Influence of ouabain and dihydroouabain on the circular dichroism of cardiac plasmalemmal microsomes.

Abstract

1. The influence of ouabain on the tertiary structure of cardiac plasmalemmal proteins was investigated by means of circular dichroism measurement. Purified plasmalemmal microsomes were obtained by sucrose gradient centrifugation. The CD-spectra of the membranal proteins were shifted to the red and the amplitudes were smaller than those of the same proteins after solubilization. 2. Ouabain induced an increase of the ellipticity bands at 210 and 222 nm of about 50% above the level yielded with microsomes after sonication. At 222 nm ouabain exhibited the half maximum effect at a concentration of 5 X 10(-9) M. The effect could, however, only be exerted if the inside of the microsomes was exposed to ouabain by sonication, thus reflecting the inside-out nature of the plasmalemmal microsomes. 3. The high specificity of the ouabain effect was underlined by the following experiments: a) Dihydroouabain, a much less cardioactive derivative of ouabain proved to be ineffective in corresponding concentrations, b) ouabain had no influence upon the CD spectrum of microsomes derived from cardiac sarcoplasmic reticulum, c) a detergent-like action of ouabain underlying the observed effect can be excluded since highly active tensides, i.e. desoxycholate and dodecylsulfate, only influence the CD spectra at concentrations exceeding 10(-3) M, d) electronmicrographs of microsomes exposed to ouabain demonstrated no alteration of either the appearance or size of the microsomes. 4. The magnitude of the observed ouabain effect indicates that a large portion of the membrane-bound proteins is involved. The number of binding sites and their isolated structural alteration induced by ouabain are not sufficient to account quantitatively for the enhanced amplitudes of the CD spctra. This suggests that ouabain evokes structural changes of membrane proteins different from actual binding sites. It seems, therefore highly improbable that changes of the Na-K-ATPase present in the plasmalemmal microsomes are responsible for the observed effect.