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.
Kobayashi
M
M
Department of Geriatrics, Endocrinology, and Metabolism, Shinshu University School of Medicine, Matsumoto, Japan.
Hashizume
K
K
Suzuki
S
S
Ichikawa
K
K
Takeda
T
T
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