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Abstract Background Sick-leave because of mental and behavioural disorders has increased considerably in Sweden since the late nineties, and especially in women. The aim of this study was to assess the level of burnout in the general working population in northern Sweden and analyse it's relation to working conditions and gender. Methods In this cross-sectional study the survey from the MONICA-study (Monitoring of Trends and Determinants in Cardiovascular Disease) in northern Sweden 2004 was used. A burnout instrument, the Shirom Melamed Burnout Questionnaire (SMBQ), was incorporated in the original survey which was sent to a random sample of 2500 individuals with a response rate of 76%. After including only actively working people, aged 25-64 years, our study population consisted of 1000 participants (497 women and 503 men). ANOVA and multiple linear regression models were used. Results The prevalence of a high level of burnout (SMBQ >4.0) was 13%. Women had a higher level of burnout than men with the most pronounced difference in the age group 35-44 years. In both sexes the level of burnout decreased with age. Demand and control at work, and job insecurity were related to burnout. In women the level of education, socioeconomic position, work object, and working varying hours were of importance. Interaction effects were found between sex and work object, and sex and working hours. In a multiple regression analysis almost half of the gender difference could be explained by work related and life situational factors. Conclusions Working life conditions contributed to the level of burnout in this actively working sample from the general population in northern Sweden. Especially in women, socioeconomic position was associated with burnout. The high level of burnout in women compared to men was partly explained by more unfavourable working conditions and life situational factors. Efforts to level out gender differences in burnout should probably focus on improving both working and socioeconomic conditions for women.

The medium-chain dehydrogenase/reductase (MDR) superfamily has more than 600,000 members in UniProt as of March 2023. As the family has been growing, the proportion of functionally characterized proteins has been falling behind. The aim of this project was to investigate the binding pockets of nine different MDR protein families based on sequence conservation patterns and three-dimensional structures of members within the respective families. A search and analysis methodology was developed. Using this, a total of 2000 eukaryotic MDR sequences belonging to nine different families were identified. The pairwise sequence identities within each of the families were 80-90 % for the mammalian sequences, like the levels observed for alcohol dehydrogenase, another MDR family. Twenty conserved residues were identified in the coenzyme part of the binding site by matching structural and conservation data of all nine protein families. The conserved residues in the substrate part of the binding pocket varied between the nine MDR families, implying divergent functions for the different families. Studying each family separately made it possible to identify multiple conserved residues that are expected to be important for substrate binding or catalysis of the enzymatic reaction. By combining structural data with the conservation of the amino acid residues in each protein, important residues in the binding pocket were identified for each of the nine MDRs. The obtained results add new positions of interest for the binding and activity of the enzyme family as well as fit well to earlier published data. Three distinct types of binding pockets were identified, containing no, one, or two tyrosine residues., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: All known attempts to isolate and characterize mammalian class V alcohol dehydrogenase (class V ADH), a member of the large ADH protein family, at the protein level have failed. This indicates that the class V ADH protein is not stable in a non-cellular environment, which is in contrast to all other human ADH enzymes. In this report we present evidence, supported with results from computational analyses performed in combination with earlier in vitro studies, why this ADH behaves in an atypical way., Results: Using a combination of structural calculations and sequence analyses, we were able to identify local structural differences between human class V ADH and other human ADHs, including an elongated β-strands and a labile α-helix at the subunit interface region of each chain that probably disturb it. Several amino acid residues are strictly conserved in class I-IV, but altered in class V ADH. This includes a for class V ADH unique and conserved Lys51, a position directly involved in the catalytic mechanism in other ADHs, and nine other class V ADH-specific residues., Conclusions: In this study we show that there are pronounced structural changes in class V ADH as compared to other ADH enzymes. Furthermore, there is an evolutionary pressure among the mammalian class V ADHs, which for most proteins indicate that they fulfill a physiological function. We assume that class V ADH is expressed, but unable to form active dimers in a non-cellular environment, and is an atypical mammalian ADH. This is compatible with previous experimental characterization and present structural modelling. It can be considered the odd sibling of the ADH protein family and so far seems to be a pseudoenzyme with another hitherto unknown physiological function.

Mammalian alcohol dehydrogenase (ADH) is a protein family divided into six classes and the number of known family members is increasing rapidly. Several primate genomes are completely analyzed for the ADH region, where higher primates (human and hominoids) have seven genes of classes ADH1-ADH5. Within the group of non-hominoids apes there have been further duplications and species with more than the typical three isozymic forms for ADH1 are present. In contrast there are few completely analyzed ADH genomes in the non-primate group of mammals, where an additional class has been identified, ADH6, that has been lost during the evolution of primates. In this study 85 mammalian genomes with at least one ADH gene have been compiled. In total more than 500 ADH amino acid sequences were analyzed for patterns that distinguish the different classes. For ADH1-ADH4 intensive investigations have been performed both at the functional and at structural levels. However, a corresponding functional protein to the ADH5 gene, which is found in most ADH genomes, has never been detected. The same is true for ADH6, which is only present in non-primates. The entire mammalian ADH family shows a broad spectrum of gene duplications and gene losses where the numbers differ from six genes (most non-primate mammals) up to ten genes (vole). Included in these sets are examples of pseudogenes and pseudoenzymes., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Alcohol dehydrogenase 5 (ADH5) is a member of the mammalian alcohol dehydrogenase family of yet undefined functions. ADH5 was first identified at the DNA level in human and deer mouse. A rat alcohol dehydrogenase structure of similar type has been isolated at the cDNA level using human ADH5 as a screening probe, where the rat cDNA structure displayed several atypical properties. mRNA for rat ADH5 was found in multiple tissues, especially in the kidney. In vitro translation experiments indicated that rat ADH5 is expressed as efficiently as ADH1 and furthermore, rat ADH5 was readily expressed in COS cells fused to Green Fluorescent Protein. However, no soluble ADH5 protein could be heterologously expressed in Escherichia coli cells with expression systems successfully used for other mammalian ADHs, including fused to glutathione-S-transferase. Molecular modelling of the enzyme indicated that the protein does not fold in a productive way, which can be the explanation why no stable and active ADH5 has been isolated. These results indicate that ADH5, while readily expressed at the mRNA level, does not behave similarly to other mammalian ADHs investigated. The results, in vitro and in silico, suggest an unstable ADH5 structure, which can explain for why no active and stable protein can be isolated. Further possibilities are conceivable: the ADH5 protein may have to interact with a stabiliser, or the gene is actually a pseudogene., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Mammalian alcohol dehydrogenase (ADH) can be divided into six classes, ADH1-ADH6, according to primary structure and function, where the classes are further subdivided into isozymes and allelic forms. With the increasing amount of available genomic data a general pattern is possible to trace within the mammalian ADH gene and protein families. The transcriptional order for the ADH genes in all mammalian genomes is the same (ADH4-ADH1-ADH6-ADH5-ADH2-ADH3), but the cluster is found on different chromosomes in different species. However, in primates only ADH1-ADH5 are present, where the loss of ADH6 may have occurred simultaneously as the split into ADH1 isoforms. ADH3, also denoted glutathione-dependent formaldehyde dehydrogenase and S-nitrosoglutathione reductase, is identified as the last gene in the ADH transcriptional order, but several pseudogenes for ADH3 have been traced at other chromosomes. The flanking genes outside the ADH genome are similar or identical for all species showing that a larger DNA region has been duplicated and further evolved. However, the only entirely completed ADH genomes are those from primates and rodents. The latest identified ADH forms, ADH5 (class V) and ADH6 (class VI), are truly different classes and both are very diverged in contrast to ADH3, which is the most conserved class of all ADHs. ADH5 and ADH6 have been identified at the gene and transcriptional levels only, and their functions are still an enigma., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Alcohol dehydrogenase 3 (ADH3) has been assigned a role in nitric oxide homeostasis due to its function as an S-nitrosoglutathione reductase. As altered S-nitrosoglutathione levels are often associated with disease, compounds that modulate ADH3 activity might be of therapeutic interest. We performed a virtual screening with molecular dockings of more than 40,000 compounds into the active site of human ADH3. A novel knowledge-based scoring method was used to rank compounds, and several compounds that were not known to interact with ADH3 were tested in vitro. Two of these showed substrate activity (9-decen-1-ol and dodecyltetraglycol), where calculated binding scoring energies correlated well with the logarithm of the k (cat)/K (m) values for the substrates. Two compounds showed inhibition capacity (deoxycholic acid and doxorubicin), and with these data three different lines for specific inhibitors for ADH3 are suggested: fatty acids, glutathione analogs, and cholic acids.

