ATP binding to solubilized (Na+ + K+)-ATPase. The abolition of subunit-subunit interaction and the maximum weight of the nucleotide-binding unit.
Membrane-bound (Na+ + K+)-ATPase from pig kidney outer medulla shows apparent heterogeneity in its ATP-binding site population when assays are carried out in the presence of K+. This finding has been interpreted as being due to interaction between (at least) two subunits, each containing an ATP-binding site. Treating the membrane-bound enzyme with the detergent, C12E8, has been shown to solubilize enzymatically active alpha beta-protomers. We show that in the dissolved enzyme all ATP-binding sites in the population are identical both in the absence and in the presence of K+, which would be consistent with an abolition of identical both in the absence and in the presence of K+, which would be consistent with an abolition of subunit-subunit interaction. This supports previous suggestions that enzyme solubilized by C12E8 is monomeric and that the membrane-bound enzyme is not. Differential extraction of enzyme-containing membranes with C12E8 yielded preparations with an ATP-binding capacity of up to 5.8 nmol per mg protein, measured by the method of Lowry et al. (Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275), with bovine serum albumin as standard. Evidence is presented that makes it likely that preparations with an ATP-binding capacity of 7.5 nmol per mg protein (as determined by the above-mentioned assay) will be obtainable. This corresponds to an alpha beta-protomer molecular weight of 133 000 which approximates closely to the minimum value found in the literature for an alpha beta-protomer (i.e., 126 000).
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D007679:Kidney Medulla / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D011485:Protein Binding; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D012995:Solubility; D013552:Swine
D046911:Macromolecular Substances; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase