Oleic acid binding and transport capacity of human red cell membrane

Resealed human red cell membranes, ghosts, bind oleate (OL) by a limited number of sites when equilibrated al 37 degrees C, pH 7.3 with OL bound to bovine serum albumin (BSA) in molar ratios below 1.5. The binding capacity is 34+/-2.2 nmol g(-1) ghosts with a dissociation equilibrium constant (37 degrees C) K-dm 1.38+/-0.15 fold K-d of albumin binding. K-dm is temperature independent and similar to 7-8 nM. Exchange efflux kinetics at 0 degrees C to buffers of various albumin concentrations ([BSA(y)]) is biexponential and is analysed in terms of a three-compartment model. Accordingly the ratio of inner to outer membrane leaflet binding sites is 0.450+/-0.018 and the rate constant of unidirectional flux from inside to outside is 0.067+/-0.01 s(-1). The rate constant of flux from the extracellular side of the membrane to BSA(y) increases with the square root of [BSA(y)] as expected of an unstirred layer effect. This provides an estimate of the dissociation rate constant of OL-BSA complex at 0 degrees C of 0.0063+/-0.0003 s(-1). Exchange efflux from ghosts containing four different [BSA(i)] obeys the expected kinetics of a three-compartment approximation of the theoretical model. Accounting for the effect of an unstirred fluid inside ghosts, the rare coefficients fit the values predicted by the parameters obtained by the studies of albumin-free ghosts. The results show that the OL transport across the membrane is mediated exclusively by the asymmetrically distributed binding sites. The differences between transport sites of three long-chain fatty acids suggest that they are protein determined microdomains of phospholipids.