Inactivation of (Na+,K+)-ATPase by beta-mercaptoethanol. Differential sensitivity to reduction of the three beta subunit disulfide bonds.

The relative rates of reduction of disulfide bonds in sheep kidney (Na+,K+)-ATPase were determined in order to test the hypothesis that cleavage of a single disulfide bond in the beta subunit is responsible for loss of ATPase activity upon treatment with high concentrations of beta-mercaptoethanol at elevated temperatures (Kawamura, M., and Nagano, K. (1984) Biochim. Biophys. Acta 774, 188-192). This was accomplished by peptide mapping of tryptic digests of the alpha and beta subunits after treatment with beta-mercaptoethanol. Alkylation with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole of all free sulfhydryls and all newly produced sulfhydryls resulting from reduction of disulfides by beta-mercaptoethanol introduced a fluorophore into those residues. The protective effect of NaCl and KCl on ATPase activity was examined with regard to the ability of those salts to protect the disulfides from reduction. The results indicated that although the disulfides in the beta subunit exhibit differential stabilities to reduction, the reduction of no single disulfide in the beta subunit can be quantitatively correlated with the loss of ATPase activity and the ability to bind ouabain. The kinetics of inhibition of ouabain binding were identical to the kinetics of inhibition of ATPase activity, suggesting a delocalized denaturation rather than reduction of a single disulfide bond as the mechanism of beta-mercaptoethanol induced inhibition. NaCl and KCl only partially protected all three beta disulfides from reduction, but completely eliminated the loss of ATPase activity and ouabain binding sites induced by beta-mercaptoethanol. The results suggest a possible folding model for the beta subunit. The alpha subunit was found to contain three disulfide bonds, but these bonds were not reduced by the beta-mercaptoethanol treatment used to inactivate the ATPase activity.