Purification and Characterization of a Cytosolic Thyroid‐Hormone‐Binding Protein (CTBP) inXenopusLiver

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 '251-labeled 3,3'5-triiodo-~-thyronine (T,). 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 7.020.1, binds T, 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. Microsequencing 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.The kinetics and nature of response to hormones and other biologically active signals that act via nuclear receptors, such as thyroid and steroid hormones, vitamin D, and retinoic acid, are determined by intracellular components with which these molecules interact before reaching their receptors. These have been well characterized for glucocorticoid hormone, progesterone and retinoids, the interaction between retinoids and cytosolic retinoic acid and retinol-binding proteins (CRABP and CRBP) being well documented, but their exact physiological function is not clear (Maden et al., 1989;Maden, 1991;Ruberte et al., 1991;Morriss-Kay, 1992). In a different type of interaction, glucocorticoids and progesterone are now known to move into the nucleus associated with a complex in the cytoplasm formed between the relevant receptor and the heat-shock protein hsp-90 (Catelli et al., 1985;Kost et al., 1989;Pratt, 1993).Although the presence of cytosolic thyroid-hormonebinding proteins (CTBPs) has been known for some time, there seems to be no concensus about their identity or their Correspondence to J. R. Tata,

Xenopus Cytosolic Thyroid Hormone-binding Protein (xCTBP) Is Aldehyde Dehydrogenase Catalyzing the Formation of Retinoic Acid

“…Purification of Native xCTBP from Liver Cytosol-xCTBP was purified as described with some modifications (11). All the following procedures were carried out at 4°C, unless otherwise noted.…”

“…Dissected tissue was minced with scissors in Barth-X amphibian Ringer, followed by several washings in the same solution. The minced tissue was homogenized in 4.5 volumes of 0.25 M sucrose, 10 mM Tris-HCl, 1 mM EDTA, 1 mM MgCl 2 , 1 mM dithiothreitol (DTT), 1 mM benzamidine hydrochloride, and 1 mM phenylmethylsulfonyl fluoride, pH 7.5, as described previously (11). After successive differential centrifugations at 3,000 ϫ g for 10 min, 12,000 ϫ g for 20 min, and 100,500 ϫ g for 60 min, a clear supernatant was obtained and stored in 10% glycerol at Ϫ85°C until its use as a cytosol.…”

“…Although there are many similarities between CTBP, CRABP, and CRBP, on the one hand, and retinoic acid receptors and thyroid hormone receptors, on the other, a major difference is the multiple types of CTBPs, unlike CRABP and CRBP (17). We have previously reported three types of xCTBPs (19), each with a distinct pattern of expression, which raises the possibility of a tissuespecific role for CTBPs. It therefore became important to characterize CTBPs in greater detail.…”

“…A unique feature of xCTBP found in adult liver is that it has a region similar to those of mammalian class 1 aldehyde dehydrogenase (ALDH1) (aldehyde:NAD ϩ oxidoreductase, EC 1.2.1.3) (11), which is one of the enzymes catalyzing the oxidation of various aliphatic and aromatic aldehydes to the corresponding acids. An important and rather specific activity of ALDH1 is to act as an enzyme catalyzing the synthesis of RA * The costs of publication of this article were defrayed in part by the payment of page charges.…”

“…Recently, CTBPs have been detected in mammalian and amphibian cells (10 -15), an interesting feature of which is that these exhibit different biochemical properties. We have earlier described a CTBP in adult Xenopus liver (xCTBP) (11) which is a 59-kDa protein with a higher affinity for T 3 than L-thyroxine (T 4 ), T 3 binding being neither Ca 2ϩ -nor NADPH-dependent, as is the case for Rana and rat CTBPs (12)(13)(14)(15).…”