Evaluation by capacitance measurements of antidiuretic hormone induced membrane area changes in toad bladder

A technique for estimating effective transepithelial capacitance in vitro was used to investigate changes in epithelial cell membrane area in response to antidiuretic hormone (ADH) exposure in toad bladder. The results indicate that transepithelial capacitance increases by about 30% within 30 min after serosal ADH addition and decreases with ADH removal. This capacitance change is not blocked by amiloride and occurs whether or not there is a transepithelial osmotic gradient. It is blocked by methohexital, a drug which specifically inhibits the hydro-osmotic response of toad bladder to ADH. We conclude that the hydro-osmotic response of toad bladder to ADH is accompanied by addition of membrane to the plasmalemma of epithelial cells. This new membrane may contain channels that are permeable to water. Stimulation of Na⁺ transport by ADH is not related to membrane area changes, but appears to reflect activation of Na⁺ channels already present in the cell membrane before ADH challenge.     

Effect of acetone feeding on alcohol dehydrogenase activity in the olive fruit fly,Bactrocera oleae

The purpose of this study is to demonstrate a clear connection between the presence of acetone in larval diet and alcohol dehydrogenase (ADH) activity in laboratory raised populations of Bactrocera oleae. ADH activity of B. oleae is depressed in acetone-impregnated diets. At the same time the change of activity is accompanied by a change in the relative proportions of the multiple forms of ADH. The bulk of activity in the most cathodally migrating form is lost, and all the activity becomes localized in the less cathodally migrating forms of the enzyme. Moreover, ADH activity, expressed in vivo, appears to drop after exposure to acetone, as shown by the fact that larvae become less sensitive to pentenol poisoning. Our results show clear selective differences imposed by acetone on three homozygous genotypes involving the ADH alleles F, S and I in B. oleae. The directions of these differences were found to vary with the fitness component under test. Acetone treatment seems to affect developmental time and larva's viability as well as allele frequencies of ADH under artificial rearing. The effect of acetone on the maintenance of ADH polymorphism in artificially reared populations of B. oleae is further discussed.     

A global perspective on genetic variation at the ADH genes reveals unusual patterns of linkage disequilibrium and diversity

Variants of different Class I alcohol dehydrogenase (ADH) genes have been shown to be associated with an effect that is protective against alcoholism. Previous work from our laboratory has shown that the two sites showing the association are in linkage disequilibrium and has identified the ADH1B Arg47His site as causative, with the ADH1C Ile349Val site showing association only because of the disequilibrium. Here, we describe an initial study of the nature of linkage disequilibrium and genetic variation, in population samples from different regions of the world, in a larger segment of the ADH cluster (including the three Class I ADH genes and ADH7). Linkage disequilibrium across approximately 40 kb of the Class I ADH cluster is moderate to strong in all population samples that we studied. We observed nominally significant pairwise linkage disequilibrium, in some populations, between the ADH7 site and some Class I ADH sites, at moderate values and at a molecular distance as great as 100 kb. Our data indicate (1) that most ADH-alcoholism association studies have failed to consider many sites in the ADH cluster that may harbor etiologically significant alleles and (2) that the relevance of the various ADH sites will be population dependent. Some individual sites in the Class I ADH cluster show Fst values that are among the highest seen among several dozen unlinked sites that were studied in the same subset of populations. The high Fst values can be attributed to the discrepant frequencies of specific alleles in eastern Asia relative to those in other regions of the world. These alleles are part of a single haplotype that exists at high (>65%) frequency only in the eastern-Asian samples. It seems unlikely that this haplotype, which is rare or unobserved in other populations, reached such high frequency because of random genetic drift alone.     

Effect of central adrenergic alpha receptor blockader IEM-611 on the activity of alcohol and aldehyde dehydrogenase in rat liver

The effect of IEM-611 (30 mg/kg) on alcohol consumption in rats under the conditions of voluntary choice between water and 15% ethanol was studied as that on alcohol dehydrogenase (ADH) in postmitochondrial supernatant and in NAD-dependent aldehyde dehydrogenases (A1DH) in liver mitochondria. Administration of IEM-611 during 6 or 12 days reduces ethanol consumption by 29 and 30%, respectively, activates ADH and appreciably decreases overall activity of NAD-dependent A1DH. At the same time the ADH/A1DH ratio increases. Activation of ADH and A1DH and the decreased ADH/A1DH ratio were disclosed in alcohol preferring rats as compared to water preferring animals. IEM-611 shifts enzymatic activity of ethanol metabolism towards the level characteristic for water preferring rats. It is suggested that variation of the ADH/A1DH ratio is one of the mechanisms responsible for the decreased ethanol consumption in rats.     

