The order of release of sodium and addition of potassium in the sodium‐potassium pump reaction mechanism.

Abstract

1. The characteristics of oligomycin inhibition of the Na‐‐K pump of human red cell membranes was investigated. Oligomycin inhibition of the pump was found to be reversible. 2. Inhibition of Na‐‐K ATPase activity was uncompetitive with respect to ATP in broken membrane preparations. In intact cells inhibition was uncompetitive with respect to both intracellular and extracellular Na. 3. Oligomycin did not significantly inhibit the K‐‐K exchange if the cells were Na‐free, but if the cells contained a small amount of Na, oligomycin inhibition of the K‐‐K exchange became significant. Taken together with the findings described above, this indicates that oligomycin combines with E1P‐Na conformation of the pump and not with any E2 conformation. 4. When measurements are made in solutions high in Na, oligomycin is a non‐competitive inhibitor with respect to external K, but in Na‐free solutions, oligomycin inhibition is uncompetitive with respect to external K. 5. These findings indicate that, in the normal operation of the pump, Na is released to the outside before K adds.

Electrophysiological Analysis of the Mutated Na,K-ATPase Cation Binding Pocket

“…Upon a conformational change back to E 1 , K ϩ is intracellularly released. In order to bring about sequential translocation of Na ϩ and K ϩ ions in the sense of a pingpong mechanism (1,2), the ion pump has to obey strict cation specificity of the phosphorylation and dephosphorylation reaction (3). The binding affinities for individual cation species change during the exposure of the binding sites from intra-to extracellular, which occurs during the E 1 P 3 E 2 P conformational change (for recent reviews, see Refs.…”

Evidence That Ser775 in the α Subunit of the Na,K-ATPase Is a Residue in the Cation Binding Pocket

“…The nature of the cation binding and occlusion sites is largely unknown. However, studies of the reaction mechanism underlying cation exchange and functional consequences of structural perturbations support the notion of a cation binding and occlusion pocket occupied consecutively by both Na ϩ and K ϩ ions during their translocation from the cytosol to extracellular milieu and vice versa (4). The structure of the binding/occlusion region must accommodate the highly distinctive cation selectivities such that Na ϩ binds with high apparent affinity at cytoplasmic sites, and K ϩ binds at extracellular sites.…”

The order of addition of sodium and release of potassium at the inside of the sodium pump of the human red cell.

“…At 36 h the cells were separated from the suspension and resealed by resuspension in a solution identical to the PCMBS solution except that PCMBS was absent, adenine was 3 0 mm, and 4 mM-dithiothreitol was included; the suspension was incubated at 37 0C for 1 h. The cells were then washed three times with unbuffered isosmotic (107 mM) MgCl2 solution or with unbuffered isosmotic (160 mM) choline chloride solution, and used for the measurement of Na efflux or K influx. The procedures for the efflux and influx measurements have been described (Sachs, 1977;Sachs, 1980). Resealed ghosts were prepared by a gel filtration method similar to that described by Kaplan (1982).…”

“…Broken red cell membranes were prepared by osmotic lysis, and ATPase activity was measured by an assay coupled to the oxidation of nicotinamide-adenine dinucleotide (NADH) (Sachs, 1980). The reaction mixture contained Tris, 21-8 mM; phosphoenolpyruvate, 1-75 mM; EDTA, 0'91 mM; dithiothreitol, 094 mM; pyruvate kinase, 1-7 u./ml; and the concentrations of Na, K, Mg and ATP indicated in the captions to the Figures and Tables.…”