Photoaffinity Labeling of the Ouabain-Binding Site on (Na+ + K+) Adenosinetriphosphatase

An ethyl diazomalonyl derivative of cymarin was synthesized in order to photoaffinity label the cardiac glycoside-binding site on (Na(+) + K(+)) adenosinetriphosphate (EC 3.6.1.3). When a noncovalent complex of the enzyme and this cardiac glycoside derivative was photolyzed, a covalent bond was formed between the ligand and the larger of the two polypeptide subunits of the enzyme. Several control experiments demonstrate that this photochemical reaction occured while the ligand was bound to the site at which it inhibits the enzyme activity. Another specific inhibitor, tentatively identified as the ethyl chloromalonyl derivative of cymarin, produced similar photoaffinity labeling of the larger subunit, demonstrating that the photolytic dissociation of the diazo group may not be responsible for the photochemical reaction. Since the cardiac glycoside-binding site, which is accessible from the outside surface of the plasma membrane, and the phosphorylation site, which is accessible from the inside surface, are both on the larger polypeptide subunit of (Na(+) + K(+)) adenosinetriphosphatase, this polypeptide has sequences exposed to both sides of the membrane.