Effects of elemental sulfur on the metabolism of the deep-sea hyperthermophilic archaeon Thermococcus strain ES-1: characterization of a sulfur-regulated, non-heme iron alcohol dehydrogenase.

The strictly anaerobic archaeon Thermococcus strain ES-1 was recently isolated from near a deep-sea hydrothermal vent. It grows at temperatures up to 91 degrees C by the fermentation of peptides and reduces elemental sulfur (S(o)) to H2S. It is shown here that the growth rates and cell yields of strain ES-1 are dependent upon the concentration of S(o) in the medium, and no growth was observed in the absence of S(o). The activities of various catabolic enzymes in cells grown under conditions of sufficient and limiting S(o) concentrations were investigated. These enzymes included alcohol dehydrogenase (ADH); formate benzyl viologen oxidoreductase; hydrogenase; glutamate dehydrogenase; alanine dehydrogenase; aldehyde ferredoxin (Fd) oxidoreductase; formaldehyde Fd oxidoreductase; and coenzyme A-dependent, Fd-linked oxidoreductases specific for pyruvate, indolepyruvate, 2-ketoglutarate, and 2-ketoisovalerate. Of these, changes were observed only with ADH, formate benzyl viologen oxidoreductase, and hydrogenase, the specific activities of which all dramatically increased in cells grown under S(o) limitation. This was accompanied by increased amounts of H2 and alcohol (ethanol and butanol) from cultures grown with limiting S(o). Such cells were used to purify ADH to electrophoretic homogeneity. ADH is a homotetramer with a subunit M(r) of 46,000 and contains 1 g-atom of Fe per subunit, which, as determined by electron paramagnetic resonance analyses, is present as a mixture of ferrous and ferric forms. No other metals or acid-labile sulfide was detected by colorimetric and elemental analyses. ADH utilized NADP(H) as a cofactor and preferentially catalyzed aldehyde reduction. It is proposed that, under So limitation, ADH reduces to alcohols the aldehydes that are generated by fermentation, thereby serving to dispose of excess reductant.     

Polymorphism of class I alcohol dehydrogenase in French, Vietnamese and Niger populations: genotyping by PCR amplification and RFLP analysis on dried blood spots.

The polymorphism of human alcohol dehydrogenase (ADH) can contribute to the explanation of the important ethnic differences towards alcohol metabolism. Its assessment at the genomic DNA level with a procedure, excluding labelled probes, consisting of PCR (Polymerase chain reaction) amplification on dried blood spots and analysis of allele-specific RFLP (Restriction fragment length polymorphism) profiles, is well adapted to extensive studies in population samples. It can emphasize the importance of ADH as a genetic marker of population. Three ethnic groups (French Caucasians, Vietnamese Orientals, Black Africans from Niger) were studied. ADH2 and ADH3 genotypes were in equilibrium according to the Hardy-Weinberg law. Important differences were noted in the distribution of ADH2 and ADH3 alleles.     

Effect of colchicine and Triton X-100 on expression of the enzyme-encoding genes in nongerminating seeds of sugarbeet (Beta vulgaris L.)

The expression of the enzyme-coding genes, controlling glucose-phosphate isomerase (GPI), malate dehydrogenase (MDH), and alcohol dehydrogenase (ADH), was examined in nongerminating seeds of sugarbeet after Triton X-100 (TX-100) and colchicine treatment. Two types of changes revealed included modification of the enzymatic loci expression (change of the isozyme electrophoretic mobility) and inactivation of standard profiles. In the MDH and GPI systems, these processes were found to be associated. Complete isozyme modification was accompanied with the disappearance of standard profiles. In the ADH system, the treatment with TX-100 and colchicine gave rise to two independent processes, including silencing of the Adh1 locus and the appearance of the ADH isozymes with abnormal electrophoretic mobility, which were probably the products of the Adh2 locus. It was suggested that the effect of TX-100 and colchicine on the expression of the enzyme-encoding genes examined depended on the intracellular localization of the encoded enzymes.     

