Showing papers on "Aldehyde dehydrogenase published in 1994"

Molecular characterization of an aldehyde/alcohol dehydrogenase gene from Clostridium acetobutylicum ATCC 824.

Abstract: A gene (aad) coding for an aldehyde/alcohol dehydrogenase (AAD) was identified immediately upstream of the previously cloned ctfA (J. W. Cary, D. J. Petersen, E. T. Papoutsakis, and G. N. Bennett, Appl. Environ. Microbiol. 56:1576-1583, 1990) of Clostridium acetobutylicum ATCC 824 and sequenced. The 2,619-bp aad codes for a 96,517-Da protein. Primer extension analysis identified two transcriptional start sites 83 and 243 bp upstream of the aad start codon. The N-terminal section of AAD shows homology to aldehyde dehydrogenases of bacterial, fungal, mammalian, and plant origin, while the C-terminal section shows homology to alcohol dehydrogenases of bacterial (which includes three clostridial alcohol dehydrogenases) and yeast origin. AAD exhibits considerable amino acid homology (56% identity) over its entire sequence to the trifunctional protein encoded by adhE from Escherichia coli. Expression of aad from a plasmid in C. acetobutylicum showed that AAD, which appears as a approximately 96-kDa band in denaturing protein gels, provides elevated activities of NADH-dependent butanol dehydrogenase, NAD-dependent acetaldehyde dehydrogenase and butyraldehyde dehydrogenase, and a small increase in NADH-dependent ethanol dehydrogenase. A 957-bp open reading frame that could potentially encode a 36,704-Da protein was identified upstream of aad. Images

Role of aldehyde metabolizing enzymes in mediating effects of aldehyde products of lipid peroxidation in liver cells.

Abstract: It is well established that many types of tumor cells have reduced lipid peroxidation capacity compared to their normal counterparts. Changes in the activity of enzymes metabolizing aldehydes produced by lipid peroxidation have also been reported in a variety of tumor cells. We have investigated the relationship between changes in lipid peroxidation and changes in aldehyde-metabolizing enzymes in normal hepatocytes and two representative rat hepatoma cell lines, McA-RH-7777 and JM2. Compared to hepatocytes, both 7777 and JM2 cells have significantly lower basal and prooxidant-induced levels of lipid peroxidation than normal hepatocytes. Using 4-hydroxynonenal (4-HNE) as substrate, both cell lines also have significantly reduced activities of alcohol dehydrogenase (ADH) and glutathione S-transferase (GST) compared to hepatocytes. JM2 cells have significantly increased aldehyde dehydrogenase (ALDH) and aldehyde reductase (ALRD) activities with 4-HNE. In 7777 cells the ALDH and ALRD activities are not different from hepatocytes. The changes in enzyme activity are inversely correlated with the sensitivity of cells to 4-HNE. JM2 cells, with increased ALDH and ALRD and decreased ADH and GST, are much more resistant to the toxic effects of 4-HNE than 7777 cells. Normal hepatocytes and JM2 cells are approximately equally resistant to 4-HNE even though hepatocytes rely primarily on GST-mediated aldehyde conjugation to metabolize 4-HNE. Coupled with previous results from our laboratories, the overall increased sensitivity of certain hepatoma cells to lipid aldehydes appears due to decreased ability of these hepatoma cells to remove toxic products of lipid peroxidation. Moreover, hepatoma cells with increased levels of aldehyde dehydrogenase and aldehyde reductase appear most like hepatocytes in their ability to metabolize lipid aldehydes.

Purification and molecular characterization of the NAD(+)-dependent acetaldehyde/alcohol dehydrogenase from Entamoeba histolytica.

Abstract: A bifunctional 95 kDa polypeptide (EhADH2) harbouring acetaldehyde dehydrogenase and alcohol dehydrogenase activities was purified to homogeneity from trophozoite extracts of the protozoan parasite Entamoeba histolytica. Kinetic studies revealed that the enzyme utilizes NAD+ rather than NADP+ as cofactor. Km values for acetyl-CoA, acetaldehyde and ethanol were found to be 0.015, 0.15 and 80 mM respectively in the presence of 0.2 mM NAD+. The primary structure of EhADH2 as deduced from respective amoebic DNA sequences showed striking similarity to the trifunctional AdhE protein of Escherichia coli and the bifunctional AAD protein of Clostridium acetobutylicum. Alignment with a number of aldehyde dehydrogenases and alcohol dehydrogenases from various species suggested that the two catalytic functions of EhADH2 are located on separate parts of the molecule. By cross-linking experiments and electron-microscopic analysis, native EhADH2 was found to be organized in a homopolymeric fashion consisting of more than 20 associated promoters which form rods about 50-120 nm in length.

