PubMed · 7484373
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V Vasiliou, S F Reuter, C A Kozak, D W Nebert. 1995. Mouse class 3 aldehyde dehydrogenases.. https://doi.org/10.1007/978-1-4615-1965-2_20
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Amino acid sequencing of an internal peptide fragment derived from purified Xenopus cytosolic thyroid hormone-binding protein (xCTBP) demonstrates high similarity to the corresponding sequence of mammalian aldehyde dehydrogenase 1 (ALDH1) (Yamauchi, K., and Tata, J. R. (1994) Eur. J. Biochem. 225, 1105-1112). Here we show that xCTBP was co-purified with ALDH and 3,3',5-triiodo-L-thyronine (T3) binding activities. By photoaffinity labeling with [125I]T3, a T3-binding site in the xCTBP was estimated to reside in amino acid residues 93-114, which is distinct from the active site of the enzyme but present in the NAD+ binding domain. The amino acid sequences deduced from the two isolated xALDH1 cDNAs (xALDH1-I and xALDH1-II) were 94.6% identical to each other and very similar to those of mammalian ALDH1 enzymes. The two recombinant xALDH1 proteins exhibit both T3 binding activity and ALDH activity converting retinal to retinoic acid (RA), which are similar to those of xCTBP. The mRNAs were present abundantly in kidney and intestine of adult female Xenopus. Interestingly, their T3 binding activities were inhibited by NAD+ and NADH but not by NADP+ and NADPH, whereas NAD+ was required for their ALDH activities. Our results demonstrate that xCTBP is identical to ALDH1 and suggest that this protein might modulate RA synthesis and intracellular level of free T3.
Thionein (T) has not been isolated previously from biological material. However, it is generated transiently in situ by removal of zinc from metallothionein under oxidoreductive conditions, particularly in the presence of selenium compounds. T very rapidly activates a group of enzymes in which zinc is bound at an inhibitory site. The reaction is selective, as is apparent from the fact that T does not remove zinc from the catalytic sites of zinc metalloenzymes. T instantaneously reverses the zinc inhibition with a stoichiometry commensurate with its known capacity to bind seven zinc atoms in the form of clusters in metallothionein. The zinc inhibition is much more pronounced than was previously reported, with dissociation constants in the low nanomolar range. Thus, T is an effective, endogenous chelating agent, suggesting the existence of a hitherto unknown and unrecognized biological regulatory system. T removes the metal from an inhibitory zinc-specific enzymatic site with a resultant marked increase of activity. The potential significance of this system is supported by the demonstration of its operations in enzymes involved in glycolysis and signal transduction.
The mammalian aldehyde dehydrogenase-3 gene (ALDH3) exhibits several aspects of tissue-specific expression. Certain normal tissues, such as the cornea, constitutively express ALDH3 at very high levels. Other tissues, such as normal liver, do not express ALDH3. In liver, ALDH3 is inducible by polycyclic aromatic hydrocarbon xenobiotics by an Ah-receptor (AhR)-mediated pathway in which a liganded AhR complexes with nuclear ARNT protein, and the complex binds to a xenobiotic response element (XRE) sequence located near -3.0 kb in the ALDH3 5' flanking region and initiates transcription. We used our recently developed rat corneal epithelium culture model (Boesch et al., J. Biol. Chem. 271, 5150-5157, 1996) to study the molecular basis of constitutive ALDH3 expression. Transient transfection assays of corneal epithelium using a battery of ALDH3 5' flanking region-CAT reporter gene constructs indicate that high constitutive ALDH3 expression involves the same cis-acting elements as xenobiotic-induced ALDH3 expression in liver. These elements include a strong basal promoter region and the XRE located near -3.0 kb. Western analysis confirms the presence of AhR and ARNT proteins in 3-methylcholanthrene-treated rat liver, as well as ARNT protein in rat corneal epithelium. No AhR protein is found in rat cornea. The -3.0-kb ALDH3 XRE region contains multiple overlapping transcription factor binding sequences, including consensus sites for AhR, ARNT, HNF1, HNF4, and C/ebp. Electrophoretic mobility shift assays (EMSAs) indicate that constitutive expression of ALDH3 in cornea involves binding of ARNT, HNF1, and HNF4 to the ALDH3-XRE in an Ah-receptor-independent, ARNT-requiring manner. Transient transfection of ALDH3-CAT reporter gene constructs possessing a mutation in either the ARNT- or HNF4-DNA binding sites of the XRE confirms the functional importance of these sequence motifs in constitutive ALDH3 expression.