Binding sites for ouabain in human and rat erythrocytes and in rat heart cells.

The presence and properties of two classes of binding sites for digitalis in erythrocytes and in heart cells are reviewed. Methods to distinguish between these two binding sites are summarized for intact cells and for their membranes. Our biochemical data do not suggest a physiological role for each class of sites. The membrane sites were modified by varying (a) the content of cholesterol or (b) the distribution of fatty acids leading to changes in the microviscosity thus affecting the degree of binding of ouabain to the two classes of sites. Oxidation of ouabain with periodate forms a di-aldehyde which could bind via Schiff base to the digitalis sites but could also attach covalently to these sites. The role of the sugar moiety in the process of ouabain binding becomes of increasing importance. A mild, controlled periodate oxidation of ouabain, especially in presence of phosphate cleaves only the bond between C-2 and C-3 of the rhamnose without affecting the steroid moiety. The periodate oxidation provided additional information for assigning a distorted chair conformation or a transient boat conformation for rhamnose in ouabain. It was also established by 1H NMR spectroscopy that this chair conformation is a 1C4 pyranose ring.