Showing papers on "Aldehyde dehydrogenase published in 1996"

Esophageal cancer and aldehyde dehydrogenase-2 genotypes in Japanese males.

Abstract: Although drinking alcohol is an established esophageal cancer risk factor, the mechanisms by which alcohol induces this high-mortality rate cancer are not clear. To help elucidate this problem and develop an implementable preventive strategy, this genetic epidemiological study focused on aldehyde dehydrogenase 2 (ALDH2), the key enzyme for elimination of acetaldehyde generated by alcohol consumption. This enzyme is polymorphic; its mutant allele, ALDH2*2, which leads to the enzyme inactivity, is prevalent in Orientals. This Japanese case-control study of ALDH2-related risk for esophageal squamous cell carcinoma included alcoholics (40 cases and 55 controls) and nonalcoholic drinkers (29 cases and 28 controls). The analysis of the results of genotyping these subjects showed that the increased risk for esophageal cancer in those with one ALDH2*2 allele was substantially higher in both alcoholics (odds ratio = 7.6; 95% confidence interval = 2.8-20.7) and nonalcoholic drinkers (odds ratio = 12.1; 95% confidence interval = 3.4-42.8). The results strongly suggest that because persons who have this mutant ALDH2*2 allele have a high concentration of blood acetaldehyde after drinking alcohol, acetaldehyde (a recognized animal carcinogen) plays a pivotal role in the pathogenesis of alcohol-related esophageal cancer in humans. These results suggest that to help lower their risk for esophageal cancer, persons with the ALDH2*2 allele should be encouraged to reduce their consumption of alcoholic beverages.

Possible Role of Liver Cytosolic and Mitochondrial Aldehyde Dehydrogenases in Acetaldehyde Metabolism

Abstract: To provide a molecular basis for understanding the possible mechanism of action of antidipsotropic agents in laboratory animals, aldehyde dehydrogenase (ALDH) isozymes were purified and characterized from the livers of hamsters and rats and compared with those from humans. The mitochondrial ALDHs from these species exhibit virtually identical kinetic properties in the oxidation and hydrolysis reactions. However, the cytosolic ALDH of human origin differs significantly from those of the rodents. Thus, for human ALDH-1, the Km value for acetaldehyde is 180 +/- 10 micromolar, whereas those for hamster ALDH-1 and rat ALDH-1 are 12 +/- 3 and 15 +/- 3 micromolar, respectively. Km values determined at pH 9.5 are virtually identical to those measured at pH 7.5. In vitro human ALDH-1 is 10 times less sensitive to disulfiram inhibition than are the hamster and rat cytosolic ALDHs. Competition between acetaldehyde and aromatic aldehydes or naphthaldehydes for the binding and catalytic sites of ALDHs shows their topography to be complex with more than one binding site. This also follows from data on substrate inhibition and activation, effects of NAD+ on ALDH-catalyzed hydrolysis of p-nitrophenyl esters, substrate specificity toward aldehydes and p-nitrophenyl esters, and inhibition by disulfiram in relation to oxidation and hydrolysis catalyzed by the ALDHs. The data further suggest that acetaldehyde cannot be considered as a "standard" ALDH substrate for studies aimed at aromatic ALDH substrates, e.g. biogenic aldehydes. Apparently, in human liver, only mitochondrial ALDH oxidizes acetaldehyde at physiological concentrations, whereas in hamster or rat liver, both the mitochondrial and cytosolic isozymes will do so.

Kinetics and specificity of human liver aldehyde dehydrogenases toward aliphatic, aromatic, and fused polycyclic aldehydes.

