Ouabain and substrate affinities of human Na+-K+-ATPase α1β1, α2β1, and α3β1when expressed separately in yeast cells

Human Na+-K+-ATPase α1β1, α2β1, and α3β1heterodimers were expressed individually in yeast, and ouabain binding and ATP hydrolysis were measured in membrane fractions. The ouabain equilibrium dissociation constant was 13–17 nM for α1β1 and α3β1at 37°C and 32 nM for α2β1, indicating that the human α-subunit isoforms have a similar high affinity for cardiac glycosides. K 0.5 values for antagonism of ouabain binding by K+ were ranked in order as follows: α2 (6.3 ± 2.4 mM) > α3(1.6 ± 0.5 mM) ≈ α1 (0.9 ± 0.6 mM), and K 0.5 values for Na+ antagonism of ouabain binding to all heterodimers were 9.5–13.8 mM. The molecular turnover for ATP hydrolysis by α1β1 (6,652 min−1) was about twice as high as that by α3β1 (3,145 min−1). These properties of the human heterodimers expressed in yeast are in good agreement with properties of the human Na+-K+-ATPase expressed in Xenopusoocytes (G Crambert, U Hasler, AT Beggah, C Yu, NN Modyanov, J-D Horisberger, L Lelievie, and K Geering. J Biol Chem275: 1976–1986, 2000). In contra...