Identification of novel glutathione conjugates of disulfiram and diethyldithiocarbamate in rat bile by liquid chromatography-tandem mass spectrometry. evidence for metabolic activation of disulfiram in vivo

Abstract: Recent studies have shown that the inhibitory effects of disulfiram and diethyldithiocarbamate (DDTC) (to which disulfiram is rapidly reduced in vivo) on the liver mitochondrial low-Km form of aldehyde dehydrogenase (ALDH) may be mediated by a reactive metabolite(s) of these compounds. In order to investigate the nature of such electrophilic intermediates in vivo, the present study was carried out with the goal of detecting and identifying their respective glutathione (GSH) conjugates in the bile of rats dosed ip with either disulfiram (75 mg kg-1) or sodium DDTC (114 mg kg-1). By means of highly selective screening strategies based on coupled liquid chromatography-tandem mass spectrometry techniques, one major and four minor GSH adducts were identified as common biliary metabolites of disulfiram and DDTC. The major conjugate, whose excretion into bile over 4 h accounted for ca. 1% of the dose of either precursor, was identified as S-(N,N-diethylcarbamoyl)glutathione (SDEG). In vitro experiments with synthetic SDEG demonstrated that this carbamate thioester derivative is chemically stable in aqueous media under physiological conditions and does not carbamoylate nucleophiles such as cysteine. Consistent with these findings, SDEG failed to inhibit yeast ALDH in vitro. The minor GSH conjugates in bile were identified as S-(N,N-diethylthiocarbamoyl)glutathione, S-(N-ethyl-carbamoyl)glutathione, S-(N-ethylthiocarbamoyl)glutathione, and S-[N-(carboxymethyl)-N- ethylcarbamoyl]glutathione, the structures of which indicate that metabolic oxidation takes place at the thiono sulfur group and at each of the carbon atoms of disulfiram and DDTC.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic polymorphism and activities of human colon alcohol and aldehyde dehydrogenases: no gender and age differences.

Abstract: Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) isoenzyme patterns from 69 (men, 47; women, 22) surgical colon mucosal specimens were identified by agarose isoelectric focusing. gamma-ADH was found to be the predominant form in the mucosa, whereas only beta-ADH was detectable in the muscle layer. ALDH1, ALDH2, and ALDH3 were detectable in the mucosa, with cytosolic ALDH1 being the major form. At pH 7.5, the ADH activities in the colon mucosae with the homozygous phenotype (exhibiting gamma 1 gamma 1) and the heterozygous phenotype (exhibiting gamma 1 gamma 1, gamma 1, gamma 2, gamma 2, gamma 2) were determined to be 183 +/- 13 and 156 +/- 30 nmol/min/g tissue, respectively. The ALDH activities in the ALDH2-active and ALDH2-inactive phenotypes were determined to be 40.2 +/- 2.3 and 34.6 +/- 2.0 nmol/min/g tissue, respectively. The lack of significant difference in the ALDH activities between these two phenotypic groups can be attributed to the very low expression of the mitochondrial ALDH2 in the colon mucosa. No significant differences in the ADH or the ALDH activities were found between the men and women studied and between the three age groups (20-40, 49-70, and 72-83 years). The ascending, transverse, descending, and sigmoid colons exhibited similar ADH and ALDH activities. The isoenzyme patterns of ADH and ALDH remained unaltered in colon carcinomas, except that a significant reduction of the enzyme activities was found in the cancer tissue as compared with the adjacent normal portions. it is concluded that human colon mucosa exhibits significant amounts of ethanol- and acetaldehyde-oxidizing activities.

Regulation of [Ah] gene battery enzymes and glutathione levels by 5,10-dihydroindeno[1,2-b]indole in mouse hepatoma cell lines.