Abstract: Human mitochondrial aldehyde dehydrogenase (ALDH-2) has a Km for acetaldehyde that is 900-fold lower than that for the cytosolic isozyme, ALDH-1. An increase in aliphatic aldehyde chain length decreases the ALDH-2 Km by up to 10-fold but decreases that of ALDH-1 by 5 orders of magnitude. As a consequence, the Km of ALDH-1 for decanal is 8 times lower than that of ALDH-2, i.e. 2.9 +/- 0.4 and 22 +/- 3 nM, respectively. Determination of these low Km values required kinetic analysis of the simultaneous enzymatic conversion of two aldehyde substrates, an approach also applied to aromatic and fused polycyclic aldehydes. For most of these substrates, maximum velocities are 5-100 times lower than those for acetaldehyde. Addition of one of these tight-binding, slow-turnover substrates to a reaction mixture containing ALDH, NAD+, and a "reference" aldehyde substrate (e.g. acetaldehyde) blocks the principal (reference) enzymatic reaction temporarily and reversibly. Once the first substrate is converted to product, the enzyme can act on the reference substrate. In terms of apparent affinity and blocking capacity, naphthalene and phenanthrene aldehydes were the most potent effectors. Other aromatic and fused polycyclic and heterocyclic aldehydes, as well as derivatives of coumarin, quinoline, indole, and pyridine, are tight-binding, slow-turnover substrates for ALDH-2 and relatively weak inhibitors of ALDH-1. The hydrophobicity of substituents of benzaldehydes, and particularly of naphthaldehydes, correlates with their binding constants toward ALDH-2. Vitamin A1 aldehydes are specific natural substrates for ALDH-1; at pH 7.5, for all-trans- and 13-cis-retinal, Km = 1.1 and 0.37 micromolar, respectively, and kcat/Km is 50-100 times higher than that for acetaldehyde. At the same time, the retinals are inhibitors of ALDH-2, all-trans-retinal being a particularly potent inhibitor (competitive Ki = 43 nM, noncompetitive Ki = 316 nM). These properties suggest that all-trans-retinal is a possible regulatory compound for ALDH-2 in vivo. The data in general point to specialized roles for both major human liver ALDH isozymes in the oxidation of bulky/hydrophobic natural compounds, with Km values in the low nanomolar range.

Bacteriocolonic Pathway for Ethanol Oxidation: Characteristics and Implications

Abstract: Alcohol ingested orally is transported to the colon by blood circulation, and after the distribution phase, intracolonic ethanol levels are equal to those in the blood. Recent studies in our laboratory suggest that in the large bowel ethanol is oxidized by a bacteriocolonic pathway. In this pathway intracolonic ethanol is at first oxidized by bacterial alcohol dehydrogenase to acetaldehyde. Then acetaldehyde is oxidized either by colonic mucosal or bacterial aldehyde dehydrogenase to acetate. Part of intracolonic acetaldehyde may also be absorbed to portal vein and be metabolized in the liver. The bacteriocolonic pathway offers a new explanation for the disappearance of a part of ethanol calories. Due to the low aldehyde dehydrogenase activity of colonic mucosa, acetaldehyde accumulates in the colon. Accordingly during ethanol oxidation highest acetaldehyde levels of the body are found in the colon and not in the liver. High intracolonic acetaldehyde may contribute to the pathogenesis of alcohol-induced diarrhoea. Because acetaldehyde is a carcinogen in experimental animals, it may also contribute to the increased risk of colon polyps and colon cancer, which have been found to be associated with heavy alcohol consumption. Intracolonic acetaldehyde may also be an important determinant of the blood acetaldehyde level and a possible hepatotoxin. In addition to acetaldehyde, gut-derived endotoxin is another potential candidate in the pathogenesis of alcohol-related liver injury. Experimental alcoholic liver injury has recently been prevented by antibiotics, and this effect was related to the prevention of endotoxin-induced activation of Kupffer's cells.

Distribution of messenger RNAs for aldehyde dehydrogenase 1, aldehyde dehydrogenase 2, and aldehyde dehydrogenase 5 in human tissues.

