S-nitrosoglutathione reductase activity of human and yeast glutathione-dependent formaldehyde dehydrogenase and its nuclear and cytoplasmic localisation

S-nitrosoglutathione (GSNO) formation represents a mechanism for storage and transport of nitric oxide. Analysis of human liver and Saccharomyces cerevisiae extracts has revealed the presence of only one enzyme able to significantly reduce GSNO, identified as glutathione-dependent formaldehyde dehydrogenase (FALDH). GSNO is the best substrate known for the human and yeast enzymes (kcat/Km = 444,400 and 350,000 mM(-1) min(-1), respectively). Although NADH is the preferred cofactor, some activity with NADPH (Km = 460 microM) can be predicted in vivo. The subcellular localization demonstrates a cytosolic and nuclear distribution of FALDH in living yeast cells. This agrees with previous results in rat, and suggests a role in the regulation of GSNO levels in the cytoplasmic and nuclear compartments of the eukaryotic cell.     

Enrichment of ligands with molecular dockings and subsequent characterization for human alcohol dehydrogenase 3

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 2 is a major hepatic enzyme for human retinol metabolism

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 μM 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. Received 12 October 2006; received after revision 6 December 2006; accepted 8 January 2007     

Medium- and short-chain dehydrogenase/reductase gene and protein families

The MDR superfamily with ~350-residue subunits contains the classical liver alcohol dehydrogenase (ADH), quinone reductase, leukotriene B4 dehydrogenase and many more forms. ADH is a dimeric zinc metalloprotein and occurs as five different classes in humans, resulting from gene duplications during vertebrate evolution, the first one traced to ~500 MYA (million years ago) from an ancestral formaldehyde dehydrogenase line. Like many duplications at that time, it correlates with enzymogenesis of new activities, contributing to conditions for emergence of vertebrate land life from osseous fish. The speed of changes correlates with function, as do differential evolutionary patterns in separate segments. Subsequent recognitions now define at least 40 human MDR members in the Uniprot database (corresponding to 25 genes when excluding close homologues), and in all species at least 10888 entries. Overall, variability is large, but like for many dehydrogenases, subdivided into constant and variable forms, corresponding to household and emerging enzyme activities, respectively. This review covers basic facts and describes eight large MDR families and nine smaller families. Combined, they have specific substrates in metabolic pathways, some with wide substrate specificity, and several with little known functions.     

Differential multiplicity of MDR alcohol dehydrogenases: enzyme genes in the human genome versus those in organisms initially studied

Screens were made for alcohol dehydrogenase (ADH) of the classical type (the MDR superfamily) in translations of human and other relevant genomes, corresponding to the organism types from which the enzyme was initially purified. Considerable multiplicities were detected in the dimeric enzymes from higher eukaryotes: seven forms in the human (plus three pseudogenes), all genes on chromosome 4, in the order class IV --> class Igamma --> class Ibeta --> class Ialpha --> class V --> class II --> class III, and eight forms in Arabidopsis thaliana (plus one pseudogene). These multiplicity patterns, and the species variability in the animal (human/mouse) and plant (Arabidopsis/pea) lines, suggest parallel but separate duplicatory events, giving rise to three families of dimeric MDR-ADH: class III, the animal non-class III, and the plant non-class III enzymes, with functions in formaldehyde elimination, in alcohol/aldehyde detoxication and in special pathways in higher eukaryotes. Multiplicity, although to a lesser extent, was also noted in tetrameric MDR-ADH, suggesting functional divergence between the dimeric and tetrameric enzymes. Combining these observations, at least five levels of divergence are reflected in the present ADH forms, corresponding to nodes at the SDR/MDR, the dimer/tetramer, the class III/non-class III, the class I/P, and the more recent class splits, each branch associated with separate functional patterns.     