Alcohol dehydrogenase 3 (ADH3) has emerged as an important regulator of protein S-nitrosation in its function as S-nitrosoglutathione (GSNO) reductase. GSNO depletion is associated with various disease conditions, emphasizing the potential value of a specific ADH3 inhibitor. The present study investigated inhibition of ADH3-mediated GSNO reduction by various substrate analogues, including medium-chain fatty acids and glutathione derivatives. The observed inhibition type was non-competitive. Similar to the Michaelis constants for the corresponding omega-hydroxy fatty acids, the inhibition constants for fatty acids were in the micromolar range and showed a clear dependency on chain length with optimal inhibitory capacity for eleven and twelve carbons. The most efficient inhibitors found were undecanoic acid, dodecanoic acid and dodecanedioic acid, with no significant difference in inhibition constant. All glutathione-derived inhibitors displayed inhibition constants in the millimolar range, at least three orders of magnitudes higher than the Michaelis constants of the high-affinity substrates GSNO and S-hydroxymethylglutathione. The experimental results as well as docking simulations with GSNO and S-methylglutathione suggest that for ADH3 ligands with a glutathione scaffold, in contrast to fatty acids, a zinc-binding moiety is imperative for correct orientation and stabilization of the hydrophilic glutathione scaffold within a predominantly hydrophobic active site.

Many carbonyl metabolizing enzymes are equally involved in xenobiotic and endogenous metabolism, but few have been investigated in terms of substrate competition and interference between different cellular pathways. Mammalian alcohol dehydrogenase 3 (ADH3) represents the key enzyme in the formaldehyde detoxification pathway by oxidation of S-hydroxymethylglutathione [HMGSH; the glutathione (GSH) adduct of formaldehyde]. In addition, several studies have established ADH3 as S-nitrosoglutathione (GSNO) reductase in endogenous NO homeostasis during the last decade. GSNO depletion associates with various diseases including asthma, and evidence for a causal relationship between ADH3 and asthma pathology has been put forward. In a recent study, we showed that ADH3-mediated alcohol oxidation, including HMGSH oxidation, is accelerated in presence of GSNO which is concurrently reduced under immediate cofactor recycling [C.A. Staab, J. Alander, M. Brandt, J. Lengqvist, R. Morgenstern, R.C. Grafström, J.-O. Höög, Reduction of S-nitrosoglutathione by alcohol dehydrogenase 3 is facilitated by substrate alcohols via direct cofactor recycling and leads to GSH-controlled formation of glutathione transferase inhibitors, Biochem. J. 413 (2008) 493-504]. Thus, considering the usually low cytosolic free NADH/NAD(+) ratio, formaldehyde may trigger and promote GSNO reduction by enzyme-bound cofactor recycling. These findings provided evidence for formaldehyde-induced, ADH3-mediated GSNO depletion with potential direct implications for asthma. Furthermore, analysis of product formation as a function of GSH concentrations suggested that, under conditions of oxidative stress, GSNO reduction can lead to the formation of glutathione sulfinamide and its hydrolysis product glutathione sulfinic acid, both potent inhibitors of glutathione transferase activity.

Alcohol dehydrogenase 3 (ADH3) is highly conserved, ubiquitously expressed in mammals and involved in essential cellular pathways. A large active site pocket entails special substrate specificities: shortchain alcohols are poor substrates, while medium-chain alcohols and particularly the glutathione adducts S-hydroxymethylglutathione (HMGSH) and S-nitrosoglutathione (GSNO) are efficiently converted under concomitant use of NAD(+)/NADH. By oxidation of HMGSH, the spontaneous glutathione adduct of formaldehyde, ADH3 is implicated in the detoxification of formaldehyde. Through the GSNO reductase activity, ADH3 can affect the transnitrosation equilibrium between GSNO and S-nitrosated proteins, arguing for an important role in NO homeostasis. Recent findings suggest that ADH3-mediated GSNO reduction and subsequent product formation responds to redox states in terms of NADH availability and glutathione levels. Finally, a dual function of ADH3 is discussed in view of its potential implications for asthma.

Gene expression of carbonyl-metabolizing enzymes (CMEs) was investigated in normal buccal keratinocytes (NBK) and the transformed buccal keratinocyte lines SVpgC2a and SqCC/Y1. Studies were performed at a serum concentration known to induce terminal squamous differentiation (TSD) in normal cells. Overall, 39 of 58 evaluated CMEs were found to be expressed at the transcript level. Together the transformed cell lines showed altered transcription of eight CME genes compared to NBK, substantiating earlier results. Serum increased transcript levels of ALDH1A3, DHRS3, HPGD and AKR1A1, and decreased those of ALDH4A1 in NBK; of these, the transformed, TSD-deficient cell lines partly retained regulation of ALDH1A3 and DHRS3. Activity measurements in crude cell lysates, including relevant enzymatic inhibitors, indicated significant capacity for CME-mediated xenobiotic metabolism among the cell lines, notably with an increase in serum-differentiated NBK. The results constitute the first evidence for differential CME gene expression and activity in non-differentiated and differentiated states of epithelial cells.

