High affinity thyroid hormone-binding protein in human kidney: Kinetic characterization and identification by photoaffinity labeling

The binding of thyroid hormones and its regulation by NADPH and NADP(+) were studied in human kidney cytosol, and a 38-kDa polypeptide (p38) was identified by photoaffinity labeling of cytosol with underivatized [I-125]T-3, SDS-PAGE, and autoradiography. The cytosolic thyroid hormone binding and p38 photolabeling were strongly activated by NADPH (maximum at 10(-7) M), whereas other nucleotides were less effective or ineffective. NADP(+) did not activate T-3 binding and p38 photolabeling, provided it was protected from conversion to NADPH by the addition of an exogenous oxidizing enzymatic system (oxidized glutathione plus glutathione reductase). Furthermore, NADP(+) inhibited NADPH activation (half-maximum inhibitory effect at similar to 2 x 10(-5) M), and oxidation of NADPH to NADP(+) induced dissociation of bound T-3. The equilibrium dissociation constant (K-d) of the NADPH-activated cytosolic T-3-binding sites was 0.3 nM, similar to the K-d of the nuclear T-3 receptors. The kidney contained 200 times more cytosolic NADPH-activated thyroid hormone-binding sites than nuclear T-3 receptors. Nonradioactive iodothyronines competed with [I-125]T-3 for both NADPH-activated binding and p38 photolabeling, with the following order of decreasing affinity: D-isomer of T-3 > T-3 > T-4 > triiodothyroacetic acid > 3'-isopropyl-3,5-diiodothyronine > rT(3). NADPH-activated T-3 binding and photolabeled p38 were also detected in human heart and liver cytosols, but not in pancreas, cultured fibroblast and erythrocyte cytosols, or plasma. Rat kidney cytosol contained a 35-kDa photolabeled polypeptide homolog to human p38. The native molecular mass of the human photolabeled protein was 50 kDa, whereas that of the rat protein was 60 kDa, as determined by nondenaturing polyacrylamide gel electrophoresis. Two-dimensional PAGE of photolabeled p38 indicated an isoelectric point of 5.3. These findings describe the molecular properties of a NADPH/NADP(+)-regulated thyroid hormone-binding protein not previously identified in human and rat kidney cytosol.