A novel NADPH-dependent cytosolic 3,5,3'-triiodo-L-thyronine-binding protein (CTBP; 5.1S) in rat liver: a comparison with 4.7S NADPH-dependent CTBP.
A novel NADPH-dependent cytosolic 3,5,3'-triiodo-L-thyronine (T3)-binding protein (CTBP) was purified by sequential fractionation of rat liver cytosol on Q-Sepharose, phenyl-Sepharose, red-Sepharose, and polyacrylamide gel electrophoresis under nondenaturing conditions. The CTBP had a sedimentation coefficient of 5.1S, a Stokes' radius of 35 A, and a calculated mol wt of 76,000. The apparently homogenous protein consisted of a dimer of a polypeptide chain with a mol wt of 38,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NADPH increased both the affinity and maximal binding capacity for T3 in the 5.1S CTBP. The maximal activity to bind T3 was obtained by 3.0 x 10(-8) M NADPH. The calculated maximal affinity constant was 2.4 x 10(9) M-1, and the maximal binding capacity was 21,000 pmol T3/mg 5.1S CTBP. The order of affinity of iodothyronine analogs to the 5.1S CTBP was as follows: D-T3 greater than L-T3 greater than L-T4 greater than triiodothyroacetic acid. The optimal pH for T3 binding was 7.2-7.5. Ca2+, Mg2+, and Mn2+ (0.1-10 mM) did not influence T3 binding to CTBP. Zn2+ (1.0 mM), however, inhibited the binding. These results suggested that 5.1S NADPH-dependent CTBP, which is distinct from 4.7S CTBP that had been purified in our laboratory from rat kidney, is present in rat liver.
Department of Geriatrics, Endocrinology, and Metabolism, Shinshu University School of Medicine, Matsumoto, Japan.
M
M
Kobayashi
K
K
Hashizume
S
S
Suzuki
K
K
Ichikawa
T
T
Takeda
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009249:NADP / Q000494:pharmacology*; D051381:Rats; D013963:Thyroid Hormones; D014284:Triiodothyronine / Q000378:metabolism / Q000494:pharmacology; D000097810:Thyroid Hormone-Binding Proteins
D002352:Carrier Proteins; D008565:Membrane Proteins; D009249:NADP; D013963:Thyroid Hormones; D014284:Triiodothyronine; D000097810:Thyroid Hormone-Binding Proteins