Abstract: BACKGROUND The distribution of aldehyde dehydrogenases in human tissues is incompletely understood, in part because of technical limitations of gel electrophoretic and other enzyme assay methods used previously and because of the instability of the enzymes. Since these enzymes participate in detoxification of endogenous and exogenous compounds, including ethanol, their tissue distribution may be relevant to the toxicology of a number of substances and to the medical consequences of alcoholism. METHODS The abundance of mRNA for aldehyde dehydrogenase 1 (ALDH1), aldehyde dehydrogenase 2 (ALDH2), and aldehyde dehydrogenase 5 (ALDH5) was determined by Northern blotting using poly A+ RNA from 16 adult human tissues and 5 fetal tissues. RESULTS The highest levels of ALDH1 mRNA were found in liver, kidney, muscle, and pancreas. ALDH2 and ALDH5 were expressed in a larger number of tissues than ALDH1, with highest levels in liver, kidney, muscle, and heart. Fetal heart, brain, liver, lung, and kidney expressed ALDH2 and ALDH5, while ALDH1 was present mainly in fetal liver, kidney, and lung. CONCLUSIONS The results are in general agreement with the distribution of enzymes studied in a limited number of tissues in the past, with the exception that the ALDH1 activity reported to exist in heart and brain may, in fact, be ALDH5. The only mRNA detected in placenta was that for ALDH5. This study extends the knowledge of the expression of these enzymes to several tissues not previously studied and establishes the tissue distribution of the new enzyme ALDH5.

The contribution of polymorphism in the alcohol dehydrogenase β subunit to alcohol sensitivity in a Japanese population

Abstract: In humans, ingested alcohol is mainly metabolized by the combination of class I alcohol dehydrogenase (ADII) and aldehyde dehydrogenase (ALDH). In Orientals, there are highly frequent polymorphisms both in the class I ADH β subunit (ADHZ) and in the low Km ALDH (ALDH2). We characterized the three genotypes of ALDH2 in a Japanese population. In the present study, we evaluated the effects of the ADH2 polymorphism in the same population (424 males and 100 females) controlling for the effects of the ALDH2 polymorphism. In the ALDH21/ALDH22 group, the frequency of facial flushing with one glass of beer was significantly higher in the ADH21/ADH22 and ADH22/ADH22 genotype than in the ADH21/ADH21 genotype. Likewise, the proportion of persons with positive results for ethanol-induced cutaneous erythema differed significantly depending on the ADH2 genotype in both the ALDH21/ALDH21 and ALDH21/ALDH22 genotypes. However, drinking habits were not significantly associated with the ADH2 genotype, suggesting that the ADH2 genotype influences the metabolism of ethanol only in the peripheral tissues.

The mutation in the mitochondrial aldehyde dehydrogenase (ALDH2) gene responsible for alcohol-induced flushing increases turnover of the enzyme tetramers in a dominant fashion.

Abstract: Deficiency in mitochondrial aldehyde dehydrogenase (ALDH2), a tetrameric enzyme, results from inheriting one or two ALDH2*2 alleles. This allele encodes a protein subunit with a lysine for glutamate substitution at position 487 and is dominant over the wild-type allele, ALDH2*1. The ALDH2*2-encoded subunit (ALDH2K) reduces the activity of ALDH2 enzyme in cell lines expressing the wild-type subunit (ALDH2E). In addition to this effect on the enzyme activity, we now report that ALDH2*2 heterozygotes had lower levels of ALDH2 immunoreactive protein in autopsy liver samples. The half-lives of ALDH2 protein in HeLa cell lines expressing ALDH2*1, ALDH2*2, or both were determined by the rate of loss of immunoreactive protein after inhibition of protein synthesis with puromycin and by pulse-chase experiments. By either measure, ALDH2E enzyme was very stable, with a half-life of at least 22 h. ALDH2K enzyme had an enzyme half-life of only 14 h. In cells expressing both subunits, most of the subunits assemble as heterotetramers, and these enzymes had a half-life of 13 h. Thus, the effect of ALDH2K on enzyme turnover is dominant. These studies indicate that the ALDH2*2 allele exerts its dominant effect both by interfering with the catalytic activity of the enzyme and by increasing its turnover. This represents the first example of a dominantly acting allele with this effect on a mitochondrial enzyme's turnover.

Overexpression of the human aldehyde dehydrogenase class I results in increased resistance to 4-hydroperoxycyclophosphamide.

