IDENTIFICATION BY PHOTOAFFINITY-LABELING OF A PYRIDINE NUCLEOTIDE-DEPENDENT TRI-IODOTHYRONINE-BINDING PROTEIN IN THE CYTOSOL OF CULTURED ASTROGLIAL CELLS
A. Beslin et al., IDENTIFICATION BY PHOTOAFFINITY-LABELING OF A PYRIDINE NUCLEOTIDE-DEPENDENT TRI-IODOTHYRONINE-BINDING PROTEIN IN THE CYTOSOL OF CULTURED ASTROGLIAL CELLS, Biochemical journal, 305, 1995, pp. 729-737
High-affinity 3,3',5-tri-iodo-L-thyronine (T-3) binding (K-d approxima
te to 0.3 nM) to the cytosol of cultured rat astroglial cells was stro
ngly activated in the presence of pyridine nucleotides. A 35 kDa pyrid
ine nucleotide-dependent T-3-binding polypeptide (35K-TBP) was photoaf
finity labelled using underivatized [I-125]T-3 in the presence of pyri
dine nucleotides and the free-radical scavenger dithiothreitol. Maximu
m 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 ac
tivates T-3 binding and 35K-TBP photolabelling, since cytosol contains
NADP(+)-reducing activity, and the activation of both processes in th
e presence of NADPH and NADP(+) was prevented by an exogenous NADPH ox
idation system. NADPH behaved as an allosteric activator of T-3 bindin
g. 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 c
oncentrations 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-thyr
onine). Molecular sieving and density-gradient centrifugation of cytos
ol 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 regul
ated by the redox state of astroglial cell compartment(s).