The affinity of rodent Na,K-ATPase alpha 1-subunit to cardiotonic steroids (CTS) is known to be approximately 1000-fold less than the affinity of Na,K-ATPase alpha 1-subunit from other mammals. The CTS-resistant isoform of Na,K-ATPase alpha 1-subunit (alpha 1R) is expressed in rodent cells, in contrast to the CTS-sensitive isoform of the alpha 1-subunit (alpha 1S), which is expressed in cells of other mammals. Earlier we have established that incubation with ouabain in concentrations that completely suppressed the activity of Na,K-ATPase alpha 1-isoforrn (alpha 1S-Na, K-ATPase) led to a death of human endothelial and smooth muscle cells but did not affect the survival of rat cells expressing alpha 1R-Na,K-ATPase. Conformational transitions that are induced by CTS-binding to Na,K-ATPase play a key role in the process of cell survival. To reveal differences in the CTS-induced conformational changes of alpha 1R- and alpha 1S-Na,K-ATPase isoforms, we used three different CTS (two cardenelids, ouabain and digoxin, and one bufadienolid, marinobufagenin) and analyzed the trypsinolysis products of alpha 1-subunits in two main conformations of Na,K-ATPase (E1 and E2-P). The treatment of the pig kidney alpha 1S-Na,K-ATPase in E1 conformation by trypsin results in a significant decrease of the amount of alpha 1S-subunit and in the appearance of protein fragments with molecular masses of about 40, 35, 23, and 19 kDa. Preincubation of Na,K-ATPase in E1 conformation with ouabain or with digoxin (1 mM) leads to a decrease of the amount of alpha 1S-subunit, increase of the amount of polypeptide fragment with molecular mass of about 40 kDa, and a significant rise of the amount of fragment with molecular mass of about 35.5 kDa, which was not found after the preincubation of the Na,K-ATPase in E1 conformation with marinobufagenin (1 mM). In the absence of CTS the trypsinolysis of alpha 1S-Na,K-ATPase in E2-P conformation results in a decrease of the amount of WS-subunit and an increase of the amount of proteolytic products with molecular mass of about 40 and 35.5 kDa. Preincubation of the Na,K-ATPase in E2-P conformation in the presence of any of the CTS studied induces the appearance of big amount of an additional peptide fragment with molecular mass of about 45 kDa. Preincubation of alpha 1RNa, K-ATPase from rat kidney with any of the CTS does not change the composition of proteolytic products and their molecular masses in either E1 or E2-P conformation. The results suggest that the structure of the CTS-binding site and a conformational response of alpha 1-Na,K-ATPase to binding of CTS is mainly determined by the primary structure of the CTS-resistant and CTS-sensitive alpha 1-subunits of the Na,K-ATPase.
