.beta. Subunit of sodium-potassium ATPase contains three disulfide bonds

Abstract

Previous studies of titratable (Na+ + K+)-ATPase sulfhydryl groups have indicated the presence of one disulfide bond per mole of holoenzyme. This single disulfide cross-link was assigned to the .beta. subunit on the basis of the difference between the number of titrated "free" sulfhydryl groups and the total number of titrated sulfhydryl groups for each subunit [Esmann, M. (1982) Biochim. Biophys. Acta 688, 251; Kawamura, M., and Nagano, K. (1984) Biochim. Biophys. Acta 694, 27]. In the present study, .beta.-subunit tryptic peptides containing disulfide cross-links were identified and purified by HPLC. Two new peptides were generated from each disulfide-bonded peptide by reduction with dithiothreitol, and the amino acid compositions of these reduced peptides were determined. The data demonstrate that there are three disulfide bonds in the native .beta. subunit; 125Cys-148Cys, 158Cys-174Cys, and 212Cys-275Cys. The number of disulfide bonds in the .beta. subunit was also estimated by titration of sulfhydryl groups with [14C]iodoacetamide. Six sulfhydryl groups were identified: two sulfhydryl groups were titrated without prior reduction, and four were identified only after reduction of the protein with dithiothreitol. These data, suggesting that the .beta. subunit contains two disulfide bonds, are inconsistent with the peptide isolation experiments, which directly identified three disulfide bonds in the .beta. subunit. This inconsistency was resolved by demonstrating that approximately 20% of each disulfide bond in the .beta. subunit was reduced prior to the start of the experiment, resulting in an underestimation of the number of disulfide-bonded sulfhydryl groups in the .beta. subunit from the titration experiments. Titration of sulfhydryl group of (Na+ + K+)-ATPase .alpha. subunit with [14C]-iodoacetamide identified 23 total sulfhydryl groups: 19 free sulfhydryl groups and 4 sulfhydryl groups that could be titrated only after reduction of the protein with dithiothreitol. In contrast to previous reports suggesting that there are no disulfide bonds in the .alpha. subunit, these data indicate that the .alpha. subunit contains two disulfide bonds.