A cytoplasmic thyroid hormone binding protein: characterization using monoclonal antibodies.
We have previously purified a cellular thyroid hormone binding protein (p58) from a human carcinoma cell line [Kitagawa, S., Obata, T., Hasumura, S., Pastan, I., & Cheng, S.-y. (1987) J. Biol. Chem. 262, 3903-3908]. In the present study, the binding characteristics, the molecular properties, and subcellular localization of p58 were further characterized. Binding of the purified p58 to thyroid hormones was examined. Analysis of binding data indicates that p58 binds to 3,3',5-triiodo-L-thyronine (T3) with a Kd of 24.3 +/- 0.3 nM and n = 0.71. p58 binds to L-thyroxine similarly as to T3. However, D-T3 and reverse-T3 bind to p58 with an affinity 4- and 20-fold less than that of T3, respectively. By use of the purified p58 as an immunogen, two hybridomas, J11 and J12, secreting monoclonal antibodies to p58 were isolated; both antibodies belong to the IgG1K subclass. J12 recognizes p58 from human, monkey, dog, hamster, and rat, but not mouse. J11 exhibits a similar species specificity except that it does not react with p58 from hamster. With these antibodies, p58 was found to be not posttranslationally modified by glycosylation, sulfation, or phosphorylation. It has a cellular degradation rate t1/2 congruent to 2.1 h. Immunocytochemical studies indicate that p58 is located in the nonmembranous cytoplasm (cytosol). These results are consistent with subcellular fractionation studies which show that greater than 95% of J11 and J12 reactivity and T3 binding activity can be found in the 110,000g supernatant.
Obata
T
T
Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892.
Fukuda
T
T
Willingham
M C
MC
Liang
C M
CM
Cheng
S Y
SY
D000818:Animals; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D008565:Membrane Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D013963:Thyroid Hormones; D000097810:Thyroid Hormone-Binding Proteins
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D008565:Membrane Proteins; D013963:Thyroid Hormones; D000097810:Thyroid Hormone-Binding Proteins