Erythrocyte membranes were prepared by osmotic lysis of fresh, 0.9% NaCl washed human erythrocytes, which was carried out by rapidly adding 10 volumes of distilled water to 1 volume of packed cells. 20,000 x g, the posthemolytic residue was washed twice with 1 mM Tris HCl containing 1 m Tris EDTA, pH 7.4, once with 10 mM Tris EDT.-\, pH 7.4, and three times with 2 mM Tris HCl, pH 7.4. The membrane residue was made up to onehalf the original packed cell volume of the washed cells and contained 4 to 5 mg of protein per ml. The preparation was frozen and thawed twice before use, to ensure complete permeability to substrates. More than 99% of the radioactivity in the T-~~P-ATP could be adsorbed on Norit A charcoal and was susceptible to hydrolysis by erythrocyte membrane ATPase.
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