Abstract: A correlation between overexpression of aldehyde dehydrogenase and resistance to oxazaphosphorines, widely used anticancer agents, has been shown. To investigate the direct role of the human aldehyde dehydrogenase class 1 (ALDH-1) in the resistance to one of these agents, 4-hydroperoxycyclophosphamide (4-HC), an active metabolite of cyclophosphamide, neomycin-selectable plasmid or retroviral constructs harboring the wild-type ALDH-1 complementary DNA in the sense orientation were transfected into K562 leukemic cell lines. Polymerase chain reaction (PCR) analysis confirmed the presence of vector DNA in the stably transfected K562 cells. Reverse transcriptase PCR and Northern and Western blot analysis showed expression of ALDH-1 mRNA and protein in the cells transfected with ALDH-1 in the sense orientation but not in cells transfected with vector alone. The activity of the expressed ALDH-1 was demonstrated using spectrophotometric assay. Stably transfected K562 cells were subjected to various doses of 4-HC, and cell viability was assayed using clonogenic cell culture in semisolid medium. Results demonstrate that K562 cells transfected with ALDH-1 in the sense orientation display increased resistance to 4-HC compared with wild-type or vector-transfected K562 cells. Furthermore, the addition of diethylaminobenzaldehyde, a specific inhibitor for ALDH-1, restored the sensitivity of the ALDH-1-expressing K562 cells to 4-HC. Thus, the data pinpoint the direct role of ALDH-1 in the protection against 4-HC cytotoxicity.

Low Km aldehyde dehydrogenase (ALDH2) polymorphism, alcohol-drinking behavior, and chromosome alterations in peripheral lymphocytes.

Abstract: Excessive drinking of alcohol is now widely known to be one of the major lifestyle choices that ca effect health. Among the various effects of alcohol drinking, cytogenetic and other genotoxic effe...

Aldehyde dehydrogenase activity and acetate production by aerobic bacteria representing the normal flora of human large intestine

Abstract: We have recently proposed the existence of a bacteriocolonic pathway for ethanol oxidation, i.e ethanol is oxidized by alcohol dehydrogenase of intestinal bacteria resulting in high intracolonic levels of reactive and toxic acetaldehyde. This study was aimed to examine aldehyde dehydrogenase (ALDH) activity, acetaldehyde consumption and production of acetate by aerobic bacteria ( n =27), representing the normal human colonic flora. Most bacterial strains did not show any membrane-associated aldehyde dehydrogenase, but possessed marked cytosolic NADP+- and NAD+-dependent aldehyde dehydrogenase activity, ranging from 155 nmol of NAD(P)H produced/min/mg of protein to zero with acetaldehyde as substrate. NADP+-linked ALDH activity was significantly higher than NAD+-linked activity in most of the tested bacteria. In addition, aerobic bacteria metabolized acetaldehyde effectively in vitro and this could be inhibited by cyanamide in nearly half of the tested strains. Production of acetate from acetaldehyde ranged from 2420 nmol/109 colony-forming units to almost negligible. In conclusion, many human aerobic colonic bacteria possess significant aldehyde dehydrogenase activity and can, consequently, produce acetate from acetaldehyde in vitro at least under the partially aerobic conditions proposed to prevail on the colonic mucosal surface. Individual variation in the capability of colonic flora to remove toxic acetaldehyde may be one factor regulating intracolonic acetaldehyde levels, as well as the rate of bacteriocolonic pathway for ethanol oxidation.

Aldehyde dehydrogenases of the rat colon: comparison with other tissues of the alimentary tract and the liver.

Abstract: Intracolonic bacteria have previously been shown to produce substantial amounts of acetaldehyde during ethanol oxidation, and it has been suggested that this acetaldehyde might be associated with alcohol-related colonic disorders, as well as other alcohol-induced organ injuries. The capacity of colonic mucosa to remove this bacterial acetaldehyde by aldehyde dehydrogenase (ALDH) is, however, poorly known. We therefore measured ALDH activities and determined ALDH isoenzyme profiles from different subcellular fractions of rat colonic mucosa. For comparison, hepatic, gastric, and small intestinal samples were studied similarly. Alcohol dehydrogenase (ADH) activities were also measured from all of these tissues. Rat colonic mucosa was found to possess detectable amounts of ALDH activity with both micromolar and millimolar acetaldehyde concentrations and in all subcellular fractions. The ALDH activities of colonic mucosa were, however, generally low when compared with the liver and stomach, and they also tended to be lower than in small intestine. Mitochondrial low K(m) ALDH2 and cytosolic ALDH with low K(m) for acetaldehyde were expressed in the colonic mucosa, whereas some cytosolic high K(m) isoenzymes found in the small intestine and stomach were not detectable in colonic samples. Cytosolic ADH activity corresponded well to ALDH activity in different tissues: in colonic mucosa, it was approximately 6 times lower than in the liver and about one-half of gastric ADH activity. ALDH activity of the colonic mucosa should, thus, be sufficient for the removal of acetaldehyde produced by colonic mucosal ADH during ethanol oxidation. It may, however, be insufficient for the removal of the acetaldehyde produced by intracolonic bacteria. This may lead to the accumulation of acetaldehyde in the colon and colonic mucosa after ingestion of ethanol that might, at least after chronic heavy alcohol consumption, contribute to the development of alcohol-related colonic morbidity, diarrhea, and cancer.