Abstract: The murine aromatic hydrocarbon ([Ah]) gene battery consists of at least six genes that code for two functionalizing (Phase I) enzymes and four non-functionalizing (Phase II) enzymes. These enzymes are induced by compounds such as aromatic hydrocarbons and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) that bind to the cytosolic Ah receptor protein. Studies in rodents indicate that certain enzymes of this battery, namely cytochrome P4501A1 (CYP1A1), UDP-glucuronosyltransferase (UGT1*06) and NAD(P)H: quinone acceptor oxidoreductase (NMO1) are induced by the synthetic antioxidant 5,10-dihydroindeno[1,2-b]indole (DHII). The induction of [Ah] gene battery enzymes and the levels of reduced glutathione (GSH) were examined in mouse Hepa-1c1c7 hepatoma wild-type cells (wt), a CYP1A1 metabolism-deficient mutant (c37) and an Ah receptor nuclear translocation-defective mutant (c4). DHII and TCDD increased the activities of ethoxyresorufin O-deethylase, an indicator of CYP1A1 activity, as well as NMO1, UGT1*06, cytosolic aldehyde dehydrogenase class 3 and glutathione S-transferase form A1 in wt cells, but had little or no induction effect in c37 or c4 cells. DHII and TCDD differed in their effects on GSH levels; while DHII increased GSH levels 3-fold in wt, but not at all in c37 or c4 cells, TCDD had no effect on GSH levels in any cell type. However, GSH levels were enhanced in both wt and c4 cells by tert-butyl hydroquinone (TBHQ). L-Buthionine S,R-sulfoximine, an inhibitor of gamma-glutamylcysteine synthetase, prevented DHII-induced increases in wt cell GSH. The increase in GSH levels occurred after 8 h, while the induction of enzymes occurred within 4 h. The induction of the higher GSH levels in wt cells by DHII and TBHQ correlated with increases in intracellular levels of the GSH precursor thiol cysteine, as well as with increased activities of gamma-glutamylcysteine synthetase, the rate-limiting enzyme of GSH synthesis. However, TBHQ-mediated GSH increases in c4 cells were accompanied by increased gamma-glutamylcysteine synthetase activity with no change in intracellular cysteine concentration. The results suggest that DHII induction of [Ah] gene battery enzymes requires a functional Ah receptor, but not the functional gene product CYP1A1. Furthermore, metabolism, possibly via CYP1A1, appears to be required for DHII to enhance intracellular levels of cysteine and GCS activity that result in higher GSH levels.

Identification of a Methylcholanthrene-induced Aldehyde Dehydrogenase in a Human Breast Adenocarcinoma Cell Line Exhibiting Oxazaphosphorine-specific Acquired Resistance

Abstract: The class-3 aldehyde dehydrogenase that is overexpressed (>100-fold) in human breast adenocarcinoma MCF-7/0 cells made resistant (>30-fold as judged by LC90s) to oxazaphosphorines, such as mafosfamide, by growing them in the presence of polycyclic aromatic hydrocarbons, e.g. , methylcholanthrene (3 µm for 5 days), was isolated and characterized. Its physical and catalytic properties were identical to those of the prototypical human stomach mucosa cytosolic class-3 aldehyde dehydrogenase, type-1 ALDH-3, except that it catalyzed, though not very rapidly, the oxidation of aldophosphamide, whereas the stomach mucosa enzyme essentially did not; hence, it was judged to be a slight variant of the prototypical enzyme. Carcinogens that are not ligands for the Ah receptor, barbiturates known to induce hepatic cytochrome P450s, steroid hormones, an antiestrogen, and oxazaphosphorines did not induce the enzyme or the largely oxazaphosphorine-specific acquired resistance. Whereas methylcholanthrene induced ( a ) resistance to mafosfamide and ( b ) class-3 aldehyde dehydrogenase activity, as well as glutathione S -transferase and DT-diaphorase activities, in the estrogen receptor-positive MCF-7/0 cells, it did not do so in two other human breast adenocarcinoma cell lines, MDA-MB-231 and SK-BR-3, each of which is estrogen receptor negative. Expression of the class-3 aldehyde dehydrogenase and the loss of sensitivity to mafosfamide by polycyclic aromatic hydrocarbon-treated MCF-7/0 cells were transient; each returned to essentially basal levels within 15 days when the polycyclic aromatic hydrocarbon was removed from the culture medium. Insensitivity to the oxazaphosphorines on the part of polycyclic aromatic hydrocarbon-treated MCF-7/0 cells was not observed when exposure to mafosfamide (30 min) was in the presence of benzaldehyde or octanal, each a relatively good substrate for cytosolic class-3 aldehyde dehydrogenases, whereas it was retained when exposure to mafosfamide was in the presence of acetaldehyde, a relatively poor substrate for these enzymes. These observations demonstrate that ligands for the Ah receptor can induce a transient, largely oxazaphosphorine-specific, acquired cellular resistance, and they are consistent with the notion that elevated levels of a cytosolic class-3 aldehyde dehydrogenase nearly identical to the prototypical type-1 class-3 aldehyde dehydrogenase expressed by human stomach mucosa account for the Ah receptor ligand-induced oxazaphosphorine-specific acquired resistance, most probably by catalyzing the detoxification of aldophosphamide.

