Robert L. Post

The linkage of sodium, potassium, and ammonium active transport across the human erythrocyte membrane☆

Abstract 1. 1. The net transport of potassium and sodium across the human erythrocyte membrane were observed at 37° in cells prepared by cold storage and fortified with nucleoside. Passive transport was minimized by adjustment of sodium and potassium concentrations in the medium. Active transport was distinguished from passive transport by the use of strophanthin, which stops active transport specifically. 2. 2. The active transport of potassium inward and sodium outward occured only at a ratio which was constant over a wide range of rates and independent of extracellular and intracellular sodium and potassium concentrations. Two atoms of potassium wer e transported inward for every three atoms of sodium that were tramsported outward. 3. 3. Ammonium appeared to substitute directly for potassium adn required a concentration 3 to 7 times greater than potassium to produce a comparable effect. 4. 4. These findings indicate that active potassium and sodium transport across the human erythrocyte membrane are parts of a single tightly-linked system.

Comparison of sources of a phosphorylated intermediate in transport ATPase

Abstract 1. 1. The relationship between the enzymatic activity and the phosphorylated intermediate of the (Na + + K + )-dependent ATPase (EC 3.1.6.3) from 11 species and 6 tissues was tested. 2. 2. The range in specific activity of the ATPase was more than 400-fold, yet the range in the ratio of the (Na + + K + )-ATPase activity to the phosphorylated intermediate was only 2-fold, and the range in the ratio of the (K + )-acylphosphatase (EC 3.6.1.7) activity of the (Na + + K + )-ATPase activity was also only 2-fold. 3. 3. The (Na + + K + )-ATPase of all sources tested required Na + for phosphorylation and its dephosphorylation was greatly accelerated in the presence of K + . 4. 4. The turnover of the phosphorylated enzyme at 0° in the absence of K + , estimated by washout of 32 P, showed a range of 3-fold. 5. 5. The phosphorylated enzymes yielded identical 32 P-labeled peptides after digestion with pepsin (EC 3.4.4.1) or pronase and electrophoresis. The 32 P-labeled peptides of the (Na + + K + )-ATPase of all sources tested were sensitive to hydroxylamine. 6. 6. The results indicate that the mechanism of the (Na + + K + )-dependent ATPase is likely to be similar in most animal species and tissues.

Isolation and characterization of a phosphorylated intermediate in the (Na+ + K+) system-dependent ATPase*

Summary 1. A phosphorylated intermediate material in the (Na + + K + )-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) transport system was solubilized by peptic digestion and.subjected to paper electrophoresis. 2. Brief digestion released one fragment which moved toward the cathode. Prolonged digestion converted this fragment into two subfragments. 3. The subfragments and the parent material were characterized as probably being acyl phosphates by their lability in strong acid and at neutral and alkaline pH, by their ease of methanolysis or ethanolysis, by their lability in the presence of hydroxylamine or molybdate, and by their response to bivalent ions. 4. The mol. wt. of the subfragments was estimated to be about 2200 by gel filtration. 5. The subfragments could be oxidized with performic acid or acetylated with acetic anhydride. 6. The subfragments were not soluble in ether.

Acetyl phosphate as a substitute for ATP in (Na− + K+)-dependent ATPase

Abstract 1. 1. In the presence of Mg 2+ and Na + , acetyl phosphate replaced ATP as an agent for phosphorylating Na + - and K + -dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3). 2. 2. The phospho-enzymes produced by both substrates were similar in the following respects: (a) electrophoretic mobility and chemical reactivity of phosphopeptides released by proteolytic digestion of the denatured enzyme, (b) the quantity of phospho-enzyme, (c) the concentration of Na + for half-maximal phosphorylation, (d) a requirement for Mg 2+ , (e) the half-life at o°. Furthermore, each substrate inhibited phosphorylation by the other, and the inhibitor ouabain enhanced the inhibitory effect of acetyl phosphate. 3. 3. In the presence of Mg 2+ and K + , acetyl phosphate, unlike ATP, also phosphorylated the enzyme. The quantity of phospho-enzyme was less and its turnover was faster. K + appeared to accelerate both phosphorylation and dephosphorylation. 4. 4. The same active site may be an intermediate in both the Na + - and K + -dependent ATPase activity and the K + -dependent acetyl phosphatase activity of this enzyme system in functionally different conformations of an active center.

Effects of vanadate on ouabain binding and inhibition of (Na+ + K+)-ATPase.

