The functional unit of the Na,K-ATPase has previously been shown to include two large polypeptide chains (mol wt ⋍ 95 000), only one of which can bind ouabain or be phosphorylated at one time. We have investigated the problem as to whether, when ouabain is bound and the enzyme is simultaneously phosphorylated from inorganic phosphate (Pi), both ligands are on the same large polypeptide chain or if they are on different 95K chains. We covalently labeled Na,K-ATPase purified from pig kidney outer medulla by using [3H]-2-nitro-5-azidobenzoylouabain ([3H]NAB-ouabain) and [32P] phosphate, solubilized it with sodium dodecyl sulfate, and isolated the 95K polypeptide chains by using polyacrylamide gel electrophoresis. Large polypeptide chains labeled with [3H]NAB-ouabain were separated from unlabeled chains by binding to a ouabain antibody and precipitation with immobilized protein A of Staphylococcus aureus. It was found that, for each chain thus separated, an equivalent amount of 32P was precipitated. In controls, when two different samples were independently labeled with [3H]NAB-ouabain or phosphorylated from 32Pi and mixed before analysis, coprecipitation of 32P with [3H]NAB-ouabain-labeled chains was not observed. The results are thus in quantitative agreement with a model in which [3H]NAB-ouabain binds at the same time to the same 95K polypeptide chain of a Na,K-ATPase functional unit that is phosphorylated from 32Pi. © 1979, American Chemical Society. All rights reserved.
