Molecular weight and stoichiometry of the sodium- and potassium-activated adenosine triphosphatase subunits.

Abstract

The molecular weight and stoichiometry of the subunits of the sodium- and potassium-activated adenosine triphosphatase. (Na,K)-ATPase, have been examined in four highly purified preparations of this enzyme: dog kidney, eel electroplax, dogfish rectal gland, and brine shrimp nauplius. The molecular weights of the subunits were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 10 different total acrylamide concentrations (%T) and by a gradient SDS-PAGE system, standardized with 16 molecular weight marker proteins of known primary structure. The molecular weight of the large (Na,K)-ATPase subunit (alpha subunit) was found to be reliably estimated by SDS-PAGE, whereas the small subunit (beta subunit) was not. The molecular weight of the alpha subunit was 97,000, 97,700, 104,200, and 97,800 for the dog, eel, dogfish, and brine shrimp (Na,K)-ATPase, respectively. The molecular weight of the beta subunit is overestimated by SDS-PAGE. Mass ratio analysis of the alpha and beta subunits separated by SDS-PAGE was determined by three techniques: total amino acid quantitation, gel scanning and dye elution of Coomassie blue-stained gels, and UV gel scanning. The former method, which provides the most reliable estimate of the mass ratio, gives an alpha/beta mass ratio of 2.41, 2.33, 2.91, and 2.44 for the dog, eel, dogfish, and brine shrimp preparations, respectively. Results of the mass ratio studies, the molecular weight analysis, and prior cross-linking studies clearly demonstrate that the quaternary structure of the (Na,K)-ATPase is an alpha 2 beta 2 tetramer in all four species. Based on all of the above data, a more reliable molecular weight estimate of the beta subunit is calculated as 40,200, 43,000, 35,800, and 40,100 for the dog, eel, dogfish, and brine shrimp preparations, respectively. These results now confirm that the molecular weight of the protein portion of the alpha subunit is near 100,000 and that of the beta subunit is near 40,000 and that the (Na,K)-ATPase holoenzyme is an alpha 2 beta 2 tetramer of molecular weight 274,000 to 280,000, regardless of species.