Solubilization, reconstitution and molecular properties of the triiodothyronine transport protein from rat erythrocyte membranes

Abstract

Samson M, Osty J, Thibout H, Blondeau J-P. Solubilization, reconstitution and molecular properties of the triiodothyronine transport protein from rat erythrocyte membranes. Eur J Endocrinol 1996;134: 660–8. ISSN 0804–4643 Triiodothyronine (T3) transport through the mammalian erythrocyte membrane is mediated by a transport system related to the aromatic amino acid transport system T. The T3-binding component of this transport system could be photolabeled with [125I]T3 as a 52-kD protein, and subsequently solubilized with non-ionic detergents. Upon purification by ion-exchange chromatography, the photolabeled 52-kD protein solubilized with octylglucoside (OG) resolved into several peaks, suggesting charge heterogeneity of labeled proteins. The saturable [125I]T3 binding to rat erythrocyte membranes was completely inhibited by non-ionic detergents at concentrations about 20 times lower than those that solubilized membrane. Therefore, detergent-free proteoliposomes were generated from the detergent-soluble extracts by treatment with a polystyrene adsorbent. Proteoliposomes prepared from OG-soluble extract contained the highest specific activity of T3 binding. The Kd of the T3 binding sites (4.5 nmol/l) and the competitive inhibition constant of tryptophan (120 μmol/l) were similar to those for native membranes. The photolabeling of the 52-kD protein in these proteoliposomes was prevented by tryptophan and T4, but not by leucine or the d-isomer of T3, corresponding to the transport specificity of system T. The 52-kD protein solubilized with OG from native membranes was partially purified by ion-exchange chromatography. The 52-kD protein was detected by photoaffinity labeling in the purified fraction only after addition of erythrocyte membrane phospholipids to generate proteoliposomes. This indicates that the association of 52-kD protein with phospholipids is critical for T3 binding. JP Blondeau, U96 INSERM, 80 Rue de Général Leclerc, 94276 Le Kremlin-Bicêtre Cedex, France