GSNO (S-nitrosoglutathione) is emerging as a key regulator in NO signalling as it is in equilibrium with S-nitrosated proteins. Accordingly, it is of great interest to investigate GSNO metabolism in terms of competitive pathways and redox state. The present study explored ADH3 (alcohol dehydrogenase 3) in its dual function as GSNOR (GSNO reductase) and glutathione-dependent formaldehyde dehydrogenase. The glutathione adduct of formaldehyde, HMGSH (S-hydroxymethylglutathione), was oxidized with a k(cat)/K(m) value approx. 10 times the k(cat)/K(m) value of GSNO reduction, as determined by fluorescence spectroscopy. HMGSH oxidation in vitro was greatly accelerated in the presence of GSNO, which was concurrently reduced under cofactor recycling. Hence, considering the high cytosolic NAD(+)/NADH ratio, formaldehyde probably triggers ADH3-mediated GSNO reduction by enzyme-bound cofactor recycling and might result in a decrease in cellular S-NO (S-nitrosothiol) content in vivo. Formaldehyde exposure affected S-NO content in cultured cells with a trend towards decreased levels at concentrations of 1-5 mM, in agreement with the proposed mechanism. Product formation after GSNO reduction to the intermediate semimercaptal responded to GSH/GSNO ratios; ratios up to 2-fold allowed the spontaneous rearrangement to glutathione sulfinamide, whereas 5-fold excess of GSH favoured the interception of the intermediate to form glutathione disulfide. The sulfinamide and its hydrolysis product, glutathione sulfinic acid, inhibited GST (glutathione transferase) activity. Taken together, the findings of the present study provide indirect evidence for formaldehyde as a physiological trigger of GSNO depletion and show that GSNO reduction can result in the formation of GST inhibitors, which, however, is prevented under normal cellular redox conditions.

In vitro models are currently not considered to be suitable replacements for animals in experiments to assess the multiple factors that underlie the development of cancer as a result of environmental exposure to chemicals. An evaluation was conducted on the potential use of normal keratinocytes, the SV40 T-antigen-immortalised keratinocyte cell line, SVpgC2a, and the carcinoma cell line, SqCC/Y1, alone and in combination, and under standardised serum-free culture conditions, to study oral cancer progression. In addition, features considered to be central to cancer development as a result of environmental exposure to chemicals, were analysed. Genomic expression, and enzymatic and functional data from the cell lines reflected many aspects of the transition of normal tissue epithelium, via dysplasia, to full malignancy. The composite cell line model develops aberrances in proliferation, terminal differentiation and apoptosis, in a similar manner to oral cancer progression in vivo. Transcript and protein profiling links aberrations in multiple gene ontologies, molecular networks and tumour biomarker genes (some proposed previously, and some new) in oral carcinoma development. Typical specific changes include the loss of tumour-suppressor p53 function and of sensitivity to retinoids. Environmental agents associated with the aetiology of oral cancer differ in their requirements for metabolic activation, and cause toxic effects to cells in both the normal and the transformed states. The results suggest that the model might be useful for studies on the sensitivity of cells to chemicals at different stages of cancer progression, including many aspects of the integrated roles of cytotoxicity and genotoxicity. Overall, the properties of the SVpgC2a and SqCC/Y1 cell lines, relative to normal epithelial cells in monolayer or organotypic culture, support their potential applicability to mechanistic studies on cancer risk factors, including, in particular, the definition of critical toxicity effects and dose-effect relationships.

Subunit interaction in sorbitol dehydrogenase (SDH) has been studied with in vitro and in silico methods identifying a vital hydrogen-bonding network, which is strictly conserved among mammalian SDH proteins. Mutation of one of the residues in the hydrogen-bonding network, Tyr110Phe, abolished the enzymatic activity and destabilized the protein into tetramers, dimers and monomers as judged from gel filtration experiments at different temperatures compared to only tetramers for the wild-type protein below 307 K. The determined equilibrium constants revealed a large difference in Gibbs energy (8 kJ/mol) for the tetramer stability between wild-type SDH and the mutated form Tyr110Phe SDH. The results focus on a network of coupled hydrogen bonds in wild-type SDH that uphold the protein interface, which is specific and favorable to electrostatic, van der Waals and hydrogen-bond interactions between subunits, interactions that are crucial for the catalytic power of SDH.

Normal and two transformed buccal keratinocyte lines were cultured under a standardized condition to explore mechanisms of carcinogenesis and tumor marker expression at transcript and protein levels. An approach combining three bioinformatic programs allowed coupling of abundant proteins and large-scale transcript data to low-abundance transcriptional regulators. The analysis identified previously proposed and suggested novel protein biomarkers, gene ontology categories, molecular networks, and functionally impaired key regulator genes for buccal/oral carcinoma.

The metabolism of all-trans- and 9-cis-retinol/ retinaldehyde has been investigated with focus on the activities of human, mouse and rat alcohol dehydrogenase 2 (ADH2), an intriguing enzyme with apparently different functions in human and rodents. Kinetic constants were determined with an HPLC method and a structural approach was implemented by in silico substrate dockings. For human ADH2, the determined K(m) values ranged from 0.05 to 0.3 microM and k(cat) values from 2.3 to 17.6 min(-1), while the catalytic efficiency for 9-cis-retinol showed the highest value for any substrate. In contrast, poor activities were detected for the rodent enzymes. A mouse ADH2 mutant (ADH2Pro47His) was studied that resembles the human ADH2 setup. This mutation increased the retinoid activity up to 100-fold. The K(m) values of human ADH2 are the lowest among all known human retinol dehydrogenases, which clearly support a role in hepatic retinol oxidation at physiological concentrations.

Alcohol dehydrogenase (ADH) catalyses the rate-determining reaction in ethanol metabolism. Genetic association studies of diverse ethnic groups have firmly demonstrated that the allelic variant ADH1B*2 significantly protects against alcoholism but that ADH1C*1, which is in linkage with ADH1B*2, produces a negligible protection. The influence of other potential candidate genes/alleles within the human ADH family, ADH1B*3 and ADH2, remains unclear or controversial. To address this question, functionalities of ADH1B3 and ADH2 were assessed at a physiological level of coenzyme and substrate range. Ethanol-oxidizing activities of recombinant ADH1B1, ADH1B2, ADH1B3, ADH1C1, ADH1C2 and ADH2 were determined at pH 7.5 in the presence of 0.5 mm NAD with 2-50 mm ethanol. The activity differences between ADH1B2 and ADH1B1 were taken as a threshold for effective protection against alcoholism and those between ADH1C1 and ADH1C2 as a threshold for null protection. Over 2-50 mm ethanol, the activities of ADH1B3 were found 2.9-23-fold lower than those of ADH1B2, largely attributed to the Km effect (ADH1B2, 1.8 mm; ADH1B3, 61 mm). Strikingly, the ADH1B3 activity was only 84% that of ADH1B1 at a low ethanol concentration, 2 mm, but increased 10-fold at 50 mm. Corrected for relative expression levels of the enzyme in liver, the hepatic ADH2 activities were estimated to be 18-97% those of ADH1B1 over 2-50 mm ethanol and were 28-140% of the activity differences between ADH1C1 and ADH1C2. The assessment based on the proposed functional window for the human ADH gene family indicates that ADH1B*3 may show some degree of protection against alcoholism and that the ADH2 functional variants appear to be negligible for this protection.

Normal oral keratinocytes (NOKs), and a Simian virus 40 T-antigen-immortalised oral keratinocyte line termed SVpgC2a, were cultured in an effort to model the human oral epithelium in vitro, including normal and dysplastic tissue. Monolayer and organotypic cultures of NOKs and SVpgC2a were successfully established in a standardised serum-free medium with high levels of amino acids, by using regular tissue culture plastic for monolayers and collagen gels containing oral fibroblasts as the base for generating tissue equivalents. NOKs express many characteristics of normal tissue, including those associated with terminal squamous differentiation. After > 150 passages, SVpgC2a cells retained an immortal, nontumourigenic phenotype that, relative to NOKs, was associated with aberrant morphology, enhanced proliferation, deficiency in terminal differentiation, proneness to apoptosis, and variably altered expression of structural epithelial markers. Transcript and protein profiling, as well as activity assays, demonstrated the expression of multiple xenobiotic-metabolising enzymes in SVpgC2a cells, some of which were higher in comparison to NOKs. A generally preserved, or even activated, ability for xenobiotic metabolism in long-term cultures of SVpgC2a cells indicated that this cell line could be useful in safety testing protocols--for example, in the development of consumer products in the oral health care field. However, SVpgC2a cells displayed some features reminiscent of a severe oral dysplasia, implying that this cell line could also to some extent serve as a model of a premalignant oral epithelium.

