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-epoxyscilliro sidin is the principal cause of poisoning of cattle by the tulip, Horn er ia pallida. The ultimate goals of this study mere to study the inte raction between 1 alpha,2 alpha-epoxyscillirosidin and ovine Na,K-ATPa se by means of inhibition and displacement binding studies, Ovine card iac Na,K-ATPase was isolated in membrane-bound form by means of deoxyc holate treatment, high-speed ultracentrifugation, NaI treatment and se lective solubilization in Lubrol. The inhibition of ovine cardiac and commercial porcine cerebral cortex Na,K-ATPase by 1 alpha,2 alpha-epox yscillirosidin and ou ab ain was studied using a discontinuous Na,K-AT Pase assay, The binding of 1 alpha,2 alpha-epoxyscillirosidin, ouabain and digoxin to the above enzymes was compared using a displacement bi nding assay with [H-3] oubain. The Lubrol-solubilized ovine cardiac Na ,K-ATPase showed a specific activity of 0.3 U/mg with no ouabain insen sitive activity, I-50 values of 2.1 x 10(-8) and 2.7 x 10(-8) mere obt ained for the inhibition of this enzyme by 1 alpha,2 alpha-epoxyscilli rosidin and ouabain, respectively, 1 alpha,2 alpha-Epoxyscillirosidin has a much higher K-D 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 stu dies with [H-3]ouabain. 1 alpha,2 alpha-Epoxyscillirosidin is a potent inhibitor of ovine cardiac Na,K-ATPase and is a slightly stronger inh ibitor of the enzyme than ouabain, The anomalous result for the displa cement of 1 alpha,2 alpha-epoxyscillirosidin from its receptor is eith er 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. (C) 1997 Elsevier S cience Ltd.