Physiological regulation of the properties of alcohol dehydrogenase II (ADH II) of <Emphasis Type="Italic">Zymomonas mobilis</Emphasis>: NADH renders ADH II resistant to cyanide and aeration

The variable cyanide-sensitivity of the iron-containing alcohol dehydrogenase isoenzyme (ADH II) of the ethanol-producing bacterium Zymomonas mobilis was studied. In aerobically grown permeabilized cells, cyanide caused gradual inhibition of ADH II, which was largely prevented by externally added NADH. Cyanide-sensitivity of ADH II was highest in cells grown under conditions of vigorous aeration, in which intracellular NADH concentration was low. Anaerobically grown bacteria, as well as those cultivated aerobically in the presence of cyanide, maintained higher intracellular NADH levels along with a more cyanide-resistant ADH II. It was demonstrated that cyanide acted as a competitive inhibitor of ADH II, competing with nicotinamide nucleotides. NADH increased both cyanide-resistance and oxygen-resistance of ADH II.     

Intracellular calcium as a modulator of transepithelial permeability to water in frog urinary bladder

The divalent cation ionophore A 23187 was used to evaluate the action of intracellular calcium on net transepithelial water movement across the isolated frog urinary bladder. Incubation with the ionophore increases the net basal water flux in a dose-dependent fashion but independent of the extracellular calcium concentration. Bladders pretreated with A 23187 and exposed thereafter to an increase in calcium concentration exhibit a water permeability that under certain conditions can be comparable to that achieved with antidiuretic hormone (ADH). Lowering the serosal calcium at the peak of the hydrosmotic responses to both ADH and A 23187 inhibited the maintenance of the net water flux. The action of a supramaximal dose of ADH is blunted in bladders pretreated with A 23187, while the hydrosmotic effects of a submaximal dose are enhanced when the ionophore is added together with the hormone. The results show that an increase in transepithelial water movement can be triggered by calcium and that serosal calcium is needed to sustain the response. This hydrosmotic response may be dependent upon the rate at which intracellular calcium concentrations change and on the absolute concentration attained. It is suggested that calcium is involved in the action of ADH on water permeability and may act as a modulator of the hydrosmotic response.     

Class II alcohol dehydrogenase (ADH2)--adding the structure

Class II alcohol dehydrogenase (ADH2) represents a highly divergent class of alcohol dehydrogenases predominantly found in liver. Several species variants of ADH2 have been described, and the rodent enzymes form a functionally distinct subgroup with interesting catalytic properties. First, as compared with other ADHs, the catalytic efficiency is low for this subgroup. Second, the substrate repertoire is unique, e.g. rodent ADH2s are not saturated with ethanol as substrate, and while omega-hydroxy fatty acids are common substrates for the human ADH1-ADH4 isoenzymes, including ADH2, these compounds function as inhibitors rather than substrates. The recently determined structure of mouse ADH2 reveals a novel substrate-pocket topography that accounts for the observed substrate specificity and may, therefore, be important for the exploration of orphan substrates of ADH2. It is possible to improve the catalytic efficiency of mouse ADH2 by an array of mutations at position 47. Residue Pro47 of the wild type ADH2 enzyme seems to strain the binding of coenzyme, which prevents a close approach between the coenzyme and substrate for efficient hydrogen transfer. Based on crystallographic and mechanistic investigations, the effects of residue replacements at position 47 are multiple, affecting the distance for hydride transfer, the pK(a) of the bound alcohol substrate as well as the affinity for coenzyme.     

Cloning and sequencing of a processed pseudogene derived from a human class III alcohol dehydrogenase gene

Current information on the molecular structure of human alcohol dehydrogenase (ADH) genes is fragmentary. To characterize all ADH genes, we have isolated 63 ADH clones from human genomic libraries made from one individual. Fifty-nine clones have been classified into five previously known loci: ADH1 (18 clones), ADH2 (20 clones), and ADH3 class I (16 clones), ADH4 class II (4 clones), and ADH5 class III (1 clone). Sequencing of one of the remaining four unclassified clones, SY lambda ADHE38, about 1.1 kb in length, shows no introns and three frameshift mutations in the coding region, with a total of 10 internal termination codons. When its deduced amino acid sequence was compared with those of the class I, class II, and class III ADHs, the proportions of identical amino acids were 56.7%, 55.5%, and 88.7%, respectively, suggesting that the processed pseudogene was derived from an ADH5 gene. The duplication event seems to have occurred about 3.5 million years ago, and the pseudogene has undergone a rapid change since then.     