Gene expression underlying cellular growth and differentiation is only partly understood. This study analyzed transcript levels of the formaldehyde-metabolizing enzyme alcohol dehydrogenase 3 (ADH3) and various growth and differentiation-related genes in human oral keratinocytes. Culture of confluent cells both with and without fetal bovine serum inhibited colony-forming efficiency and induced a squamous morphology. Confluency alone decreased the transcript levels of ADH3, the proliferation markers cell division cycle 2 (CDC2) and proliferating cell nuclear antigen (PCNA), and the basal cell marker cytokeratin 5 (K5), but increased transcripts for the suprabasal differentiation markers involucrin (INV) and small proline-rich protein 1B (SPR1). These changes were variably influenced by serum, i.e., loss of CDC2 and PCNA was inhibited, loss of K5 promoted, increase of SPR1 transcripts inhibited, and increase of INV promoted. The extent and onset of the effects implied that ADH3 transcription serves as a proliferation marker and that confluency with or without serum exposure can serve to selectively analyze proliferative and differentiated cellular states.

ADH2 is a member of one of the six classes of mammalian alcohol dehydrogenases, which catalyze the reversible oxidation of alcohols using NAD(+) as a cofactor. Within the ADH2 class, the rodent enzymes form a subgroup that exhibits low catalytic activity with all substrates that were examined, as compared to other groups, such as human ADH2. The low activity can be ascribed to the rigid nature of the proline residue at position 47 as the activity can be increased by approximately 100-fold by substituting Pro47 with either His (as found in human ADH2), Ala, or Gln. Mouse ADH2 follows an ordered bi-bi mechanism, and hydride transfer is rate-limiting for oxidation of benzyl alcohols catalyzed by the mutated and wild-type enzymes. Structural studies suggest that the mouse enzyme with His47 has a more closed active site, as compared to the enzyme with Pro47, and hydride transfer can be more efficient. Oxidation of benzyl alcohol catalyzed by all forms of the enzyme is strongly pH dependent, with pK values in the range of 8.1-9.3 for turnover numbers and catalytic efficiency. These pK values probably correspond to the ionization of the zinc-bound water or alcohol. The pK values are not lowered by the Pro47 to His substitution, suggesting that His47 does not act as a catalytic base in the deprotonation of the zinc ligand.

Human alcohol dehydrogenase 3/glutathione-dependent formaldehyde dehydrogenase was shown to rapidly and irreversibly catalyse the reductive breakdown of S-nitrosoglutathione. The steady-state kinetics of S-nitrosoglutathione reduction was studied for the wild-type and two mutated forms of human alcohol dehydrogenase 3, mutations that have previously been shown to affect the oxidative efficiency for the substrate S-hydroxymethylglutathione. Wild-type enzyme readily reduces S-nitrosoglutathione with a kcat/Km approximately twice the kcat/Km for S-hydroxymethylglutathione oxidation, resulting in the highest catalytic efficiency yet identified for a human alcohol dehydrogenase. In a similar manner as for S-hydroxymethylglutathione oxidation, the catalytic efficiency of S-nitrosoglutathione reduction was significantly decreased by replacement of Arg115 by Ser or Lys, supporting similar substrate binding. NADH was by far a better coenzyme than NADPH, something that previously has been suggested to prevent reductive reactions catalysed by alcohol dehydrogenases through the low cytolsolic NADH/NAD+ ratio. However, the major products of S-nitrosoglutathione reduction were identified by electrospray tandem mass spectrometry as glutathione sulfinamide and oxidized glutathione neither of which, in their purified form, served as substrate or inhibitor for the enzyme. Hence, the reaction products are not substrates for alcohol dehydrogenase 3 and the overall reaction is therefore irreversible. We propose that alcohol dehydrogenase 3 catalysed S-nitrosoglutathione reduction is of physiological relevance in the metabolism of NO in humans.

Mammalian alcohol dehydrogenases (ADHs), including ADH1-ADH5/6, interact extensively in the oxidation and reduction of alcohols and aldehydes. ADH1 and ADH2 are involved in several metabolic pathways besides the oxidation of ethanol and have also been shown to be involved in drug transformations. The ADH2 enzymes show further complexity among the species, e.g. in enzymatic characteristics where the rodent forms essentially lack ethanol-oxidizing capacity. ADH3 (glutathione-dependent formaldehyde dehydrogenase) has been shown to catalyze the reductive breakdown of S-nitrosoglutathione, indicating involvement in nitric oxide metabolism. Mass spectrometry identified the major enzymatic product as glutathione sulfinamide. This reductive breakdown directly interferes with the formaldehyde scavenging that has been proposed to be the physiological action of ADH3. The human ADH5 and rodent ADH6 seem to be the corresponding enzymes due to their similar behavior. None of these latter ADHs have so far been assigned to any function. They can be expressed as recombinant proteins but no enzymatic activity has been detected.

Aims: Celecoxib is a novel selective cyclooxygenase-2 inhibitor, which is subject to extensive hepatic metabolism. The aims of the present in vitro investigation were 1) to compare the rate of celecoxib hydroxylation by different genetic variants of cytochrome P450 2C9 (CYP2C9), and 2) to identify the enzyme(s) involved in the formation of the major metabolite carboxycelecoxib., Methods: Hydroxycelecoxib formation was studied in human liver microsomes from 35 genotyped livers, as well as in yeast microsomes with recombinant expression of different P450 variants. Carboxycelecoxib formation was studied in liver microsomes incubated in the absence or presence of liver cytosol. The metabolites were identified and quantified by h.p.l.c. In addition, hydroxycelecoxib oxidation by different variants of recombinant human alcohol dehydrogenase (ADH1-3) was analysed by spectrophotometric monitoring of NADH generation from NAD+., Results: The intrinsic clearance of celecoxib hydroxylation was significantly lower for yeast-expressed CYP2C9.3 (0.14 ml min-1 nmol-1 enzyme) compared with CYP2C9.1 (0.44 ml min-1 nmol-1 enzyme). In human liver microsomes, a significant 2-fold decrease in the rate of hydroxycelecoxib formation was evident in CYP2C9*1/*3 samples compared with CYP2C9*1/*1 samples. There was also a marked reduction (up to 5.3 times) of hydroxycelecoxib formation in a liver sample genotyped as CYP2C9*3/*3. However, the CYP2C9*2 samples did not differ significantly from CYP2C9*1 in any of the systems studied. Inhibition experiments with sulphaphenazole (SPZ) or triacetyloleandomycin indicated that celecoxib hydroxylation in human liver microsomes was mainly dependent on CYP2C9 and not CYP3A4. The further oxidation of hydroxycelecoxib to carboxycelecoxib was completely dependent on liver cytosol and NAD+. Additional experiments showed that ADH1 and ADH2 catalysed this reaction in vitro with apparent K m values of 42 micro m and 10 micro m, respectively, whereas ADH3 showed no activity., Conclusions: The results confirm that CYP2C9 is the major enzyme for celecoxib hydroxylation in vitro and further indicate that the CYP2C9*3 allelic variant is associated with markedly slower metabolism. Furthermore, it was shown for the first time that carboxycelecoxib formation is dependent on cytosolic alcohol dehydrogenase, presumably ADH1 and/or ADH2.