Mouse Microsomal Class 3 Aldehyde Dehydrogenase: AHD3 cDNA Sequence, Inducibility by Dioxin and Clofibrate, and Genetic Mapping

Abstract: We have cloned and sequenced the mouse AHD3 cDNA, which codes for the Class 3 microsomal aldehyde dehydrogenase (ALDH3m). The cDNA is 2,997 bp in length excluding the poly(A)+ tail, and has 5' and 3' non-translated regions of 113 bp and 1,429 bp, respectively. The deduced amino acid sequence consists of 484 amino acids, including the first methionine (Mr = 53,942), and contains a hydrophobic segment at the carboxyl terminus which is the putative membrane anchor. The mouse AHD3 protein was found to be: 95% similar to the rat microsomal ALDH3m protein, 65% identical to the mouse, rat and human cytosolic ALDH3c protein, and <28% similar to the rat Class 1 and Class 2 ALDH and methylmalonate-semialdehyde dehydrogenase proteins. Southern hybridization analysis of mouse cDNA probed with the full-length AHD3 cDNA revealed that the Ahd3 gene likely spans less than a total of 25 kb. The mouse Ahd3 gene is very tightly linked to the Ahd4 gene on chromosome 11. Mouse AHD3 mRNA levels are increased by dioxin in mouse Hepa-1c1c7 hepatoma wild-type (wt) cells but not in the Ah receptor nuclear translocator (ARNT)-defective (c4) mutant line, indicating that the induction process is mediated by the Ah (aromatic hydrocarbon) dioxin-binding receptor. AHD3 mRNA levels are also inducible by clofibrate in both the wt and c4 lines. AHD3 mRNA levels are not elevated in the CYP1A1 metabolism-deficient c37 mutant line or as part of the oxidative stress response found in the untreated 14CoS/14CoS mouse cell line. These data indicate that, although inducible by dioxin, the Ahd3 gene does not qualify as a member of the aromatic hydrocarbon [Ah] gene battery.

Detoxification of reactive aldehydes in mitochondria: effects of age and dietary restriction.

Abstract: Previously, we proposed that reactive aldehydic products generated from lipid peroxidation might be the deleterious cause of mitochondrial dysfunction during aging. Our present study focuses on the roles that aging and dietary restriction (DR) play in the elimination of 4-hydroxynonenal (HNE) in rat liver by exploring three enzymatic systems: aldehyde dehydrogenase (ALDH), glutathione S-transferase (GST), and alcohol dehydrogenase (ADH). Results show that the main pathways of HNE elimination in mitochondria are through ALDH-catalyzed oxidation, and the GST-catalyzed conjugation of HNE. Findings also show that age reduces both ALDH and GST activities; mitochondrial HNE oxidation by ALDH declines at 18 and 24 months of age, and the glutathione conjugation of HNE reduces at 24 months of age. However, these enzymatic processes were found to be well-preserved in DR animals throughout their life span, supporting the evidence of less HNE accumulation in the membranes of restricted rats. These findings are consistent with our earlier proposal that indicates an age-associated decrease in mitochondrial detoxification as a major underlying process for malondialdehyde and lipofuscin accumulation in older animals. They also indicate that the prevention of the age-associated decrease in aldehyde detoxification by DR may be an important mechanism underlying enhanced aldehyde elimination, thus minimizing the functional deterioration observed in mitochondria of old animals.

