Erythrocyte Membrane Lipids and Rh Antigen Activity

Abstract

The Rh antigens have never been totally and reproducibly obtained in free solution. Indirect evidence, based among other observations on the reversible loss of Rh antigen activity of lyophilized Rh-positive erythrocyte membranes with mercurial sulfhydryl reagents, suggests that the activity may depend on membrane protein. With the hypothesis that this membrane protein may be associated perhaps structurally with membrane lipid, studies were undertaken on the effects of organic solvents on lyophilized membrane Rh antigen activity. The antigen activity was abolished after extensive extraction with 100% 1-butanol, but could be regenerated to approximately 50% of the unextracted membranes by the addition of certain lipids. The present investigations establish that phospholipids are the only class of lipids which result in such regeneration. The binding of 14C-labeled lecithin of biological origin was measured in benzene solution, in sonicated aqueous dispersions, and in 5% methanol-ether dispersions. Regeneration of Rh activity was also followed, which showed that the binding of aqueous sonicated phospholipids (liposomes) was associated with the best regeneration and the maximum labeled lecithin bound. The binding of 14C-fatty acids also occurred under similar conditions, but no associated regeneration of Rh antigen activity was observed. Similarly, an extensive series of nonphosphorus-containing lipids failed to regenerate the antigen activity. Synthetic dipalmitoyl lecithin was found to bind to the 1-butanol-extracted membranes, but not to result in regenerated activity. Labeled phosphorylcholine showed little binding to these membranes, and large amounts of unlabeled phosphorylcholine only mildly depressed the binding of labeled lecithin. This suggests that the binding of the lecithin is predominantly hydrophobic. A graded effect of different phospholipids was noted in regard to the extent of antigen regeneration. Thus, the Rh antigen activity is dependent on the presence of bound phospholipid, containing at least one unsaturated fatty acid, with neither the polar nor the nonpolar portion of the molecule alone satisfying this requirement.