Crystal structure of the vertebrate NADP(H)-dependent alcohol dehydrogenase (ADH8)

The amphibian enzyme ADH8, previously named class IV-like, is the only known vertebrate alcohol dehydrogenase (ADH) with specificity towards NADP(H). The three-dimensional structures of ADH8 and of the binary complex ADH8-NADP(+) have been now determined and refined to resolutions of 2.2A and 1.8A, respectively. The coenzyme and substrate specificity of ADH8, that has 50-65% sequence identity with vertebrate NAD(H)-dependent ADHs, suggest a role in aldehyde reduction probably as a retinal reductase. The large volume of the substrate-binding pocket can explain both the high catalytic efficiency of ADH8 with retinoids and the high K(m) value for ethanol. Preference of NADP(H) appears to be achieved by the presence in ADH8 of the triad Gly223-Thr224-His225 and the recruitment of conserved Lys228, which define a binding pocket for the terminal phosphate group of the cofactor. NADP(H) binds to ADH8 in an extended conformation that superimposes well with the NAD(H) molecules found in NAD(H)-dependent ADH complexes. No additional reshaping of the dinucleotide-binding site is observed which explains why NAD(H) can also be used as a cofactor by ADH8. The structural features support the classification of ADH8 as an independent ADH class.     

Class II alcohol dehydrogenase (ADH2) — adding the structure

Class II alcohol dehydrogenase (ADH2) represents a highly divergent class of alcohol dehydrogenases predominantly found in liver. Several species variants of ADH2 have been described, and the rodent enzymes form a functionally distinct subgroup with interesting catalytic properties. First, as compared with other ADHs, the catalytic efficiency is low for this subgroup. Second, the substrate repertoire is unique, e.g. rodent ADH2s are not saturated with ethanol as substrate, and while ω-hydroxy fatty acids are common substrates for the human ADH1–ADH4 isoenzymes, including ADH2, these compounds function as inhibitors rather than substrates. The recently determined structure of mouse ADH2 reveals a novel substrate-pocket topography that accounts for the observed substrate specificity and may, therefore, be important for the exploration of orphan substrates of ADH2. It is possible to improve the catalytic efficiency of mouse ADH2 by an array of mutations at position 47. Residue Pro47 of the wild type ADH2 enzyme seems to strain the binding of coenzyme, which prevents a close approach between the coenzyme and substrate for efficient hydrogen transfer. Based on crystallographic and mechanistic investigations, the effects of residue replacements at position 47 are multiple, affecting the distance for hydride transfer, the pK_a of the bound alcohol substrate as well as the affinity for coenzyme.     

Alcohol dehydrogenase (ADH) isozymes in the AdhN/AdhN strain ofPeromyscus maniculatus (ADH− deermouse) and a possible role of class III ADH in alcohol metabolism

Although the Adh^N/Adh^N strain ofPeromyscus maniculatus (so-called ADH^? deermouse) has been previously considered to be deficient in ADH, we found ADH isozymes of Classes II and III but not Class I in the liver of this strain. On the other hand, the Adh^F/Adh^F strain (so-called ADH⁺ deermouse), which has liver ADH activity, had Class I and III but not Class II ADH in the liver. In the stomach, Class III and IV ADHs were detected in both deermouse strains, as well as in the ddY mouse, which has the normal mammalian ADH system with four classes of ADH. These ADH isozymes were identified as electrophoretic phenotypes on the basis of their substrate specificity, pyrazole sensitivity, and immunoreactivity. Liver ADH activity of the ADH^? strain was barely detectable in a conventional ADH assay using 15 mM ethanol as substrate; however, it increased markedly with high concentrations of ethanol (up to 3M) or hexenol (7 mM). Furthermore, in a hydrophobic reaction medium containing 1.0M t-butanol, liver ADH activity of this strain at low concentrations of ethanol (<100 mM) greatly increased (about sevenfold), to more than 50% that of ADH⁺ deermouse. These results were attributable to the presence of Class III ADH and the absence of Class I ADH in the liver of ADH^? deermouse. It was also found that even the ADH⁺ strain has low liver ADH activity (<40% that of the ddY mouse) with 15 mM ethanol as substrate, probably due to low activity in Class I ADH. Consequently, liver ADH activity of this strain was lower than its stomach ADH activity, in contrast with the ddY mouse, whose ADH activity was much higher in the liver than in the stomach, as well as other mammals. Thus, the ADH systems in both ADH^? and ADH⁺ deermouse were different not only from each other but also from that in the ddY mouse; the ADH^? strain was deficient in only Class I ADH, and the ADH⁺ strain was deficient in Class II ADH and down-regulated in Class I ADH activity. Therefore, Class III ADH, which was found in both strains and activated allosterically, may participate in alcohol metabolism in deermouse, especially in the ADH^? strain.     

