The Journal of General Physiology, Vol 81, 401-420, Copyright © 1983 by The Rockefeller University Press
[3H]Ouabain binding and Na+, K+-ATPase in resealed human red cell ghosts
DG Shoemaker and PK Lauf
The interaction of the cardiac glycoside [3H]ouabain with the Na+, K+ pump
of resealed human erythrocyte ghosts was investigated. Binding of
[3H]ouabain to high intracellular Na+ ghosts was studied in high
extracellular Na+ media, a condition determined to produce maximal ouabain
binding rates. Simultaneous examination of both the number of ouabain
molecules bound per ghost and the corresponding inhibition of the Na+,
K+-ATPase revealed that one molecule of [3H]ouabain inhibited one Na+,
K+-ATPase complex. Intracellular magnesium or magnesium plus inorganic
phosphate produced the lowest ouabain binding rate. Support of ouabain
binding by adenosine diphosphate (ADP) was negligible, provided synthesis
of adenosine triphosphate (ATP) through the residual adenylate kinase
activity was prevented by the adenylate kinase inhibitor Ap5A. Uridine
5'-triphosphate (UTP) alone did not support ouabain binding after
inhibition of the endogenous nucleoside diphosphokinase by trypan blue and
depletion of residual ATP by the incorporation of hexokinase and glucose.
ATP acting solely at the high- affinity binding site of the Na+, K+ pump
(Km approximately 1 microM) promoted maximal [3H]ouabain binding rates.
Failure of 5'-adenylyl-beta- gamma-imidophosphate (AMP-PNP) to stimulate
significantly the rate of ouabain binding suggests that phosphorylation of
the pump was required to expose the ouabain receptor.
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