Effect of fatty acids on H+ transport activity of the reconstituted uncoupling protein

A detailed study on the activation of H+ transport by reconstituted uncoupling proteins from brown adipose tissue is given, including the influence of chain lengths and of other structural modifications, concentration dependence, and the influence of nucleotides. Uncoupling protein was reconstituted with phosphatidylcholine in such a way as to keep H+ transport with endogenous fatty acids at a minimum. Using excess of polystyrene beads on reconstitution avoided the complications arising from the use of albumin. Both delta psi-driven H+ uptake and H+ efflux systems are used by changing the polarity. Fatty acids stimulate H+ uptake up to 6-fold and H+ efflux more than 10-fold. There is no competition between the inhibition by nucleotides (GTP) and fatty acids. Also, the binding of GTP and ATP is not affected. Only fatty acids starting from C8 activate, reaching a maximum at C14. However, unsaturated homologous of C18 (oleic, linoleic, etc.) are fully active. Hydrophilic substitutions by hydroxyl, CO2H, bromo, doxyl groups also permit good activation, probably due to improved uptake into the lipid phase. The hydrophobic moiety exhibits a low specificity. Blockage of carboxyl by esterification abolishes the activation. Maximum activation requires high total concentrations of 200-300 microM. The distribution of fatty acids between proteoliposomes and solution was determined. The activation mode of fatty acids is discussed either as regulatory activators or as cofactors in H+ translocation involving their carboxyl groups. Two alternatives are considered, namely that the fatty acids carboxyl group is at the translocation center or in the channel facilitating H+ transfer to the constituent H(+)-translocating carboxyl groups.