Peroxovanadate inhibits Ca2+ release from mitochondria

Mitochondria contain a specific Ca²⁺ release pathway which operates when oxidized mitochondrial pyridine nucleotides are hydrolyzed. NAD⁺ hydrolysis and therefore Ca²⁺ release is possible when some vicinal thiols are cross-linked. Here we report that the thiol oxidant peroxovanadate inhibits the specific Ca²⁺ release pathway. In mitochondria, peroxovanadate causes a complete loss of reduced glutathione, which is not accompanied by formation of glutathione disulfide, and a partial loss of protein thiols. In model reactions, peroxovanadate oxidizes reduced glutathione predominantly to the sulfonate derivative, but does not react with glutathione disulfide. When the vicinal thiols relevant for Ca²⁺ release are cross-linked, Ca²⁺ release is no longer inhibited by peroxovanadate. Conversely, pretreatment of mitochondria with peroxovanadate makes them insensitive to compounds promoting the disulfide state. These results suggest that peroxovanadate inhibits the prooxidant-induced Ca²⁺ release from mitochondria by (i) depleting mitochondria of reduced glutathione and (ii) oxidizing the vicinal thiols relevant for Ca²⁺ release to a state higher than disulfide, presumably the sulfonate state. The findings provide further insight into the regulation of Ca²⁺ release from intact mitochondria, and may be relevant for a better understanding of the action of peroxovanadate in cells, where the compound can be insulin mimetic. Received 28 March 2002; received after revision 8 May 2002; accepted 15 May 2002     

Genotype-isopropanol interaction in theAdh locus ofDrosophila buzzatii

Summary Drosophila buzzatii, when reared in a medium with isopropanol, shows a significant band interconversion in ADH zymograms and a general lowering of ADH activity. Changes in activity are greater inAdh ^F homozygotes than inAdh ^S homozygotes and generate a significant genotype-isopropanol interaction. These mechanisms are relevant to an explanation of the high naturalAdh polymorphism of this species.This work has been partly supported by a fellowship from the Instituto Nacional para la Asistencia y Promoción del Estudiante (INAPE). We are much indebted to the personnel of the Departamento de Bioquímica, Facultad de Farmacia, Universidad de Santiago de Compostela, for their help in using the facilities at that Department.     

Biochemical polymorphism and the 2,3-diphosphoglycerate in the sheep red blood cells

Summary There was no significant difference in the level of 2,3-DPG in the red blood cells of sheep of different haemoglobin types (Hb A and Hb B) or potassium types (HK and LK). However, low glutathione (GSH^L) sheep had significantly higher (p<0.01) level of 2,3-DPG in their red blood cells than high glutathione (GSH^H) sheep. There was also significant effect of interactions between glutathione, haemoglobin and potassium types (p<0.05) and glutathione and haemoglobin types (p<0.01) on red cell 2,3-DPG levels.     

The effect of NADH and NAD+ on the proteolysis of lactate dehydrogenase isozymes by trypsin

Résumé L'effet de la NADH et NAD⁺ sur la protéolyse de la lacticodéhydrogénase isoenzyme 1 (LDH 1) et isoenzyme 5 (LDH 5) par la trypsine fut étudié. Les résultats montrent que la NAD⁺ exerce une protection contre la protéolyse de la LDH 1. Mais ni la NADH ni la NAD⁺ ne protègent contre la protéolyse de la LDH 5.     

Chromate reduction inStreptomyces

Summary Streptomyces species 3M grew in peptone yeast extract medium with 1000 g/ml K₂Cr₂O₇. Incubation of the chromate with different cell fractions in the presence of NADH and NADPH resulted in a decrease of Cr⁶⁺ in the reaction mixture. The level of Cr⁶⁺ was reduced by 82.7% by a particulate cell fraction obtained by centrifugation at 105,000×g for 1 h, in the presence of NADH. The reducing enzyme was associated with this cell fraction. The enzyme was constitutive and reduced Cr⁶⁺ to Cr³⁺.     

Nicotinoprotein (NAD+ -containing) alcohol dehydrogenase: structural relationships and functional interpretations

The primary structure of nicotinoprotein alcohol dehydrogenase (ADH) from Amycolatopsis methanolica was determined and used for modelling against known ADH structures, and for evaluation of the coenzyme binding. The results establish the medium-chain dehydrogenase/reductase nature of the nicotinoprotein ADH. Its subunit model and that of the human class Ibeta ADH subunit structure are similar, with mean a carbon deviations of 0.95 A, but they differ in seven loops. Nicotinoprotein ADH occupies a phylogenetic position intermediate between the dimeric and tetrameric ADH families. Two of the differing loops are important for coenzyme binding in the nicotinoprotein model, where one (with a Thr271Arg exchange towards the traditional enzyme) may suggest a slight rotation of the coenzyme adenine ring in the nicotinoprotein, and the other, with an Asn288 insertion, may suggest an extra hydrogen bond to its nicotinamide ribose, favouring stronger binding of the coenzyme. Combined with previous data, this suggests differences in the details of the tight coenzyme binding in different nicotinoproteins, but a common mode for this binding by loop differences.     