Differential corneal sensitivity to ultraviolet light among inbred strains of mice. Correlation of ultraviolet B sensitivity with aldehyde dehydrogenase deficiency.

Abstract: Adult male mice from four inbred albino strains (SJL/J, NZW/BL, BALB/c HeA, and SWR/J) were subjected to ultraviolet radiation (UVR) exposure (302 nm peak wavelength, intensity 398 microW/cm2) for 3.25 min and photographed 4 days postexposure to assess corneal clouding. Corneal extracts from control (unexposed) mice from each strain, were also monitored for aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) activity and soluble protein content. The SWR/J strain exhibited more extensive corneal clouding after UV exposure than did the other strains, and control SWR/J mice exhibited a low activity variant phenotype for the major ocular ALDH AHD-4, and decreased levels of soluble protein in corneal extracts. These data support earlier proposals for a major role for ALDH in assisting the cornea in protecting the eye against UVR-induced tissue damage.

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.

Alcohol dehydrogenase: enzymology and metabolism.

Abstract: Alcohol dehydrogenase (ADH), the principal enzyme responsible for ethanol oxidation, constitutes a complex family in humans. Based on structural and kinetic features, ADH can be divided into five classes. Low-Km class I beta-ADH and gamma-ADH show genetic polymorphism among racial populations. The allozymes exhibit distinct maximal activities due to single amino acid exchanges at different sites in the coenzyme-binding domain. Class IV mu-ADH also shows ethnic variability: it is detected in the stomach mucosa of Caucasians but not detectable in about 70% of Orientals. Class I, II, IV and V ADH isozymes exhibit tissue-specific distribution. Approximately 50% of Orientals lack the activity of the mitochondrial low-Km aldehyde dehydrogenase (ALDH2). Ethanol- and acetaldehyde-oxidizing activities of the liver, lung, and gastrointestinal tract appear to be correlated with their isozyme patterns of ADH and ALDH and with the allozymes. The frequencies of the alleles ADH(2)2 and ADH(3)1, coding for the high-Vmax beta 2- and gamma 1-ADH respectively, and of the mutant ALDH(2)2 in the Oriental subjects with alcoholism or alcoholic cirrhosis are significantly lower than those in healthy controls. These genotyping results support the current notion that genetic variation in ADH and ALDH may influence drinking behavior and susceptibility for alcoholism and possibly alcohol-induced organ injury by modulating the rate of metabolism of ethanol and acetaldehyde.

Aldehyde dehydrogenase and cytotoxicity of purified bovine serum amine oxidase and spermine in Chinese hamster ovary cells

Abstract: Bovine serum amine oxidase (EC 1.4.3.6) catalyses the oxidative deamination of polyamines giving rise to the corresponding aldehydes, ammonia, and hydrogen peroxide. It has been suggested that the ...

Genetic polymorphisms of alcohol metabolizing enzymes related to alcohol sensitivity and alcoholic diseases

Abstract: Indiciduals with the atypical aldehyde dehydrogenase ALDH22 allele, both homozygous and heterozygous status, are alcohol sensitive and have a markedly reduced risk of developing alcoholic diseases. Genetic abnormalities of the ALDH1 locus are also associated with alcohol flushing. The ALDH3 and ALDHx , loci are polymorphic and their variations may affect the development of alcoholic diseases. The variations of alcohol dehydrogenase ADH2 and ADH3 loci have no profound effects on alcohol sensitivity. The newly identified ADH6 gene has hormone response elements, and it may cause the gender difference in alcoholic problems.

Genetic organization of Acetobacter for acetic acid fermentation.

Abstract: Plasmid vectors for the acetic acid-producing strains ofAcetobacter andGluconobacter were constructed from their cryptic plasmids and the efficient transformation conditions were established. The systems allowed to reveal the genetic background of the strains used in the acetic acid fermentation. Genes encoding indispensable components in the acetic acid fermentation, such as alcohol dehydrogenase, aldehyde dehydrogenase and terminal oxidase, were cloned and characterized. Spontaneous mutations at high frequencies in the acetic acid bacteria to cause the deficiency in ethanol oxidation were analyzed. A new insertion sequence element, IS1380, was identified as a major factor of the genetic instability, which causes insertional inactivation of the gene encoding cytochromec, an essential component of the functional alcohol dehydrogenase complex. Several genes including the citrate synthase gene ofA. aceti were identified to confer acetic acid resistance, and the histidinolphosphate aminotransferase gene was cloned as a multicopy suppressor of an ethanol sensitive mutant. Improvement of the acetic acid productivity of anA. aceti strain was achieved through amplification of the aldehyde dehydrogenase gene with a multicopy vector. In addition, spheroplast fusion of theAcetobacter strains was developed and applied to improve their properties.

