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

Ammetric titration with silver nitrate revealed the presence in pig kidney Na+,K(+)-ATPase of five disulfide bonds and twenty free cysteine residues, most of which are masked. Complete alkylation of all of free SH groups was found possible only after preliminary digestion of the membrane-bound Na+,K(+)-ATPase. A fraction of disulfide-containing peptides involving three fragments of the alpha-subunit polypeptide chain, namely: Cys452-Lys461, Ile507-Lys519, Val545-Phe558, has been isolated from the tryptic digest alkylated with 4-vinylpyridine. Reduction of S-S bonds with beta-mercaptoethanol and alkylation of the released cysteine residues with radiolabeled iodoacetic acid indicated that three above fragments contained cysteine residues that are involved in the formation of two disulfide bonds.