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Am J Physiol Cell Physiol 281: C1355-C1364, 2001;
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Vol. 281, Issue 4, C1355-C1364, October 2001

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

Jochen Müller-Ehmsen1,3, Padmaja Juvvadi1, Curtis B. Thompson1, Lusine Tumyan1, Michelle Croyle2, Jerry B. Lingrel2, Robert H. G. Schwinger3, Alicia A. McDonough1, and Robert A. Farley1

1 Department of Physiology and Biophysics, Keck School of Medicine, University of Southern California, Los Angeles, California 90033; 2 Department of Molecular Genetics, Biochemistry, and Microbiology, College of Medicine, University of Cincinnati, Cincinnati, Ohio 45267-0524; and 3 Laboratory of Muscle Research and Molecular Cardiology, Clinic III of Internal Medicine, University of Cologne, 50924 Cologne, Germany

Human Na+-K+-ATPase alpha 1beta 1, alpha 2beta 1, and alpha 3beta 1 heterodimers 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 alpha 1beta 1 and alpha 3beta 1 at 37°C and 32 nM for alpha 2beta 1, indicating that the human alpha -subunit isoforms have a similar high affinity for cardiac glycosides. K0.5 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) approx  alpha 1 (0.9 ± 0.6 mM), and K0.5 values for Na+ antagonism of ouabain binding to all heterodimers were 9.5-13.8 mM. The molecular turnover for ATP hydrolysis by alpha 1beta 1 (6,652 min-1) was about twice as high as that by alpha 3beta 1 (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 Xenopus oocytes (G Crambert, U Hasler, AT Beggah, C Yu, NN Modyanov, J-D Horisberger, L Lelievie, and K Geering. J Biol Chem 275: 1976-1986, 2000). In contrast to Na+ pumps expressed in Xenopus oocytes, the alpha 2beta 1 complex in yeast membranes was significantly less stable than alpha 1beta 1 or alpha 3beta 1, resulting in a lower functional expression level. The alpha 2beta 1 complex was also more easily denatured by SDS than was the alpha 1beta 1 or the alpha 3beta 1 complex.

sodium pump; cardiac glycosides; heterologous expression


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