IDENTIFICATION BY PHOTOAFFINITY-LABELING OF A PYRIDINE NUCLEOTIDE-DEPENDENT TRI-IODOTHYRONINE-BINDING PROTEIN IN THE CYTOSOL OF CULTURED ASTROGLIAL CELLS

High-affinity 3,3',5-tri-iodo-L-thyronine (T-3) binding (K-d approximate to 0.3 nM) to the cytosol of cultured rat astroglial cells was strongly activated in the presence of pyridine nucleotides. A 35 kDa pyridine nucleotide-dependent T-3-binding polypeptide (35K-TBP) was photoaffinity labelled using underivatized [I-125]T-3 in the presence of pyridine nucleotides and the free-radical scavenger dithiothreitol. Maximum activations of T-3 binding and 35K-TBP photolabelling were obtained at approx. 1 x 10(-7) M NADP(+) or NADPH, or 1 x 10(-4) M NADH. NAD(+) and other nucleotides were without effect. NADPH is the form which activates T-3 binding and 35K-TBP photolabelling, since cytosol contains NADP(+)-reducing activity, and the activation of both processes in the presence of NADPH and NADP(+) was prevented by an exogenous NADPH oxidation system. NADPH behaved as an allosteric activator of T-3 binding. The NADPH oxidation system promoted the release of bound T-3 in the absence of any change in the total concentration of the hormone. The 35K-TBP photolabelling and [I-125]T-3 binding were similarly inhibited by non-radioactive T-3 (half-maximum effect at 0.5-1.0 nM T-3). The concentrations of iodothyronine analogues that inhibited both processes were correlated (3,3',5-tri-iodo-D-thyronine greater than or equal to T-3 > L-thyroxine > tri-iodothyroacetic acid > 3,3'5'-tri-iodo-L-thyronine). Molecular sieving and density-gradient centrifugation of cytosol identified a 65 kDa T-3-binding entity, which included the 35K-TBP. These results indicate that 35K-TBP is the cytosolic entity involved in the pyridine nucleotide-dependent T-3 binding, and suggest that the sequestration and release of intracellular thyroid hormones are regulated by the redox state of astroglial cell compartment(s).