PURIFICATION, MOLECULAR-CLONING, AND FUNCTIONAL EXPRESSION OF THE HUMAN NICODINAMIDE-ADENINE DINUCLEOTIDE PHOSPHATE-REGULATED THYROID HORMONE-BINDING PROTEIN

The kidney and several other thyroid hormone-responsive tissues contai n a NADP-regulated thyroid hormone (TH)-binding protein (THBP), with a n apparent molecular mass of 36 kDa on SDS-PAGE, responsible for most of the intracellular high-affinity T-3 and T-4 binding. THBP was purif ied to homogeneity from human kidney cytosol and used to generate prot eolytic peptides. Microsequencing of four peptides revealed identity t o amino acid sequences deduced from a human cDNA homolog to a cDNA enc oding kangaroo mu-crystallin. This protein is a major structural kanga roo lens protein with no known function in other species. A full-sized cDNA (TH5.9) was isolated by 5'- and 3'-rapid amplification of cDNA e nds using a human brain cDNA library and gene-specific PCR primers, co nfirming identity to the previously cloned human cDNA. The TH5.9 cDNA encodes a 314-residue protein (theoretical mol wt = 33,775) with signi ficant homologies (40 to 60%) with two bacterial enzymes: lysine cyclo deaminase and ornithine cyclodeaminase. The TH5.9 cDNA was expressed i n Escherichia coli as a glutathione S-transferase (GST) fusion protein . Purified GST fusion protein, but not GST, bound T-3 specifically wit h high affinity [dissociation constant (K-d) = 0.5 nM] in the presence of NADPH, and was labeled by UV-driven cross-linking of underivatized [I-125]T-3. T-3 binding and photoaffinity labeling of GST fusion prot ein were activated by NADPH [activation constant (K-act) = 10(-8) M], but not by NADH. The expressed protein displays the appropriate bindin g properties, indicating that TH5.9 cDNA encodes the NADP-regulated TH BP characterized in human tissues.