Evidence for the presence of two active forms of cytosolic 3,5,3'-triiodo-L-thyronine (T3)-binding protein (CTBP) in rat kidney. Specialized functions of two CTBPs in intracellular T3 translocation.
Cytosolic NADPH-dependent 3,5,3'-triiodo-L-thyronine (T3)-binding protein (CTBP) purified from rat kidney was further characterized in its T3 binding and its interaction with nuclei. Pretreatment of the CTBP with NADP induced dithiothreitol (DTT)-dependent T3 binding. The DTT-dependent T3 binding was increased by NADP in a concentration-dependent manner, and the maximal binding was obtained by 0.1 microM NADP. Higher concentrations of NADP (more than 0.1 microM), however, reduced T3 binding. NAD also induced DTT-dependent T3 binding, but was very low compared to that induced by NADP. NADPH and NADH did not produce DTT-dependent T3 binding. This NADP-activated, DTT-dependent T3 binding was characterized as follows: Ka for T3 binding was 1.8 x 10(9) M-1, and the maximal binding capacity was 15,000 pmol/mg of protein in the CTBP activated by 0.1 microM NADP. The molecular weight of the CTBP was 58,000 (4.7 S). A complex of [125I]T3 and CTBP (NADP.DTT.CTBP.[125I]T3), which was made from the CTBP pretreated with NADP and DTT, did not bind to DNA. However, the complex bound to the nuclei prepared from rat kidney. Treatment of the nuclei with 0.38 M KCl and with DNase I did not lead to loss of the binding activity for the complex. Treatment of nuclei with 0.5 M NaCl led to the loss of the activity for binding the complex. A complex of [125I]T3 and NADPH-activated CTBP did not bind these nuclear preparations. These results suggested that the active form of CTBP is present in two different forms: one is NADPH-activated, which plays a role as a reservoir for cytoplasmic T3, and the other is NADP-activated, which plays a role as a T3 carrier protein that transfers T3 from cytoplasm to nucleus.
Hashizume
K
K
Department of Gerontology, Endocrinology and Metabolism, Shinshu University School of Medicine, Matsumoto, Japan.
Miyamoto
T
T
Ichikawa
K
K
Yamauchi
K
K
Sakurai
A
A
Ohtsuka
H
H
Kobayashi
M
M
Nishii
Y
Y
Yamada
T
T
D000818:Animals; D001692:Biological Transport; D002021:Buffers; D002352:Carrier Proteins / Q000032:analysis* / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002606:Charcoal; D003600:Cytosol / Q000378:metabolism*; D004229:Dithiothreitol; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000032:analysis* / Q000378:metabolism; D008970:Molecular Weight; D009249:NADP / Q000502:physiology; D051381:Rats; D013963:Thyroid Hormones; D014284:Triiodothyronine / Q000378:metabolism*; D000097810:Thyroid Hormone-Binding Proteins
D002021:Buffers; D002352:Carrier Proteins; D002606:Charcoal; D004229:Dithiothreitol; D046911:Macromolecular Substances; D008565:Membrane Proteins; D009249:NADP; D013963:Thyroid Hormones; D014284:Triiodothyronine; D000097810:Thyroid Hormone-Binding Proteins