PURIFICATION AND CHARACTERIZATION OF RAT-BRAIN CYTOSOLIC 3,5,3'-TRIIODO-L-THYRONINE-BINDING PROTEIN - EVIDENCE FOR BINDING-ACTIVITY DEPENDENT ON NADPH, NADP AND THIOREDOXIN

A rat brain cytosolic 3,5,3'-triiodo-L-thyronine-(T3)-binding protein (CTBP) was purified using, successively, carboxymethyl-Sephadex, DEAE-Spherodex, T3 - Sepharose-4B affinity chromatography and Sephacryl S-200. The molecular mass determined by SDS/PAGE wa 58 kDa. The binding characteristics determined by Scatchard analysis revealed a single class of binding sites with a K(a) of 1.56 nM-1 and a maximal binding capacity of 7500 nmol T3/g protein. The relative binding affinities of iodothyronine analogues were D-T3 > L-T3 > L-T4 > 3,3'-5-triiodothyroacetic acid > reverse T3. The optimum pH for binding was 7.5. Purified brain CTBP was reversibly inactivated by charcoal. NADPH, NADP and thioredoxin restored binding activity to a level higher than that of the control; this effect was concentration dependent. Maximal activation was observed at 25 nM NADPH. NADP was effective only in the presence of 1 mM dithiothreitol; maximal activity was obtained at 10 nM NADP. At concentrations higher than 50 nM NADP, the binding gradually decreased. Thioredoxin in the presence of 1 mM dithiothreitol activated CTBP; maximal binding was obtained with 4 muM thioredoxin. In the presence of NADPH, NADP or thioredoxin the maximal binding capacity increased 2-4 times and the K(a) was 2.6 nM-1. These results show that the activity of purified cytosolic brain T3-binding protein may be modulated by NADPH, NADP or thioredoxin.