Sulfonates are low-affinity ligands for the GDP-binding site of brown-fat mitochondria

Abstract

In order to study the function of the brown-fat specific uncoupling protein thermogenin (UCP), the effect of certain sulfonates on [ 3H]GDP binding to the GDP-binding site of brown adipose tissue mitochondria was studied. The affinity of [ 3H]GDP for the site was 1.3 μM in the normal sucrose medium, but the apparent K D was increased to ≈ 20 μM in 100 mM hexanesulfonate medium. This increase in apparent K D was found to be due to a competitive binding of hexanesulfonate to the GDP-binding site; the affinity of hexanesulfonate was only 13 mM but this was sufficient to affect the apparent affinity of GDP under experimental conditions. Also in KCl-medium, the affinity of GDP was high (≈ 3 μM), but both in a benzenesulfonate medium and in a para-aminobenzenesulfonate (sulfanilate) medium, the apparent affinity was lower (≈ 12 μM); as benzenesulfonate is well transported by thermogenin but sulfanilate is not, the reduction in affinity was unrelated to transport. In agreement with earlier data (Jezek, P. and Garlid, K.D. (1990) J. Biol. Chem. 265, 19303–19311), the potency of GDP to inhibit transport was dependent on the species transported; the fact that GDP potency was lower for benzenesulfonate transport (EC 50 = 324 μM) than for Cl − transport (EC 50 = 32 μM) could adequately be explained by the competitive interaction of benzenesulfonate with the GDP-binding site, but this effect could only partly explain the even lower potency of GDP to inhibit hexanesulfonate transport (EC 50 = 4074 μM). It was concluded that these types of substrate for thermogenin-mediated transport may directly interact with the GDP-binding site, but that this effect could only partly explain in the dependence of GDP potency on substrate species.