The human alcohol dehydrogenase system is comprised of multiple forms that catalyse the oxidation/reduction of a large variety of alcohols and aldehydes. A transition that results in an Ile308Val substitution was identified in the human ADH2 gene by single-strand conformation polymorphism analysis. Screening a Swedish population revealed that Val308 was the most frequent allele (73%), and site-directed mutagenesis was used to obtain both allelozymes, which were expressed in Escherichia coli for characterisation. Thermostability was assayed by activity measurements and circular dichroism spectroscopy. The results showed that the 308Val substitution decreases protein stability, as compared to the Ile308 variant, an effect also demonstrated during prolonged storage. Ethanol, octanol, 12-hydroxydodecanoic acid and all-trans retinol were used as model substrates and, generally, slightly higher Km values were observed with Val at position 308. Finally, homology modelling, from mouse ADH2, further supported the decreased stability of the Val308 variant and located position 308 in the subunit interface of the molecule and in the vicinity of the active-site pocket entrance. In conclusion, the Ile308Val substitution represents a novel functional polymorphism within the human alcohol dehydrogenase gene cluster that may affect the metabolism of ethanol and other substrates.

The ADH3 gene encodes alcohol dehydrogenase 3 (ADH3)/glutathione-dependent formaldehyde dehydrogenase, the ancestral and most conserved form of alcohol dehydrogenase. ADH3 is expressed in all tissues examined and the enzyme is essential for formaldehyde scavenging. We have screened the promoter region including exon 1 and exons 5, 6 and 7 of the ADH3 gene for allelic variants. Using 80 samples of genomic DNA from Swedes as template, the various parts of the gene were PCR amplified and subsequently analyzed on single strand conformation polymorphism (SSCP) gels. No abnormal migration patterns could be detected by SSCP analysis of exons 5, 6 and 7 while for the promoter region, a large number of the samples displayed differences in SSCP gel migration patterns. Cloning and sequence analysis revealed four possible base pair exchanges in the promoter region. Two transitions were found at position -197 and -196, GG --> AA, one at position -79, G --> A and finally, close to the transcription start site, a fourth transition was found at position +9, C --> T. An allele specific PCR method was developed and allele frequencies were determined in three populations: Chinese, Spanish and Swedish. GG-197,-196 and AA-197,-196 alleles were common in all three populations, G-79 and A-79 were common in Swedes and Spaniards but only A-79 was found among Chinese. T+9 was the most rare allele with an allele frequency of 1.5% in Swedes. Finally, promoter activity assessments and electrophoretic mobility shift assays demonstrated that the C+9 --> T+9 exchange resulted in a significant transcriptional decrease in HeLa cells and a decreased binding of nuclear proteins. These base pair exchanges may have an effect on the expression of the enzyme and thereby influence the capacity of certain individuals to metabolize formaldehyde.

Enzymes involved in various protective and metabolic processes of carbonyl compounds were analysed utilising a micro-array method in a three-stage in vitro model for oral carcinogenesis involving cultured normal, immortalised and malignant human oral keratinocytes. A complete transcript profiling of identified carbonyl-metabolising enzymes belonging to the ADH, ALDH, SDR and AKR families is presented. Expression of 17 transcripts was detected in normal, 14 in immortalized and 19 in malignant keratinocytes of a total of 12,500 genes spotted on the micro-array chip. For the detected transcripts, about half were changed by cell transformation, and for the various enzyme families, differences in expression patterns were observed. The detected AKR transcripts displayed a conserved pattern of expression, indicating a requirement for the keratinocyte phenotype, while most of the detected SDRs displayed changed expression at the various stages of malignancy. The importance of multiple experiments in using a microarray technique for reliable results is underlined and, finally, the strength of the method in detecting co-expressed enzymes in metabolic pathways is exemplified by the detection of the formaldehyde-scavenging pathway enzymes and the polyol pathway enzymes.

The human oral epithelium is a target for damage from the inhalation of formaldehyde. However, most experimental studies on this chemical have relied on laboratory animals that are obligatory nose breathers, including rats and mice. Therefore, in vitro model systems that mimic the structure of the human oral epithelium and which retain normal tissue-specific metabolic competence are desirable. Based on the established role of alcohol dehydrogenase 3 (ADH3), also known as glutathione-dependent formaldehyde dehydrogenase, as the primary enzyme catalysing the detoxification of formaldehyde, the aim of this study was to investigate the expression of ADH3 in organotypic epithelia regenerated with normal (NOK), immortalised (SVpgC2a) and malignant (SqCC/Y1) human oral keratinocytes. Organotypic epithelia, usually consisting of 5-10 cell layers, were produced at the air-liquid interface of collagen gels containing human oral fibroblasts, after culture for 10 days in a standardised serum-free medium. Immunochemical staining demonstrated uniform expression of ADH3 in these organotypic epithelia, as well as in the epithelial cells of oral tissue. The specificity of the ADH3 antiserum was ascertained from the complete neutralisation of the immunochemical reaction with purified ADH3 protein. Assessment of the staining intensities indicated that the expression levels were similar among the regenerated epithelia. Furthermore, the regenerated epithelia showed similar ADH3 expression to the epithelium in oral tissue. Therefore, a tissue-like expression pattern for ADH3 can be generated from the culture of various oral keratinocyte lines in an organotypic state. Similar expression levels among the various cell lines indicate the preservation of ADH3 during malignant transformation, and therefore that NOK, SVpgC2a and SqCC/Y1 represent functional models for in vitro studies of formaldehyde metabolism in human oral mucosa.

Mammalian alcohol dehydrogenase (ADH) constitutes a complex system with different forms and extensive multiplicity (ADH1-ADH6) that catalyze the oxidation and reduction of a wide variety of alcohols and aldehydes. The ADH1 enzymes, the classical liver forms, are involved in several metabolic pathways beside the oxidation of ethanol, e.g. norepinephrine, dopamine, serotonin and bile acid metabolism. This class is also able to further oxidize aldehydes into the corresponding carboxylic acids, i.e. dismutation. ADH2, can be divided into two subgroups, one group consisting of the human enzyme together with a rabbit form and another consisting of the rodent forms. The rodent enzymes almost lack ethanol-oxidizing capacity in contrast to the human form, indicating that rodents are poor model systems for human ethanol metabolism. ADH3 (identical to glutathione-dependent formaldehyde dehydrogenase) is clearly the ancestral ADH form and S-hydroxymethylglutathione is the main physiological substrate, but the enzyme can still oxidize ethanol at high concentrations. ADH4 is solely extrahepatically expressed and is probably involved in first pass metabolism of ethanol beside its role in retinol metabolism. The higher classes, ADH5 and ADH6, have been poorly investigated and their substrate repertoire is unknown. The entire ADH system can be seen as a general detoxifying system for alcohols and aldehydes without generating toxic radicals in contrast to the cytochrome P450 system., (Copyright 2001 National Science Council, ROC and S. Karger AG, Basel)

The human ADH5 gene was reported to lack the last exon compared to other mammalian ADHs and consequently should be expressed as a truncated protein. Here we show with PCR amplification of 3'-cDNA ends that the ADH5 gene harbors the "missing" exon. Besides a cDNA identical to the published sequence, we found full-length transcripts that contained additional codons for eight amino acid residues. Northern blot analysis established the full-length variant as the major transcript with the strongest signal from adult liver. Sequence analysis of genomic DNA confirmed that the ADH5 gene displays composite internal/terminal exons, which can be differentially processed; i.e., 3'-end generation is a result of competition between polyadenylation and splicing., (Copyright 2000 Academic Press.)

