Ovine cardiac Na,K-ATPase: Isolation by means of selective solubilization in lubrol and the effect of 1α,2α-epoxyscillirosidin on this enzyme

Abstract

The inhibition of cardiac Na,K-ATPase by 1α,2α-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 1α,2α-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 1α,2α-epoxyscillirosidin and ouabain was studied using a discontinuous Na,K-ATPase assay. The binding of 1α,2α-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. I 50 values of 2.1 × 10 −8 and 2.7 × 10 −8 were obtained for the inhibition of this enzyme by 1α,2α-epoxyscillirosidin and ouabain, respectively. 1α,2α-Epoxyscillirosidin has a much higher K D value (1.5 × 10 −7 M), however, than ouabain (9.5 × 10 −9 M) and digoxin (1.7 × 10 −8 M) in displacement binding studies with [ 3H]ouabain. 1α,2α-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 1α,2α-epoxyscillirosidin from its receptor is either a result of different affinities that K + has for the enzyme-ouabain and enzyme-1α,2α-epoxyscillirosidin complexes or because of different complex stabilities of these complexes.