Localization of cytosolic aldehyde dehydrogenase in the developing chick retina : in situ hybridization and immunohistochemical analyses

Abstract: Cytosolic aldehyde dehydrogenase (ALDH) mRNA is present at high levels in the undifferentiated chick retina Tissue maturation is accompanied by a 20-25x decrease in transcript levels To determine the spatial and temporal distribution pattern of the ALDH transcript and its encoded protein in the developing retina, in situ hybridization and immunohistochemical analyses were carried out using chick embryos at different stages of development The ALDH transcript and protein were detected at the earliest stage tested, in the inner layer of the optic cup of stage 14 (day 2) embryos Both the ALDH transcript and protein were found in the dorsal retina of chick embryos from stage 18 (day 3) to day 16 of incubation Accumulation of the ALDH protein in the neurites of ganglion cells could readily be detected at early developmental stages Staining of this ganglion fiber layer was strong in the dorsal retina and could be followed up to and into the optic nerve By day 11, ALDH mRNA was located primarily in the ciliary margin and in the inner nuclear layer of the dorsal retina In addition to these areas, the ALDH protein was also found in the inner plexiform and optic nerve fiber layers These results suggest that environmental or transcriptional factors involved in the regulation of the ALDH gene are restricted to the dorsal retina at early developmental stages and that there is a requirement for the compartmentalization of the ALDH transcript/protein in the undifferentiated chick retina

S-methyl-N,N-diethylthiocarbamate sulfoxide and S-methyl-N,N-diethylthiocarbamate sulfone, two candidates for the active metabolite of disulfiram

Abstract: The mechanism of action of disulfiram involves inhibition of hepatic aldehyde dehydrogenase (ALDH). Although disulfiram inhibits ALDH in vitro, it is believed that the drug is too short-lived in vivo to inhibit the enzyme directly. The ultimate inhibitor is thought to be a metabolite of disulfiram. In this study, we examined the effects of S-methyl-N,N-diethylthiocarbamate (MeDTC) sulfoxide and S-methyl-N,N-diethylthiocarbamate sulfone (confirmed and proposed metabolites of disulfiram, respectively) on rat liver mitochondrial low K(m) ALDH. MeDTC sulfoxide and MeDTC sulfone, in 10-min incubations with detergent-solubilized mitochondria, inhibited ALDH activity with an IC50 (mean +/- SD) of 0.93 +/- 0.04 and 0.53 +/- 0.11 microM, respectively, compared with 7.4 +/- 1.0 microM for the parent drug disulfiram. Inhibition by MeDTC sulfone and MeDTC sulfoxide, both at 0.6 microM, was time-dependent, following apparent pseudo-first-order kinetics with a t1/2 of inactivation of 3.5 and 8.8 min, respectively. Dilution of ALDH inhibited by either sulfoxide or sulfone did not restore activity, an indication of irreversible inhibition. Addition of glutathione (50 to 1000 microM) to ALDH before the inhibitors did not alter the inhibition by MeDTC sulfoxide. In contrast, the inhibition by MeDTC sulfone was decreased > 10-fold (IC50 = 6.3 microM) by 50 microM of glutathione and almost completely abolished by 500 microM of glutathione. The cofactor NAD, in a concentration-dependent manner, protected ALDH from inhibition by MeDTC sulfoxide and MeDTC sulfone. In incubations with intact mitochondria, the potency of the two compounds was reversed (IC50 of 9.2 +/- 3.6 and 0.95 +/- 0.30 microM for the MeDTC sulfone and sulfoxide, respectively). Our results suggest that MeDTC sulfone is highly reactive with normal cellular constituents (e.g., glutathione), which may protect ALDH from inhibition, unless this inhibitor is formed very near the target enzyme. In contrast, MeDTC sulfoxide is a better candidate for the ultimate active metabolite of disulfiram, because it is more likely to be sufficiently stable to diffuse from a distant site of formation, such as the endoplasmic reticulum, penetrate the mitochondria, and react with ALDH located in the mitochondrial matrix.

Aldehyde dehydrogenase from adult human brain that dehydrogenates gamma-aminobutyraldehyde: purification, characterization, cloning and distribution.

