Transport of fatty acids across the membrane of human erythrocyte ghosts.

Abstract

Human erythrocyte ghosts were used for studying the mechanism of uptake and membrane transport of fatty acids. Hemoglobin-free ghosts were prepared and loaded with substrates such as CoA and/or ATP, and their ability for transporting and activating radiolabelled palmitic acid was tested further. Uptake of radiolabelled palmitic acid by CoA- and ATP-loaded ghosts exceeded that observed with ghosts loaded only with ATP, the latter being greater than that measured with non-loaded ghosts. Acyl-CoA was synthesized in CoA- and ATP-loaded ghosts upon incubation with radiolabelled palmitic acid. Both CoA and ATP were needed within the ghosts to permit acyl-CoA synthesis, suggesting that the acyl-CoA synthetase is located in and is bound to the inner layer of the membrane. The rate of acyl-CoA synthesis was saturable with increasing concentration of palmitic acid in the incubation mixture, and kinetic parameters were calculated. The rate of acyl-CoA synthesis in CoA- and ATP-loaded ghosts upon incubation with radiolabelled palmitic acid was markedly decreased when increasing albumin concentration in the incubation medium up to a molar ratio albumin/fatty acid of one to one. It is not easy to distinguish experimentally fatty acids located in the outer layer and the inner layer of the membrane and the data of this paper suggest that acyl-CoA synthesis by an enzyme located in the inner layer could be used as a measure of the acyl groups which have been translocated across the membrane of erythrocyte ghosts.