title: Monomer of sodium and potassium ion activated adenosinetriphosphatase displays complete enzymatic function.
abstract: The distribution of sodium and potassium ion activated adenosinetriphosphatase [(Na+ + K+)-ATPase] among the various oligomeric forms present in a given solution is assessed unambiguously by cross-linking with glutaraldehyde. Purified enzyme dissolved in a solution of a nonionic detergent, octaethylene glycol dodecyl ether, remains dispersed and unaggregated after removal of the bulk of the detergent. Increases in the aggregation of the enzyme, which have been previously observed upon the addition of substrates to such a solution, are found to be due to changes in ionic strength rather than a consequence of the initiation of turnover. Furthermore, conditions are described that produce solutions containing stable, enzymatically active mixtures of the smaller oligomers of the asymmetric unit, alpha beta. Cross-linking by glutaraldehyde while the enzyme is turning over demonstrates that at least one of these oligomers is responsible for the observed enzymatic activity. A determination of which oligomers are present in each fraction from a glycerol gradient demonstrates that the profiles of the enzymatic activity and the concentration of monomer coincide. In addition, the monomer can form the sodium-dependent, phosphorylated intermediate of the mechanism for the enzyme. Finally, a preparation of (Na+ + K+)-ATPase, dissolved in solutions of the same nonionic detergent, can be prepared in which the predominant species (greater than 85%) is the monomer. The enzyme in this solution exhibits high specific activity, and its apparent Michaelis constants for the cationic substrates are very similar to those of the purified, membrane-bound enzyme. It is concluded from these results that a monomer of the alpha beta asymmetric unit is fully capable of catalyzing (Na+ + K+)-ATPase activity, and hence active transport, in the native enzyme. A reassessment of proposed molecular mechanisms for active transport is made in light of these discoveries.
mesh_terms: D000818:Animals; D001693:Biological Transport, Active; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents; D003902:Detergents / Q000494:pharmacology; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D005976:Glutaral / Q000494:pharmacology; D007679:Kidney Medulla / Q000201:enzymology; D046911:Macromolecular Substances; D008566:Membranes / Q000201:enzymology; D010766:Phosphorylation; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
chemical_list: D003432:Cross-Linking Reagents; D003902:Detergents; D046911:Macromolecular Substances; D000254:Sodium-Potassium-Exchanging ATPase; D005976:Glutaral