Abstract: Enzyme purification and characterization, cDNA cloning and Northern blot analysis were the techniques utilized during this investigation to determine the identity and occurrence of the aldehyde dehydrogenase that metabolizes gamma-aminobutyraldehyde in adult human brain. The purification yielded one major protein which was active with gamma-aminobutyraldehyde. It had the physico-chemical and kinetic properties of the human liver E3 isoenzyme of aldehyde dehydrogenase (EC 1.2.1.3), and also interacted with an anti-(liver E3 isoenzyme) antibody. Tryptic peptides derived from the purified brain protein matched the amino acid sequence of the liver E3 isoenzyme. Employing liver E3 cDNA, a human cerebellar cDNA library was screened and a 2.0 kb cDNA fragment was isolated. The cerebellar cDNA yielded a derived primary structure which differed from the liver E3 amino acid sequence by a single serine-to-cysteine substitution at position 88 (position 84 in the liver sequence). Thus the gamma-amino-butyraldehyde-metabolizing enzyme from human brain can be identified as E3', a variant of the E3 isoenzyme. The catalytic properties of the brain variant were indistinguishable from those of E3, and so the functional importance of this variant is at present unknown. The distribution of this enzyme in brain was investigated by Northern blot analysis, which demonstrated the presence of E3' mRNA in all regions of the human brain. mRNA levels were variable in the different brain areas, with the highest levels in the spinal cord and the lowest in the occipital pole.

Effect of UVB radiation on corneal aldehyde dehydrogenase

Abstract: Purpose. A Class 3 aldehyde dehydrogenase happens to be a major soluble protein constituent of the cornea. Its role is conjectured to be manifold: to protect the tissue from oxidative damage by eliminating the toxic aldehydes produced upon lipid perox-idation under oxidative stress, to act as an UV-absorber, and to maintain the level of the coenzyme NADH in the cornea. We have studied the effect of UVB on the structure and enzyme activity of corneal aldehyde dehydrogenase.Methods. Aldehyde dehydrogenase was irradiated at 295 nm for varying periods of time and change in its enzyme activity assayed. The structural changes in the molecule accompanying irradiation were monitored using fluorescence and circular dichroism spectroscopy, and its hydrodynamic behavior and surface hydrophobicity studied using gel filtration chromatog-raphy and binding of the hydrophobic fluorophore ANS. The protective ability of aldehyde dehydrogenase in preventing aggregation of photolabile proteins, such as Γ-crystallin of the ey...

Genetic analysis of the phenobarbital regulation of the cytochrome P-450 2b-9 and aldehyde dehydrogenase type 2 mRNAs in mouse liver

Abstract: The aim of this study was to investigate the effect of the genetic background on the phenobarbital inducibility of cytochrome P-450 2b-9, cytochrome P-450 2b-10 and aldehyde dehydrogenase type 2 mRNAs in mice. We analysed the basal expression and the phenobarbital inducibility of both cytochrome P-450 mRNAs by semi-quantitative specific reverse transcription-PCR analyses in five inbred mouse strains (A/J,BALB/cByJ,C57BL/6J, DBA/2J and SWR/J). Male mice constitutively expressed cytochrome P-450 2b-9 and cytochrome P-450 2b-10 mRNAs, but a number of differences in their response to phenobarbital were observed. In all these mouse strains, phenobarbital induced cytochrome P-450 2b-10 mRNA whereas it could have either a positive or a negative effect on cytochrome P-450 2b-9 expression, depending on the strain and the sex of the mice. Specifically, phenobarbital increased cytochrome P-450 2b-9 expression in C57BL/6J males while it decreased it in DBA/2J mice. Interestingly, dexamethasone was able to mimic the phenobarbital effect on both cytochromes P-450 in these two strains. Aldehyde dehydrogenase type 2 mRNA was always induced by phenobarbital, except in the C57BL/6J strain. Genetic analysis revealed that the phenobarbital-inducible phenotype was either a semi-dominant or a recessive trait in F1 animals from a C57BL/6J x DBA/2J cross for the cytochrome P-450 2b-9 and the aldehyde dehydrogenase type 2 genes, respectively. This study suggests that the genetic basis for phenobarbital induction in mice depends on the target gene, and that more than one regulatory step would by involved in this response pathway.