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(/sup 3/H)Ouabain binding and Na+, K+-ATPase in resealed human red cell ghosts

Journal Article · · J. Gen. Physiol.; (United States)
DOI:https://doi.org/10.1085/jgp.81.3.401· OSTI ID:5849878
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The interaction of the cardiac glycoside (/sup 3/H)ouabain with the Na+, K+ pump of resealed human erythrocyte ghosts was investigated. Binding of (/sup 3/H)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 (/sup 3/H)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 (/sup 3/H)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.
Research Organization:
Department of Physiology, Duke University Medical Center, Durham, North Carolina
OSTI ID:
5849878
Journal Information:
J. Gen. Physiol.; (United States), Journal Name: J. Gen. Physiol.; (United States) Vol. 81:3; ISSN JGPLA
Country of Publication:
United States
Language:
English

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Related Subjects

551001* -- Physiological Systems-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACID ANHYDRASES
ALKALI METAL COMPOUNDS
ATP
ATP-ASE
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY FLUIDS
CARBOHYDRATES
CARDIAC GLYCOSIDES
CARDIOTONICS
CARDIOVASCULAR AGENTS
CELL CONSTITUENTS
CELL MEMBRANES
CHEMICAL BONDS
DRUGS
ENZYMES
ERYTHROCYTES
GLYCOSIDES
HYDROLASES
INHIBITION
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
MATERIALS
MEMBRANE TRANSPORT
MEMBRANES
NUCLEOTIDES
ORGANIC COMPOUNDS
OUABAIN
PHOSPHOHYDROLASES
POTASSIUM COMPOUNDS
SODIUM COMPOUNDS
STROPHANTHINS
TRACER TECHNIQUES
TRITIUM COMPOUNDS