Purification and characterization of NADPH-dependent cytosolic 3,5,3'-triiodo-L-thyronine binding protein in rat kidney.
The NADPH-dependent cytosolic 3,5,3'-triiodo-L-thyronine(T3)-binding protein (CTBP) has been purified over 30,000-fold from rat kidney by using charcoal extraction, Mono Q-Sepharose, Blue Sepharose CL-6B, and Sephacryl S-200 column chromatography. Purified CTBP had a sedimentation coefficient of 4.7 S, Stokes radius of 32.5A, and calculated molecular weight of 58,000. The apparently homogeneous protein consisted of a single polypeptide chain with Mr of 58,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Scatchard analysis of T3 binding showed that NADPH increases maximal binding capacity without changes in the affinity constant (Ka = 2.43 X 10(9) M-1). Double reciprocal analysis of NADPH and binding capacity gave maximal binding capacity of 16,400 pmol/mg of CTBP, Mr = 58,000. The order of affinity of iodothyronine analogues to purified CTBP was as follows: L-T3 = D-T3 greater than triiodothyroacetic acid greater than L-thyroxine. [125I]T3 bound to purified CTBP spontaneously dissociated from CTBP at 20 degrees C (t 1/2 = 22 min) in the absence of NADPH, whereas the dissociation was not observed in the presence of NADPH. The optimal pH for T3 binding was 7.2-7.5 Na+, K+, Ca2+, and Mg2+ (0-200 mM) did not influence T3 binding to CTBP. The purified CTBP did not bind to DNA and was not adsorbed to concanavalin A-Sepharose.
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
Kobayashi
M
M
Sakurai
A
A
Ohtsuka
H
H
Nishii
Y
Y
Yamada
T
T
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002606:Charcoal; D003600:Cytosol / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D009249:NADP / Q000502:physiology*; D011188:Potassium; D051381:Rats; D011919:Rats, Inbred Strains; D012964:Sodium; D013963:Thyroid Hormones; D014284:Triiodothyronine / Q000378:metabolism*; D000097810:Thyroid Hormone-Binding Proteins
D002352:Carrier Proteins; D002606:Charcoal; D008565:Membrane Proteins; D009249:NADP; D011188:Potassium; D012964:Sodium; D013963:Thyroid Hormones; D014284:Triiodothyronine; D000097810:Thyroid Hormone-Binding Proteins