Document Detail

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.
MedLine Citation:
PMID:  11546674     Owner:  NLM     Status:  MEDLINE    
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 equilibrium dissociation constant was 13-17 nM for alpha(1)beta(1) and alpha(3)beta(1) at 37 degrees C and 32 nM for alpha(2)beta(1), indicating that the human alpha-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: alpha(2) (6.3 +/- 2.4 mM) > alpha(3) (1.6 +/- 0.5 mM) approximately alpha(1) (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 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 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(2)beta(1) complex in yeast membranes 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.
J Müller-Ehmsen; P Juvvadi; C B Thompson; L Tumyan; M Croyle; J B Lingrel; R H Schwinger; A A McDonough; R A Farley
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  American journal of physiology. Cell physiology     Volume:  281     ISSN:  0363-6143     ISO Abbreviation:  Am. J. Physiol., Cell Physiol.     Publication Date:  2001 Oct 
Date Detail:
Created Date:  2001-09-07     Completed Date:  2001-10-18     Revised Date:  2007-11-15    
Medline Journal Info:
Nlm Unique ID:  100901225     Medline TA:  Am J Physiol Cell Physiol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  C1355-64     Citation Subset:  IM    
Department of Physiology and Biophysics, Keck School of Medicine, University of Southern California, Los Angeles, California 90033, USA.
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MeSH Terms
Adenosine Triphosphate / metabolism
Amino Acid Sequence
Cloning, Molecular
Enzyme Inhibitors / pharmacology*
Gene Expression Regulation, Enzymologic
Isoenzymes / genetics*,  metabolism*
Microsomes / enzymology
Ouabain / pharmacology*
Saccharomyces cerevisiae
Sodium-Potassium-Exchanging ATPase / genetics*,  metabolism*
Substrate Specificity
Grant Support
5 R01 HL-28573-17/HL/NHLBI NIH HHS; GM-28673/GM/NIGMS NIH HHS; P01 HL-41496-12/HL/NHLBI NIH HHS
Reg. No./Substance:
0/Enzyme Inhibitors; 0/Isoenzymes; 56-65-5/Adenosine Triphosphate; 630-60-4/Ouabain; EC ATPase

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