Because formaldehyde exposure has been shown to induce pathological changes in human oral mucosa, eg, micronuclei, the potential enzymatic defense by alcohol dehydrogenase 3 (ADH3)/glutathione-dependent formaldehyde dehydrogenase was characterized in oral tissue specimens and cell lines using RNA hybridization and immunological methods as well as enzyme activity measurements. ADH3 mRNA was expressed in basal and parabasal cell layers of oral epithelium, whereas the protein was detected throughout the cell layers. ADH3 mRNA and protein were further detected in homogenates of oral tissue and various oral cell cultures, including, normal, SV40T antigen-immortalized, and tumor keratinocyte lines. Inhibition of the growth of normal keratinocytes by maintenance at confluency significantly decreased the amount of ADH3 mRNA, a transcript with a determined half-life of 7 hours. In contrast, decay of ADH3 protein was not observed throughout a 4-day period in normal keratinocytes. In samples from both tissue and cells, the ADH3 protein content correlated to oxidizing activity for the ADH3-specific substrate S:-hydroxymethylglutathione. The composite analyses associates ADH3 mRNA primarily to proliferative keratinocytes where it exhibits a comparatively short half-life. In contrast, the ADH3 protein is extremely stable, and consequently is retained during the keratinocyte life span in oral mucosa. Finally, substantial capacity for formaldehyde detoxification is shown from quantitative assessments of alcohol- and aldehyde-oxidizing activities including K:(m) determinations, indicating that ADH3 is the major enzyme involved in formaldehyde oxidation in oral mucosa.

The structure of mouse class II alcohol dehydrogenase (ADH2) has been determined in a binary complex with the coenzyme NADH and in a ternary complex with both NADH and the inhibitor N-cyclohexylformamide to 2.2 A and 2.1 A resolution, respectively. The ADH2 dimer is asymmetric in the crystal with different orientations of the catalytic domains relative to the coenzyme-binding domains in the two subunits, resulting in a slightly different closure of the active-site cleft. Both conformations are about half way between the open apo structure and the closed holo structure of horse ADH1, thus resembling that of ADH3. The semi-open conformation and structural differences around the active-site cleft contribute to a substantially different substrate-binding pocket architecture as compared to other classes of alcohol dehydrogenase, and provide the structural basis for recognition and selectivity of alcohols and quinones. The active-site cleft is more voluminous than that of ADH1 but not as open and funnel-shaped as that of ADH3. The loop with residues 296-301 from the coenzyme-binding domain is short, thus opening up the pocket towards the coenzyme. On the opposite side, the loop with residues 114-121 stretches out over the inter-domain cleft. A cavity is formed below this loop and adds an appendix to the substrate-binding pocket. Asp301 is positioned at the entrance of the pocket and may control the binding of omega-hydroxy fatty acids, which act as inhibitors rather than substrates. Mouse ADH2 is known as an inefficient ADH with a slow hydrogen-transfer step. By replacing Pro47 with His, the alcohol dehydrogenase activity is restored. Here, the structure of this P47H mutant was determined in complex with NADH to 2.5 A resolution. His47 is suitably positioned to act as a catalytic base in the deprotonation of the substrate. Moreover, in the more closed subunit, the coenzyme is allowed a position closer to the catalytic zinc. This is consistent with hydrogen transfer from an alcoholate intermediate where the Pro/His replacement focuses on the function of the enzyme., (Copyright 2000 Academic Press.)

Alcohol dehydrogenase (ADH) constitutes a complex enzyme system with different forms and extensive multiplicity. A combination of constant and variable properties regarding function, multiplicity and structure of ADH is highlighted for the human system and extended to ADH forms in general. Future perspectives suggest continued studies in specific directions for distinction of metabolic, regulatory and pharmacogenetic roles of ADH., (Copyright 2000 S. Karger AG, Basel)

3beta-Hydroxy (iso) bile acids are formed during enterohepatic circulation from 3alpha-hydroxy bile acids and constitute normal compounds in plasma but are virtually absent in bile. Isoursodeoxycholic acid (isoUDCA) is a major metabolite of UDCA. In a recent study it was found that after administration of isoUDCA, UDCA became the major acid in bile. Thus, epimerization of the 3beta-hydroxy to a 3alpha-hydroxy group, catalyzed by 3beta-hydroxysteroid dehydrogenases (HSD) and 3-oxo-reductases must occur. The present study aims to characterize the human liver bile acid 3beta-HSD. Human liver cytosol and recombinant alcohol dehydrogenase (ADH) betabeta and gammagamma isozymes were subjected to native polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing. Activity staining with oxidized nicotinamide adenine dinucleotide (NAD(+)) or oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)) as cofactors and various iso bile acids as substrates was used to screen for 3beta-HSD activity. Reaction products were identified and quantified by gas chromotography/mass spectrometry (GC/MS). Computer-assisted substrate docking of isoUDCA to the active site of a 3-dimensional model of human class I gammagamma ADH was performed. ADH gammagamma isozyme was identified as the iso bile acid 3beta-HSD present in human liver cytosol, with NAD(+) as a cofactor. Values for k(cat)/K(m) were in the rank order isodeoxycholic acid (isoDCA), isochenodeoxycholic acid (isoCDCA), isoUDCA, and isolithocholic acid (isoLCA) (0.10, 0.09, 0.08, and 0. 05 min(-1) x micromol/L(-1), respectively). IsoUDCA fits as substrate to the 3-dimensional model of the active-site of ADH gammagamma. ADH gammagamma isozyme was defined as the only bile acid 3beta-HSD in human liver cytosol. Hydroxysteroid dehydrogenases are candidates for the binding and transport of 3alpha-hydroxy bile acids. We assume that ADH gammagamma isozyme is involved in cytosolic bile acid binding and transport processes as well.

