Indentification by photoaffinity labeling of 3,5-diiodo-L-thyronine-binding proteins in rat liver cytosol

In this study, we obtained evidence for the presence of cytosolic-binding proteins for 3,5-diiodo-L-thyronine (3,5-T-2). UV irradiation of rat liver cytosol with [I-125]3,5-T-2 resulted in specific covalent attachment of I-125 to three polypeptides with apparent molecular masses of 86, 66, and 38 kDa. The photoaffinity labeling of all three proteins was strongly inhibited (by about 90%) when the reaction was carried out in the presence of a 10-fold excess of unlabeled 3,5-T-2 or T-3. However, whereas inhibition by 3,5-T-2 was nicotinamide adenine dinucleotide phosphate reduced (NADPH) independent, T-3 inhibited only in the presence of NADPH. The 38-kDa protein, which showed the greatest affinity for 3,5-T-2, was partially purified by preparative fast-performance liquid chromatography. Its binding activity was optimal at pH 7.4, stable between 0 and 37 C, and already maximal after 5-10 min of incubation. The finding that a 38-kDa cytosolic-binding protein binds 3,5-T-2 in the absence of NADPH, but T-3 only in a NADPH-dependent manner, suggests that it may serve to regulate intracellular T-3/3,5-T-2 translocation in a way that depends on the nicotinamide adenine dinucleotide phosphate/ NADPH ratio.