Location of disulfide bonds in the Na+, K(+)-ATPase alpha subunit

For localizing S-S bonds in the pig kidney Na+,K(+)-ATPase alpha subunit, cystine-containing peptides (V-1, VII-1, and VII-2), obtained in our previous study from the enzyme's tryptic digest, were analysed. Chemical modification of the cystine-containing peptides performed at cysteine residues involved successive alkylation, first with radioactive iodoacetic acid and then with ABD-F in the absence and presence of a reducing agent, respectively. Cysteinyl peptides were isolated by HPLC, their amino acid sequences determined, and two disulfide bonds: Cys452-Cys456 and Cys511-Cys549 were localized by identification of fluorescent cysteine residues.