Effects of vanadate on ouabain binding and inhibition of sodium and potassium adenosine triphosphatase (Na+ + K+)-ATPase) were investigated under various ionic conditions. 1. Vanadate facilitated ouabain binding to (Na+ + K+)-ATPase in the presence of Mg2+ and this facilitation was partially reversed by catechol. 2. Vanadate antagonized the ability of high concentrations of NaCl to inhibit ouabain binding in the presence of magnesium. 3. Ouabain binding to the vanadate-enzyme complex, formed from magnesium and vanadate, was more sensitive to depression by potassium than that to the phosphoenzyme formed from magnesium and inorganic phosphate. 4. Preincubation of (Na+ + K+)-ATPase with vanadate in the presence of magnesium initially formed a potassium-insensitive complex as shown by a rapid initial rate of ouabain binding. However, within 5 min potassium overcame the vanadate potentiation of ouabain binding regardless of the order in which it was added to the reaction mixture. 5. Under conditions of enzyme turnover, vanadate failed to antagonize the inhibitory power of ouabain despite the presence of a high concentration of potassium. This suggests a possible relationship between the sensitivity of the sodium pump in various tissues to the cardiac glycosides and intracellular vanadate concentrations.

Further characterization of a phosphorylated intermediate in (Na+ + K+)-dependent ATPase

Abstract 1. A radioactive acyl phosphate intermediate in the (Na+ + K+)-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) of guinea-pig kidney was treated with proteolytic enzymes. The soluble products were separated by paper electrophoresis at pH 2.1. 2. Differential digestion with pepsin (EC 3.4.4.1) yielded a sequence of six fragments. Their molecular weights ranged from about 10000 to about 2200 as estimated by gel filtration. 3. All fragments were positively charged and contained at least one sulfhydryl group and no disulfide bond. 4. Hydrolysis of the intermediate or the peptic fragments with pronase gave a positively charged fragment with a molecular weight of 380. This fragment contained no sulfhydryl group. At least part of the positive charge was not that of histidine or lysine. The pronase fragment was not arginyl phosphate. 5. The results provide further evidence that the acyl phosphate group is attached to a protein containing one or more sulfhydryl and basic groups.

Adenosine Triphosphate Synthesis by Sodium, Potassium Adenosine Triphosphatase

To characterize the mechanism of the sodium and potassium ion pump of plasma membranes of animal cells, we have investigated the sodium and potassium ion-transport adenosine triphosphatase (Na, K-ATPase). This enzyme expresses the activity of the pump in preparations of broken or leaky membranes. We have investigated specifically the sequence of addition and release of ligands and have become interested in the interaction-free energies of their binding (WEBER, 1974). In particular, binding of Na+ appears to increase the free energy of hydrolysis of a phosphate group at an active site on the enzyme. To distinguish between effects due to binding and those due to translocation of ions, we have preferred to work with leaky membranes, across which concentration gradients do not persist. It has also been an advantage to conduct the hydrolytic reaction of this enzyme in the backward direction. In a first step, the enzyme was phosphorylated from inorganic phosphate (Pi). In a second step, addition of ADP with a high concentration of Na+ produced ATP.

Round Table Discussion Groups

The round-table group on (Na+ + K+)-ATPase discussed topics including the following: 1. Loss of dependence on K+ in the gills of euryhaline fish after adaptation of the fish from salt water to fresh water. Is there a replacement of one enzyme by another or controlled alteration of a single enzyme? Are there many varieties of Na+-dependent ATPase in general? Is there hormonal control of (Na+ + K+)-ATPase, particularly via aldosterone? 2. Mutagenic variation in ouabain-binding affinity of cells in tissue culture. 3. Variation in characteristics of erythrocyte (Na+ + K+)-ATPase in genetically related varieties of sheep with a high or low concentration of K+ in their red cells. 4. The relationship of (Na+ + K+)-ATPase to the transport of Na+ across epithelial tissue. 5. The mechanism of the therapeutic action of cardioactive steroids in the relief of congestive heart disease according to the hypothesis of Baker, Blaustein, Hidgkin and Steinhardt. 6. Purification of (Na+ + K+)-ATPase. Is there one or more than one peptide chain in the enzyme? 7. The reaction sequence of phosphorylation and dephosphonylation of this enzyme and remarkable effects of ethanol on the kinetics. Is there adequate evidence that so-called E1-P is necessarily a precursor of so-called E2-P?