The metabolic interaction between ethanol and serotonin (5-hydroxytryptamine) via alcohol dehydrogenase (ADH; EC 1.1.1.1) was studied in tissue homogenates of Sprague-Dawley rats by following the transfer of deuterium from deuterated ethanol over endogenous NADH to 5-hydroxytryptophol (5HTOL). Homogenates of whole brain, lung, spleen, kidney, liver, stomach, jejunum, ileum, colon, and caecum were incubated in the presence of [2H2]ethanol and 5-hydroxyindole-3-acetaldehyde (5HIAL), and the [2H]5HTOL formed was identified and quantified using gas chromatography-mass spectrometry. ADH activity was most abundant in liver, kidney, and within the gastrointestinal tract. The highest incorporation of deuterium was obtained in homogenates of kidney, lung, and colon, whereas in brain, which contains very low ADH activity, no incorporation could be demonstrated. Addition of extra NAD+ (2.4 mM) increased the formation of [2H]5HTOL 2.6-fold in liver homogenates, but only 1.2-fold in kidney homogenates. 4-Methylpyrazole, a potent inhibitor of class I ADH, inhibited the 5HIAL reduction in homogenates of lung, kidney, jejunum, ileum, and colon, and caused a marked drop in 5HTOL oxidation in all tissues except stomach and spleen. These results demonstrate that in the rat a metabolic interaction between ethanol and serotonin via the ADH pathway may take place in several tissues besides the liver, which is the main tissue for ethanol detoxification.

Mice and rats were found to possess class II alcohol dehydrogenases with novel enzymatic and structural properties. A cDNA was isolated from mouse liver and the encoded alcohol dehydrogenase showed high identity (93.1%) with the rat class II alcohol dehydrogenase which stands in contrast to the pronounced overall variability of the class II line. The two heterologously expressed rodent class II enzymes exhibited over 100-fold lower catalytic efficiency (k(cat)/K(m)) for oxidation of alcohols as compared with other alcohol dehydrogenases and were not saturated with ethanol. Hydride transfer limited the rate of octanol oxidation as indicated by a deuterium isotope effect of 4.8. The mutation P47H improved hydride transfer and turnover rates were increased to the same level as for the human class II enzyme. Michaelis constants for alcohols and aldehydes were decreased while they were increased for the coenzyme. The rodent class II enzymes catalyzed reduction of p-benzoquinone with about the same maximal turnover as for the human form. This activity was not affected by the P47H mutation while a S182T mutation increased the K(m) value for benzoquinone 10-fold. omega-Hydroxy fatty acids were catalyzed extremely slow but functioned as potent inhibitors by binding to the enzyme-NAD(+) complex. All these data indicate that the mammalian class II alcohol dehydrogenase line is divided into two structurally and functionally distinct subgroups.

The alcohol dehydrogenase (ADH) gene family encodes enzymes that metabolize a wide variety of substrates, including ethanol, retinol, other aliphatic alcohols, hydroxysteroids, and lipid peroxidation products. Studies on 19 vertebrate animals have identified ADH orthologs across several species, and this has now led to questions of how best to name ADH proteins and genes. Seven distinct classes of vertebrate ADH encoded by non-orthologous genes have been defined based upon sequence homology as well as unique catalytic properties or gene expression patterns. Each class of vertebrate ADH shares <70% sequence identity with other classes of ADH in the same species. Classes may be further divided into multiple closely related isoenzymes sharing >80% sequence identity such as the case for class I ADH where humans have three class I ADH genes, horses have two, and mice have only one. Presented here is a nomenclature that uses the widely accepted vertebrate ADH class system as its basis. It follows the guidelines of human and mouse gene nomenclature committees, which recommend coordinating names across species boundaries and eliminating Roman numerals and Greek symbols. We recommend that enzyme subunits be referred to by the symbol "ADH" (alcohol dehydrogenase) followed by an Arabic number denoting the class; i.e. ADH1 for class I ADH. For genes we recommend the italicized root symbol "ADH" for human and "Adh" for mouse, followed by the appropriate Arabic number for the class; i.e. ADH1 or Adh1 for class I ADH genes. For organisms where multiple species-specific isoenzymes exist within a class, we recommend adding a capital letter after the Arabic number; i.e. ADH1A, ADH1B, and ADH1C for human alpha, beta, and gamma class I ADHs, respectively. This nomenclature will accommodate newly discovered members of the vertebrate ADH family, and will facilitate functional and evolutionary studies.

An ethanol-active medium-chain dehydrogenase/reductase (MDR) alcohol dehydrogenase was isolated and characterized from Escherichia coli. It is distinct from the fermentative alcohol dehydrogenase and the class III MDR alcohol dehydrogenase, both already known in E. coli. Instead, it is reminiscent of the MDR liver enzyme forms found in vertebrates and has a K(m) for ethanol of 0.7 mM, similar to that of the class I enzyme in humans, however, it has a very high k(cat), 4050 min(-1). It is also inhibited by pyrazole (K(i) = 0.2 microM) and 4-methylpyrazole (K(i)= 44 microM), but in a ratio that is the inverse of the inhibition of the human enzyme. The enzyme is even more efficient in the reverse direction of acetaldehyde reduction (K(m) = 30 microM and k(cat) = 9800 min(-1)), suggesting a physiological function like that seen for the fermentative non-MDR alcohol dehydrogenase. Growth parameters in complex media with and without ethanol show no difference. The structure corresponds to one of 12 new alcohol dehydrogenase homologs present as ORFs in the E. coli genome. Together with the previously known E. coli MDR forms (class III alcohol dehydrogenase, threonine dehydrogenase, zeta-crystallin, galactitol-1-phosphate dehydrogenase, sensor protein rspB) there is now known to be a minimum of 17 MDR enzymes coded for by the E. coli genome. The presence of this bacterial MDR ethanol dehydrogenase, with a structure compatible with an origin separate from that of yeast, plant and animal ethanol-active MDR forms, supports the view of repeated duplicatory origins of alcohol dehydrogenases and of functional convergence to ethanol/acetaldehyde activity. Furthermore, this enzyme is ethanol inducible in at least one E. coli strain, K12 TG1, with apparently maximal induction at an enthanol concentration of approximately 17 mM. Although present in several strains under different conditions, inducibility may constitute an explanation for the fairly late characterization of this E. coli gene product.

Alcohols and aldehydes in the metabolic pathways of ethanol and serotonin are substrates for alcohol dehydrogenases (ADH) of class I and II. In addition to the reversible alcohol oxidation/aldehyde reduction, these enzymes catalyse aldehyde oxidation. Class-I gammagamma ADH catalyses the dismutation of both acetaldehyde and 5-hydroxyindole-3-acetaldehyde (5-HIAL) into their corresponding alcohols and carboxylic acids. The turnover of acetaldehyde dismutation is high (kcat = 180 min-1) but saturation is reached first at high concentrations (Km = 30 mm) while dismutation of 5-HIAL is saturated at lower concentrations and is thereby more efficient (Km = 150 microm; kcat = 40 min-1). In a system where NAD+ is regenerated, the oxidation of 5-hydroxytryptophol to 5-hydroxyindole-3-acetic acid proceeds with concentration levels of the intermediary 5-HIAL expected for a two-step oxidation. Butanal and 5-HIAL oxidation is also observed for class-I ADH in the presence of NADH. The class-II enzyme is less efficient in aldehyde oxidation, and the ethanol-oxidation activity of this enzyme is competitively inhibited by acetate (Ki = 12 mm) and 5-hydroxyindole-3-acetic acid (Ki = 2 mm). Reduction of 5-HIAL is efficiently catalysed by class-I gammagamma ADH (kcat = 400 min-1; Km = 33 microm) in the presence of NADH. This indicates that the increased 5-hydroxytryptophol/5-hydroxyindole-3-acetic acid ratio observed after ethanol intake may be due to the increased NADH/NAD+ ratio on the class-I ADH.

