Ouabain and substrate affinities of human Na+-K+-ATPase alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) when expressed separately in yeast cells

Human Na+-K+-ATPase alpha (1)beta (1), alpha (2)beta (1), and alpha (3)beta (1) heterodimers were expressed individually in yeast, and ouabain binding and ATP hydrolysis were measured in membrane fractions. The ouabain equili brium dissociation constant was 13-17 nM for alpha (1)beta (1) and alpha (3 )beta (1) at 37 degreesC and 32 nM for alpha (2)beta (1), indicating that t he human alpha -subunit isoforms have a similar high affinity for cardiac g lycosides. K-0.5 values for antagonism of ouabain binding by K+ were ranked in order as follows: alpha (2) (6.3 +/-2.4 mM) > alpha (3) (1.6 +/-0.5 mM) approximate to alpha (1) (0.9 +/-0.6 mM), and K-0.5 values for Na+ antagon ism of ouabain binding to all heterodimers were 9.5-13.8 mM. The molecular turnover for ATP hydrolysis by alpha (1)beta (1) (6,652 min(-1)) was about twice as high as that by alpha (3)beta (1) (3,145 min(-1)). These propertie s of the human heterodimers expressed in yeast are in good agreement with p roperties of the human Na+-K(+)ATPase expressed in Xenopus oocytes (G Cramb ert, U Hasler, AT Beggah, C Yu, NN Modyanov, J-D Horisberger, L Lelievie, a nd K Geering. J Biol Chem 275: 1976-1986, 2000). In contrast to Na+ pumps e xpressed in Xenopus oocytes, the alpha (2)beta (1) complex in yeast membran es was significantly less stable than alpha (1)beta (1) or alpha (3)beta (1 ), resulting in a lower functional expression level. The alpha (2)beta (1) complex was also more easily denatured by SDS than was the alpha (1)beta (1 ) or the alpha (3)beta (1) complex.