MOLECULAR-CLONING AND IMMUNOLOGICAL CHARACTERIZATION OF THE GAMMA-POLYPEPTIDE, A SMALL PROTEIN ASSOCIATED WITH THE NA,K-ATPASE

The gamma subunit of the Na,K-ATPase is a small membrane protein that copurifies with the alpha and beta subunits of the enzyme. Strong evid ence that the gamma subunit is a component of the Na,K-ATPase comes fr om studies indicating that the subunit is involved in forming the site for cardiac glycoside binding. We have isolated and characterized the cDNAs coding the gamma subunit from several species. The gamma subuni t is a highly conserved protein consisting of 58 amino acids with a mo lecular weight of 6500. Hydropathy analysis reveals the presence of a single hydrophobic domain that is sufficient to cross the membrane. Th ere are no sites for N-linked glycosylation. Northern blot analysis re vealed that the gamma subunit mRNA is expressed in a tissue-specific f ashion and is present in all tissues characterized. Gamma-specific ant ibodies have been used to verify that the sequenced protein is the sam e protein labeled by [H-3]nitroazidobenzoyl-ouabain (NAB-ouabain), and that this protein, the gamma subunit of the Na,K-ATPase, has a distri bution pattern along nephron segments that is identical with the alpha subunit. In addition, coimmunoprecipitation of the alpha, beta and ga mma subunits demonstrate specific association of the subunits. These r esults are consistent with the notion that the gamma subunit is specif ically associated with and may be an important component of the Na,K-A TPase.