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Identification of 3,5-Diiodo-L-Thyronine-Binding Proteins in Rat Liver Cytosol by Photoaffinity Labeling

Dipartimento di Scienze Biologiche ed Ambientali (M.M., E.S., F.G.), Università degli Studi del Sannio, I-82100 Benevento, Italy; Dipartimento di Fisiologia Generale ed Ambientale (A.Lo.), Università degli Studi di Napoli "Federico II," I-80134 Naples, Italy; Dipartimento di Scienze della Vita (A.La.), Seconda Università degli Studi di Napoli, I-81100 Caserta, Italy; and Department of Internal Medicine (T.J.V.), Erasmus MC, 3000 DR Rotterdam, The Netherlands

Address all correspondence and requests for reprints to: Professor Antonia Lanni, Dipartimento di Scienze della Vita, Seconda Università degli Studi di Napoli, Via Vivaldi 43, 81100 Caserta, Italy. E-mail: antonia.lanni{at}unina2.it.

In this study, we obtained evidence for the presence of cytosolic-binding proteins for 3,5-diiodo-L-thyronine (3,5-T₂). UV irradiation of rat liver cytosol with [¹²⁵I]3,5-T₂ resulted in specific covalent attachment of ¹²⁵I 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-T₂ or T₃. However, whereas inhibition by 3,5-T₂ was nicotinamide adenine dinucleotide phosphate reduced (NADPH) independent, T₃ inhibited only in the presence of NADPH. The 38-kDa protein, which showed the greatest affinity for 3,5-T₂, 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-T₂ in the absence of NADPH, but T₃ only in a NADPH-dependent manner, suggests that it may serve to regulate intracellular T₃/3,5-T₂ translocation in a way that depends on the nicotinamide adenine dinucleotide phosphate/NADPH ratio.

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