Two classes of ouabain binding sites in ferret heart and two forms of Na+-K+-ATPase
In partially purified Na+-K+-adenosinetriphosphatase (ATPase) obtained from ferret heart, ouabain produced a monophasic inhibition curve; however, the curve spanned over 5 logarithmic units, indicating the presence of more than one classes of enzyme. (/sup 3/H)ouabain binding studies revealed high-and low-affinity binding sites in approximately equal abundance, with apparent dissociation constants of 10 and 230 nM, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of phosphoenzyme formed from (gamma-/sup 32/P)ATP showed two distinct K+-sensitive bands of approximately 100,000 molecular weight. Phosphoenzyme formation from the high-molecular-weight alpha(+) form was selectively inhibited by N-ethylmaleimide. Ouabain caused a 50% inhibition of phosphorylation of the alpha(+) form at 40 nM and the lower-molecular-weight alpha form at 300 nM. In papillary muscle preparations, 1-30 nM ouabain produced a modest positive inotropic effect that reached an apparent plateau at 30 nM. Further increases in ouabain concentrations, however, produced additional and prominent inotropic effects at 0.1-10 microM. These results indicate for the first time in cardiac muscle that the high- and low-affinity ouabain binding sites are associated with the alpha(+) and alpha forms of the Na+-K+-ATPase, respectively, and that binding of ouabain to either of these sites causes enzyme inhibition and the positive inotropic effect.
- Research Organization:
- Michigan State Univ., East Lansing
- OSTI ID:
- 6501466
- Journal Information:
- Am. J. Physiol.; (United States), Journal Name: Am. J. Physiol.; (United States)
- Country of Publication:
- United States
- Language:
- English
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MUSCLES
CONTRACTION
OUABAIN
BIOCHEMICAL REACTION KINETICS
RECEPTORS
AFFINITY
ATP
ATP-ASE
ELECTROPHORESIS
IN VITRO
ISOENZYMES
MAMMALS
MOLECULAR WEIGHT
MYOCARDIUM
PHOSPHORUS 32
PHOSPHORYLATION
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ACID ANHYDRASES
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BODY
CARBOHYDRATES
CARDIAC GLYCOSIDES
CARDIOTONICS
CARDIOVASCULAR AGENTS
CARDIOVASCULAR SYSTEM
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
DRUGS
ENZYMES
GLYCOSIDES
HEART
HYDROLASES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LABELLED COMPOUNDS
LIGHT NUCLEI
MEMBRANE PROTEINS
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANS
PHOSPHOHYDROLASES
PHOSPHORUS ISOTOPES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
STROPHANTHINS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques