A new variant of the γ subunit of renal Na,K-ATPase -: Identification by mass spectrometry, antibody binding, and expression in cultured cells

The gamma subunit is a specific regulator of Na,K-ATPase expressed mainly in kidney, On SDS-polyacryylamide gel electrophoresis, gamma runs as a doublet, but the origin and significance of the doublet is obscure. Mass spectrometry of the gamma chains of rat kidney Na,K-ATPase shows that gamma(a) (upper) has a mass of 7184.0 +/- 1 Da (carbamidomethyl cysteine), corresponding closely to that for the published sequence without the initiator methionine, while gamma(b) (lower) has a mass of 7337.9 +/- 1Da Tryptic peptide mapping and sequencing by mass spectrometry reveals that the seven N-terminal residues of gamma(a), TELSANH, are replaced by Ac-MDRWYL in gamma(b), but otherwise the chains are identical. Antibodies raised against peptides TELSANHC and MDRWYLC recognize either gamma(a) or gamma(b) of the Na,K-ATPase, respectively, gamma(a) or gamma(b) cDNAs have been expressed in human embryonic kidney and HeLa cells, The major bands expressed correspond to gamma(a), or gamma(b) of renal Na,K-ATPase, Additional minor bands seen after transfection, namely gamma(a)' in human embryonic kidney and gamma(b)' in HeLa, are presumably cell-specific modifications, The present work clarifies earlier uncertainty regarding doublets seen in kidney and in transfected cells. In particular, the results show that renal Na,K-ATPase contains two variants of the gamma sub unit with different sequences but otherwise are unmodified. We discuss the possible functional significance of the two variants.