Short-chain dehydrogenases/reductases (SDR) and medium-chain dehydrogenases/reductases (MDR) are protein families originally distinguished from characterisations of alcohol dehydrogenase of these two types. Screening of completed genome sequences now reveals that both these families are large, wide-spread and complex. In Escherichia coli alone, there are no fewer than 17 MDR forms, identified as open reading frames, considerably extending previously known MDR relationships in prokaryotes and including ethanol-active alcohol dehydrogenase. In entire databanks, 1056 SDR and 537 MDR forms are currently known, extending the multiplicity further. Complexity is also large, with several enzyme activity types, subgroups and evolutionary patterns. Repeated duplications can be traced for the alcohol dehydrogenases, with independent enzymogenesis of ethanol activity, showing a general importance of this enzyme activity.

Human class I alcohol dehydrogenase was mutated at positions 57 and 115, exchanging for Asp and Arg respectively, in an attempt to introduce glutathione-dependent formaldehyde dehydrogenase characteristics. In addition, class III alcohol dehydrogenase, identical to glutathione-dependent formaldehyde dehydrogenase, was mutated at position 115, introducing Ser or Lys. The attempted class transformation was partly successful considering a higher affinity for 12-hydroxydodecanoate and a lower affinity for ethanol that was monitored for the class I mutant. However, the class I mutant displayed neither glutathione-dependent formaldehyde dehydrogenase activity nor fatty acid activation of alcohol oxidation. Interestingly, both class III mutants showed reduced activities for S-hydroxymethylglutathione and 12-hydroxydodecanoate through increased Km, values. Overall results show that it is not possible, by single point mutations, to completely transform enzyme characteristics between these two classes of alcohol dehydrogenase.

cDNAs coding for class II alcohol dehydrogenase were isolated from a rabbit-liver cDNA library. Deduced amino acid sequences show that isozymic forms of rabbit class II alcohol dehydrogenase exist, with a positional identity of 88.4%. A high variability in structure of class II alcohol dehydrogenase between the species is also reflected in function. The rabbit II-1 isozyme shows common characteristics with the human enzyme, but has a lower Km value for ethanol, 4.2 mM. The II-2 isozyme shows restriction for aliphatic alcohols longer than pentanol. For shorter alcohols the II-2 form has similar Km values as the II-1 isozyme, 5.5 mM for ethanol, but is a low activity variant with a 10-fold decrease in k(cat) values compared with II-1. Nevertheless, II-2 has a higher specificity for benzoquinone than II-1 due to a lower Km value, 80 microM compared with 1 mM, and is in this sense more like the human class II enzyme. In addition a rabbit class III alcohol dehydrogenase cDNA was isolated that encodes a typical class III enzyme/glutathione-dependent formaldehyde dehydrogenase. The finding of isozymic forms of class II alcohol dehydrogenase is in line with the evolution of the system of medium-chain alcohol dehydrogenases with different enzymes, different classes and different isozymes and further underline the complexity of the entire mammalian alcohol dehydrogenase system.

We have previously shown that the rat Na(+)-K(+)-ATPase alpha 1-isoform is phosphorylated at Ser-943 by protein kinase A (PKA) and at Ser-23 by protein kinase C (PKC), which in both cases results in inhibition of enzyme activity. We now present evidence that suggests that the phosphorylation of Ser-943 by PKA modulates the response of Na(+)-K(+)-ATPase to PKC. Rat Na(+)-K(+)-ATPase alpha 1 or a mutant in which Ser-943 was changed to Ala-943 was stably expressed in COS cells. The inhibition of enzyme activity measured in response to treatment with the phorbol ester, phorbol 12,13-dibutyrate (PDBu; 10(-6) M), was significantly reduced in the cells expressing the Ala-943 mutant compared with that observed in cells expressing wild-type enzyme. In contrast, for cells expressing Na(+)-K(+)-ATPase alpha 1 in which Ser-943 was mutated to Asp-943, the effect of PDBu was slightly enhanced. The PDBu-induced inhibition was not mediated by activation of the adenosine 3',5'-cyclic monophosphate/PKA system and was not achieved via direct phosphorylation of Ser-943. Sp-5,6-DCI-cBIMPS, a specific PKA activator, increased the phosphorylation of Ser-943, and this was associated with an enhanced response to PDBu. Thus the effect of PKC on rat Na(+)-K(+)-ATPase alpha 1 is determined not only by the activity of PKC but also by the state of phosphorylation of Ser-943.

Tissue distribution of the five identified classes of human alcohol dehydrogenase was studied by assessment of mRNA levels in 23 adult and four fetal tissues. Alcohol dehydrogenase of class I was found in most tissues, brain and placenta excluded, but expression levels among tissues differed widely. The distribution pattern of class III transcripts was consistent with those of housekeeping enzymes while, in contrast, class IV transcripts were found only in stomach. Transcripts of multiple length were detected for most classes and were due to different gene products arising through the use of different poly-A signals or transcription from different gene loci. Both class II and class V showed a pattern of liver-enriched expression. However, low mRNA levels were detected also in stomach, pancreas and small intestine for class II, and in fetal kidney and small intestine for class V. Significantly higher levels of class V transcripts were present in fetal liver when compared with levels in adult liver, which suggests that human class V is a predominantly fetal alcohol dehydrogenase.

Human alcohol dehydrogenases of class I and class II but not class III catalyse NAD+-dependent aldehyde oxidation in addition to the NADH-dependent aldehyde reduction. The two reactions are coupled, i.e. the enzymes display dismutase activity. Dismutase activity of recombinantly expressed human class I isozymes beta1beta1 and gamma2gamma2, class II and class III alcohol dehydrogenases was assayed with butanal as substrate by gas chromatographic-mass spectrometric quantitations of butanol and butyric acid. The class I gamma2gamma2 isozyme showed a pronounced dismutase activity with a high kcat, 1300 min(-1), and a moderate Km, 1.2 mM. The class I beta1beta1 isozyme and the class II alcohol dehydrogenase showed moderate catalytic efficiencies for dismutase activity with lower kcat values, 60-75 min(-1). 4-Methylpyrazole, a potent class I ADH inhibitor, inhibited the class I dismutation completely, but cyanamide, an inhibitor of mitochondrial aldehyde dehydrogenase, did not affect the dismutation. The dismutase reaction might be important for metabolism of aldehydes during inhibition or lack of mitochondrial aldehyde dehydrogenase activity.

A serine protease has been isolated and characterized from Bacillus subtilis, strain RT-5 (a thermostable soil isolate from the Tharparkar desert of Pakistan) able to grow at 55 degrees C. The primary structure was established by a combination of protein and DNA-sequence analyses. The amino-acid sequence, inhibition pattern and solubility properties identify the enzyme as a subtilisin. It has 43 amino-acid replacements toward subtilisin BPN' and as much as 83 replacements toward another subtilisin, confirming that strain variabilities are extensive between different subtilisin forms. However, the structure is identical to one of unknown functional properties deduced from DNA and is closely related to mesentericopeptidase but that homologue is not thermostable. From comparisons with that form and with subtilisin BPN', it is concluded that replacements of Ala --> Ser at positions 85 and 89, Ser --> Ala at position 88 and Asp or Ser --> Asn at position 259 may promote thermostability.