Determination of sidedness of COOH-terminus of Na⁺/K⁺ ATPase alpha subunit by vectorial labeling

Abstract

Extensive studies have been performed on structure-function relationship of the Na⁺ /K⁺-ATPase. Nevertheless, current ideas on basic processes underlying coupled ATP hydrolysis, energy transduction and cation transport are deficient in molecular details. This is mainly due to the lack of reliable data on the spatial protein structure (1). The three-dimensional structure of the Na⁺/K⁺-ATPase cannot yet be solved by X-ray analysis. However, significant information on the spatial organization of the protein molecule may be derived by other experimental approaches such as limited proteolysis, affinity modification, labeling by permeable and impermeable reagents, analysis with specific antibodies and spectroscopic methods. A combination of these techniques has been used to identify functional domains and to probe the folding of subunit polypeptide chains in the membrane. It is generally accepted that the beta subunit spans the membrane only once (1,4). In contrast, the transmembrane folding of the catalytic alpha subunit has been the subject of considerable controversy. Several topological models differ in the number and location of the membrane-spanning segments within the COOH-terminal half of the polypeptide chain and therefore the orientation of the COOH-terminus (2,3,5,7).