PURIFICATION AND CHARACTERIZATION OF A CYTOSOLIC THYROID-HORMONE-BINDING PROTEIN (CTBP) IN XENOPUS LIVER

A variety of cytosolic thyroid-hormone-binding proteins with different characteristics have previously been reported. Here, we first describ e the thyroid-hormone-binding characteristics of adult Xenopus liver c ytosol, then a novel procedure for purifying cytosolic thyroid-hormone -binding protein (CTBP) from Xenopus liver (xCTBP). The procedure cons ists of combining preparative isoelectrofocusing, FPLC cation-exchange chromatography, HPLC hydrophobic-interaction chromatography and ultra violet light cross-linking of I-125-labeled 3,3'5-triiodo-L-thyronine (T-3). The isolated xCTBP thus prepared retained all the characteristi cs of the major thyroid-hormone-(TH)-binding component of the unfracti onated cytosol. It is a monomeric protein of approximately 59 kDa with an isoelectric point of 7.0 +/- 0.1, binds T-3 with a higher affinity than its analogs with a K-d of approximately 9 nM, and is sensitive t o sulfhydryl agents but not to NADPH. In several respects, xCTBP diffe rs from most CTBP-like preparations from other sources described hithe rto. Microsequencing of a 23-amino-acid peptide generated from xCTBP b y cyanogen bromide digestion revealed 92-100% identity of a 23-amino-a cid sequence of several mammalian (amino acids 236-258) and avian (ami no acids 245-267) cytosolic aldehyde dehydrogenases (ALDH); xCTBP also exhibited significant similarity of amino acid composition with rat A LDH. This novel finding of sequence identity between a CTBP and ALDH, and the diversity of CTBPs from different sources, suggest that a vari ety of cytosolic proteins, depending on the species and tissue, can fu nction as thyroid-hormone-binding proteins.