Purification and Characterization of a Cytosolic Thyroid‐Hormone‐Binding Protein (CTBP) in Xenopus Liver

Abstract

A variety of cytosolic thyroid‐hormone‐binding proteins with different characteristics have previously been reported. Here, we first describe the thyroid‐hormone‐binding characteristics of adult Xenopus liver cytosol, then a novel procedure for purifying cytosolic thyroid‐hormone‐binding protein (CTBP) from Xenopus liver (xCTBP). The procedure consists of combining preparative isoelectrofocusing, FPLC cation‐exchange chromatography, HPLC hydrophobic‐interaction chromatography and ultraviolet light cross‐linking of 125I‐labeled 3,3′5–triiodo‐L‐thyronine (T3). The isolated xCTBP thus prepared retained all the characteristics of the major thyroid‐hormone‐(TH)‐binding component of the unfractionated cytosol. It is a monomeric protein of approximately 59 kDa with an isoelectric point of 7.0±0.1, binds T3 with a higher affinity than its analogs with a Kd of approximately 9 nM, and is sensitive to sulfhydryl agents but not to NADPH. In several respects, xCTBP differs from most CTBP‐like preparations from other sources described hitherto. Microse‐quencing of a 23‐amino‐acid peptide generated from xCTBP by cyanogen bromide digestion revealed 92–100% identity of a 23‐amino‐acid sequence of several mammalian (amino acids 236–258) and avian (amino acids 245–267) cytosolic aldehyde dehydrogenases (ALDH); xCTBP also exhibited significant similarity of amino acid composition with rat ALDH. This novel finding of sequence identity between a CTBP and ALDH, and the diversity of CTBPs from different sources, suggest that a variety of cytosolic proteins, depending on the species and tissue, can function as thyroid‐hormone‐binding proteins. Copyright © 1994, Wiley Blackwell. All rights reserved