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

Unite de Recherche sur la Glande Thyroide et la Regulation Hormonale, Institut National de la Sante et de la Recherche Medicale, Le Kremlin-Bicetre, France.

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 mumol/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.

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