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 |
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Authors: | Müller-Ehmsen J Juvvadi P Thompson C B Tumyan L Croyle M Lingrel J B Schwinger R H McDonough A A Farley R A |
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Institution: | Department of Physiology and Biophysics, Keck School of Medicine, University of Southern California, Los Angeles, California 90033, USA. |
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Abstract: | HumanNa+-K+-ATPase11,21, and 31heterodimers were expressed individually in yeast, and ouabainbinding and ATP hydrolysis were measured in membrane fractions. Theouabain equilibrium dissociation constant was 13-17 nM for11 and 31at 37°C and 32 nM for 21, indicatingthat the human -subunit isoforms have a similar high affinity forcardiac glycosides. K0.5 values for antagonism of ouabain binding by K+ were ranked in order as follows:2 (6.3 ± 2.4 mM) > 3(1.6 ± 0.5 mM) 1 (0.9 ± 0.6 mM),and K0.5 values for Na+ antagonismof ouabain binding to all heterodimers were 9.5-13.8 mM. Themolecular turnover for ATP hydrolysis by11 (6,652 min1) was abouttwice as high as that by 31 (3,145 min1). These properties of the human heterodimersexpressed in yeast are in good agreement with properties of the humanNa+-K+-ATPase expressed in Xenopusoocytes (G Crambert, U Hasler, AT Beggah, C Yu, NN Modyanov, J-DHorisberger, L Lelievie, and K Geering. J Biol Chem275: 1976-1986, 2000). In contrast to Na+ pumpsexpressed in Xenopus oocytes, the21 complex in yeast membranes wassignificantly less stable than 11 or31, resulting in a lower functionalexpression level. The 21 complex was also more easily denatured by SDS than was the11 or the31 complex. |
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