Ovine cardiac Na,K-ATPase: isolation by means of selective solubilization in Lubrol and the effect of 1 alpha,2 alpha-epoxyscillirosidin on this enzyme.
The inhibition of cardiac Na,K-ATPase by 1 alpha,2 alpha-epoxyscillirosidin is the principal cause of poisoning of cattle by the tulip, Homeria pallida. The ultimate goals of this study were to study the interaction between 1 alpha,2 alpha-epoxyscillirosidin and ovine Na,K-ATPase by means of inhibition and displacement binding studies. Ovine cardiac Na,K-ATPase was isolated in membrane-bound form by means of deoxycholate treatment, high-speed ultracentrifugation, NaI treatment and selective solubilization in Lubrol. The inhibition of ovine cardiac and commercial porcine cerebral cortex Na,K-ATPase by 1 alpha,2 alpha-epoxyscilirosidin and ouabain was studied using a discontinuous Na,K-ATPase assay. The binding of 1 alpha,2 alpha-epoxyscillirosidin, ouabain and digoxin to the above enzymes was compared using a displacement binding assay with [3H] oubain. The Lubrol-solubilized ovine cardiac Na,K-ATPase showed a specific activity of 0.3 U/mg with no ouabain insensitive activity. I50 values of 2.1 x 10(-8) and 2.7 x 10(-8) were obtained for the inhibition of this enzyme by 1 alpha,2 alpha-epoxyscillirosidin and ouabain, respectively. 1 alpha,2 alpha-Epoxyscillirosidin has a much higher KD value (1.5 x 10(-7) M), however, than ouabain (9.5 x 10(-9) M) and digoxin (1.7 x 10(-8) M) in displacement binding studies with [3H]ouabain. 1 alpha,2 alpha-Epoxyscillirosidin is a potent inhibitor of ovine cardiac Na,K-ATPase and is a slightly stronger inhibitor of the enzyme than ouabain. The anomalous result for the displacement of 1 alpha,2 alpha-epoxyscillirosidin from its receptor is either a result of different affinities that K+ has for the enzyme ouabain and enzyme-1 alpha,2 alpha-epoxyscillirosidin complexes or because of different complex stabilities of these complexes.
Venter
P A
PA
Department of Biochemistry, University of Port Elizabeth, South Africa.
Naudé
R J
RJ
Oelofsen
W
W
Swan
G E
GE
D000818:Animals; D001667:Binding, Competitive; D002301:Cardiac Glycosides / Q000494:pharmacology*; D002417:Cattle; D002540:Cerebral Cortex / Q000201:enzymology; D002770:Cholenes / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004795:Enzyme Stability; D009206:Myocardium / Q000201:enzymology*; D010042:Ouabain / Q000378:metabolism; D011092:Polyethylene Glycols; D012756:Sheep; D012974:Sodium Iodide / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D013552:Swine
D002301:Cardiac Glycosides; D002770:Cholenes; D004791:Enzyme Inhibitors; C110052:epoxyscillirosidin; D011092:Polyethylene Glycols; C005092:lubrol; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase; D012974:Sodium Iodide