Identification of 3,5-diiodo-L-thyronine-binding proteins in rat liver cytosol by photoaffinity labeling.
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 [(125)I]3,5-T(2) resulted in specific covalent attachment of (125)I 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.
Moreno
Maria
M
Dipartimento di Scienze Biologiche ed Ambientali, Università degli Studi del Sannio, I-82100 Benevento, Italy.
Silvestri
Elena
E
Lombardi
Assunta
A
Visser
Theo J
TJ
Goglia
Fernando
F
Lanni
Antonia
A
D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004104:Diiodothyronines / Q000378:metabolism* / Q000494:pharmacology; D007457:Iodine Radioisotopes; D008099:Liver / Q000378:metabolism*; D008297:Male; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D019920:Photoaffinity Labels; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar
D002352:Carrier Proteins; D004104:Diiodothyronines; D007457:Iodine Radioisotopes; D019920:Photoaffinity Labels; D009249:NADP; C030103:3,5-diiodothyronine