IDENTIFICATION BY PHOTOAFFINITY-LABELING OF A MEMBRANE THYROID HORMONE-BINDING PROTEIN ASSOCIATED WITH THE TRIIODOTHYRONINE TRANSPORT-SYSTEM IN RAT ERYTHROCYTES

Photoaffinity labeling with underivatized T3 was used to identify T3-binding proteins in the membrane of rat erythrocytes. UV irradiation of ghosts and peripheral protein-depleted membranes in the presence of [I-125]T3 resulted in the covalent attachment of I-125 to membrane proteins (analyzed by polyacrylamide gel electrophoresis and autoradiography). In the presence of the free radical scavenger dithiothreitol, I-125 was selectively incorporated into a 45,000 mol wt band (p45) that was an integral membrane polypeptide. p45 photolabeling was half-inhibited by 14 nM unlabeled T3. This concentration is similar to the K(m) for T3 transport in rat erythrocytes and the K(d) of the high affinity T3-binding sites under equilibrium binding conditions in the rat erythrocyte membrane. T4 and tryptophan also strongly inhibited p45 labeling, whereas the D-isomer of T3 was less efficient, and leucine had no effect. This corresponds to the specificity of the system T-related T3 transport system and T3-binding sites of rat erythrocytes. The SH-reagent N-ethylmaleimide prevented p45 labeling, unless T3 was present to protect the T3 transport activity and the high affinity T3-binding sites from inactivation. No saturable labeling of p45 or other polypeptides was detected in membranes prepared from human erythrocytes, which have very low T3 transport activity and no measurable high affinity T3-binding sites. p45 is not disulfide linked and is not a degradation product of higher mol wt polypeptides. Substrates and specific inhibitors of known erythrocyte membrane transporters did not alter p45 photolabeling, indicating that p45 is not functionally related to these transporters. We conclude that the photoaffinity-labeled T3-binding protein p45 has the properties expected of the T3-binding component of the T3 transport system in rat erythrocytes.