Identification of chloroacetaldehyde dehydrogenase involved in 1,2-dichloroethane degradation.

Abstract: The degradation of 1,2-dichloroethane and 2-chloroethanol by Xanthobacter autotrophicus GJ10 proceeds via chloroacetaldehyde, a reactive and potentially toxic intermediate. The organism produced at least three different aldehyde dehydrogenases, of which one is plasmid encoded. Two mutants of strain GJ10, designated GJ10M30 and GJ10M41 could no longer grow an 2-chloroethanol and were found to lack the NAD-dependent aldehyde dehydrogenase that is the predominant protein in wild-type cells growing on 2-chloroethanol. Mutant GJ10M30, selected on the basis of its resistance to 1,2-dibromoethane, also had lost haloalkane dehalogenase activity and Hg2+ resistance, indicating plasmid loss. From a gene bank of strain GJ10, different clones that complemented one of these mutants were isolated. In both transconjugants, the aldehyde dehydrogenase that was absent in the mutants was overexpressed. The enzyme was purified and was a tetrameric protein of 55-kDa subunits. The substrate range was rather broad, with the highest activity measured for acetaldehyde. The K-m value for chloroacetaldehyde was 160 mu M, higher than those for other aldehydes tested. It is concluded that the ability of GJ10 to grow with 2-chloroethanol is due to the high expression level of an aldehyde dehydrogenase with a rather low activity for chloroacetaldehyde.

In vivo pharmacodynamic studies of the disulfiram metabolite S-methyl N,N-diethylthiolcarbamate sulfoxide: inhibition of liver aldehyde dehydrogenase.

Abstract: S-methyl N,N-diethylthiolcarbamate sulfoxide (DETC-MeSO) is proposed to be the metabolite of disulfiram responsible for the in vivo inhibition of liver low Km, aldehyde dehydrogenase (ALDH) in the rat. Studies were conducted in male Sprague-Dawley rats and also in vitro using both rat liver mitochondrial and purified bovine mitochondrial low Km ALDH to investigate further the pharmacodynamic and pharmacokinetic characteristics of DETC-MeSO. Administration of DETC-MeSO to rats produced a rapid and maximal inhibition of liver mitochondrial low Km, ALDH within 2 hr, which was still inhibited 30% after 168 hr. After DETC-MeSO treatment, the maximum plasma concentration of DETC-MeSO was reached within 0.5 hr, with DETC-MeSO being undetectable 2 hr after DETC-MeSO dosing. Although a trace amount of DETC-Me was detected in the plasma 0.5 hr after DETC-MeSO administration to rats, this disappeared within 1 hr. When rats were treated with disulfiram, the maximal plasma concentration of DETC-MeSO was found within 2 hr, with only a very small quantity of DETC-MeSO still detectable after 8 hr. Rats also were given the disulfiram metabolites diethyldithiocarbamate (DDTC), diethyldithiocarbamate-methyl ester (DDTC-Me), and S-methyl N,N-diethylthiolcarbamate (DETC-Me), and plasma analyzed for DETC-MeSO 2 hr after the administration of these metabolites. DETC-MeSO was detected in plasma, further illustrating that DETC-MeSO can be found in plasma after the administration of either disulfiram, or the subsequent in vivo metabolites DDTC, DDTC-Me, or DETC-Me. Rats treated with DETC-MeSO and then challenged with ethanol exhibited an increase in blood acetaldehyde similar to that found from previous studies with disulfiram. In in vitro studies, incubation of rat liver mitochondria with 0.75 μM DETC-MeSO inhibited rat liver mitochondrial low Km ALDH maximally within 2 hr, whereas purified bovine mitochondrial low Km ALDH was inhibited 90% and 99%, respectively, when incubated for 1 hr with 2 and 6 μM DETC-MeSO. These studies with rats and purified bovine ALDH show that DETC-MeSO is a potent inhibitor of mitochondrial low Km ALDH both in vivo and in vitro, providing additional evidence that DETC-MeSO is the metabolite to which disulfiram must be bioactivated for liver ALDH to be inhibited.