Stimulation of sodium transport by antidiuretic hormone in bullfrog intestine

Summary Antidiuretic hormone (ADH) increased the potential difference (PD) and shortcircuit current (SCC) across the small intestine of the bullfrog. This effect was independent of those produced by amiloride or high calcium but was masked by a theophylline-induced effect. Net active sodium (Na⁺) absorption accounted for the observed electrical changes.This work was supported by Whitehall Foundation, grant No. 78 156 ck-1 DSR No.     

Identification and characterisation of two allelic forms of human alcohol dehydrogenase 2

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.     

Drug modification of silver-induced sodium transport across toad skin

Summary Stimulation of active Na⁺ transport in the toad skin by antidiuretic hormone (ADH) and p-chloromecuribenzoate (P-CMB) was blunted by the presence of silver (Ag⁺). Amiloride inhibited active Na⁺ transport, equivalently, whether Ag⁺ was present or not.This work was supported by the Nephrology Training grant from the National Institute of Arthritis and Metabolic Diseases (1-TO1-AM-05697-02 and –03) and by Whitehall Foundation grant No. 78-156 ck-1 DSR.     

Glutathione plays different roles in the induction of the cytotoxic effects of inorganic and organic arsenic compounds in cultured BALB/c 3T3 cells

The cytotoxicity of arsenic compounds towards BALB/c 3T3 cells in culture was investigated, together with the role of glutathione (GSH) in the induction of the cytotoxic effects. The rank order of cytotoxicity was as follows: arsenite (As³⁺)>arsenate (As⁵⁺)>dimethylarsinic acid (DMAA)>methylarsonic acid (MAA)>trimethylarsine oxide (TMAO). Arsenobetaine, arsenocholine and the tetramethylarsonium ion were less toxic. Depletion of GSH enhanced the cytotoxic effects of As³⁺, As⁵⁺, MAA and TMAO, while the cytotoxicity of DMAA was markedly reduced by depletion of GSH. These results suggest that GSH plays a role in protecting the cells against the toxic effects of As³⁺, As⁵⁺, MAA and TMAO while it is involved in the induction of the cytotoxic effects of DMAA.     

Genetic variation at the alcohol dehydrogenase locus inDrosophila melanogaster: A third ubiquitous allele

Summary A 3rd allele at theAdh locus,Adh ^FCh.D., has been found at polymorphic frequencies in natural populations ofD. melanogaster. The ADH-FChD enzyme has properties distinct from those of the 2 more common forms of ADH. TheAdh polymorphism should now be analyzed as a triallelic system.     

Comparison of the production rates of bacteria in the rumen estimated by using labelled live and formaldehyde treated mixed bacterial cells

Summary The bacteria production rates in the rumen have been estimated by injecting¹⁴C- and³⁵S-labelled mixed rumen bacteria, either live or killed by treatment with formaldehyde, into the rumen and applying isotope dilution technique. The rate of bacteria production when estimated by using either live- or dead-(protected-)labelled bacterial cells were comparable.     

Some properties of external NADH oxidation by human placental mitochondria

Summary Isolated human term placenta mitochondria catalyse oxidation of external NADH in the presence of cytochrome c. This reaction is insensitive to the respiratory chain inhibitors such as rotenone and antimycin A, and is not coupled to phosphorylation. Comparison of the effect of Mg⁺⁺ ion on NADH plus cytochrome c oxidation by human term placental, human skeletal muscle and rat skeletal mitochondria showed that Mg⁺⁺ ion exerts an inhibitory effect in the case of human mitochondria and a stimulatory effect in the case of rat skeletal muscle mitochondria.This work has been supported by a grant from Ministry of Higher Education Science and Technology within the project No. 01.02.     

Activity tests of alcohol dehydrogenases in wheat,rye and triticale

Summary The relative band staining intensities of ADH isoenzymes in wheat and triticale suggest alloploid genome interactions. Rye ADH is scarcely affected by anti-wheat-ADH. Despite the evolutionary divergence of their Adh genes, ADH monomers of wheat and rye form enzymatically active heterodimers in triticale.Dedicated to Prof. Dr V. Schwartz, Tübingen, on the occasion of his 70th birthday.I thank Miss Hiltrud Wambach and Miss Rita Lippert for technical assistance.