Solubilization, reconstitution and molecular properties of the triiodothyronine transport protein from rat erythrocyte membranes.
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.
Samson
M
M
Unité de Recherche sur la Glande Thyroïde et la Régulation Hormonale, Institut National de la Santé et de la Recherche Médicale, Le Kremlin-Bicêtre, France.
Osty
J
J
Thibout
H
H
Blondeau
J P
JP
D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000097:blood*; D002845:Chromatography; D003902:Detergents / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000737:chemistry*; D007700:Kinetics; D008297:Male; D008563:Membrane Lipids / Q000097:blood; D008565:Membrane Proteins / Q000097:blood*; D008970:Molecular Weight; D010743:Phospholipids / Q000097:blood; D010782:Photolysis; D051381:Rats; D017208:Rats, Wistar; D012995:Solubility; D013963:Thyroid Hormones; D014284:Triiodothyronine / Q000378:metabolism*; D000097810:Thyroid Hormone-Binding Proteins
D000345:Affinity Labels; D002352:Carrier Proteins; D003902:Detergents; D008563:Membrane Lipids; D008565:Membrane Proteins; D010743:Phospholipids; D013963:Thyroid Hormones; D014284:Triiodothyronine; D000097810:Thyroid Hormone-Binding Proteins