Opportunities for natural selection on DNA and protein at the Adh locus in Drosophila melanogaster

A study was made of environmental and genetic factors affecting the quantity and disposition of the alcohol dehydrogenase (ADH) protein in Drosophila melanogaster. It was found that the amount of enzyme per fly is greatly influenced by the environmental conditions in which it develops. A critical factor is the concentration of yeast in the medium. A high concentration of yeast can double the quantity of ADH. The yeast appears to act through the provision of protein, and the protein to act through the provision of threonine, which is already known to induce ADH in fungi. Various genetic factors affect the quantity of enzyme. Males have more ADH than females. Files homozygous for the Fast allele have more ADH than those homozygous for the slow allele, and the difference is greater in females than in males. One particular line (ve), homozygous for Slow, has approximately half the normal quantity of enzyme, and the quantity segregates with the electrophoretic allele. Lines differ in the relative amounts of ADH in the gut (including Malpighian tubules) and the fat body. In general it seems that slow lines have relatively more enzyme in the fat body. In a cross between ve and a line homozygous to Fast, the difference in tissue distribution segregated with the electrophoretic allele. It is argued, but not demonstrated, that the differences in quantity and tissue distribution are due to nucleotide substitutions in noncoding regions close to, or within, the structural gene. It seems likely that the observed environmental and genetic differences in the quantity and disposition of ADH will influence the relative selective values of the electrophoretic genotypes.     

A tightly bound quinone functions in the ubiquinone reaction sites of quinoprotein alcohol dehydrogenase of an acetic acid bacterium, Gluconobacter suboxydans

Quinoprotein alcohol dehydrogenase (ADH) of acetic acid bacteria is a membrane-bound enzyme that functions as the primary dehydrogenase in the ethanol oxidase respiratory chain. It consists of three subunits and has a pyrroloquinoline quinone (PQQ) in the active site and four heme c moieties as electron transfer mediators. Of these, three heme c sites and a further site have been found to be involved in ubiquinone (Q) reduction and ubiquinol (QH2) oxidation respectively (Matsushita et al., Biochim. Biophys. Acta, 1409, 154-164 (1999)). In this study, it was found that ADH solubilized and purified with dodecyl maltoside, but not with Triton X-100, had a tightly bound Q, and thus two different ADHs, one having the tightly bound Q (Q-bound ADH) and Q-free ADH, could be obtained. The Q-binding sites of both the ADHs were characterized using specific inhibitors, a substituted phenol PC16 (a Q analog inhibitor) and antimycin A. Based on the inhibition kinetics of Q2 reductase and ubiquinol-2 (Q2H2) oxidase activities, it was suggested that there are one and two PC16-binding sites in Q-bound ADH and Q-free ADH respectively. On the other hand, with antimycin A, only one binding site was found for Q2 reductase and Q2H2 oxidase activities, irrespective of the presence of bound Q. These results suggest that ADH has a high-affinity Q binding site (QH) besides low-affinity Q reduction and QH2 oxidation sites, and that the bound Q in the QH site is involved in the electron transfer between heme c moieties and bulk Q or QH2 in the low-affinity sites.     

Antidiuretic action of prolactin in the rat with diabetes insipidus.

Rats with hereditary hypothalamic diabetes insipidus, devoid of endogenous ADH, exhibited a prompt antidiuresis when injected subcutaneously or intraarterially with ovine prolactin. The antidiuresis was accompanied by a decrease in free water clearance and an increase in urine osmolality without a change in osmolal clearance or creatinine excretion. Measurement of PAH and insulin clearances indicated that prolactin had no effect on renal plasma flow or glomerular filtration rate. Prolactin injection caused a transient decrease in urinary sodium excretion, but proximal tubular sodium reabsorption, estimated by lissamine green transit time, was unaffected. The antidiuretic effect of prolactin could not be attributed to ADH contamination of the ovine prolactin preparation. Kidney cyclic AMP content was increased significantly 5 min after injection of prolactin. Thus, prolactin has an antidiuretic effect similar to that which occurs as a result of ADH action on the kidney and does not require either the release or the presence of ADH in order to cause the antidiuresis. Further, the impaired water excretion cannot be attributed to an increase in proximal tubular sodium reabsorption or to alteration of renal hemodynamics. It is suggested that prolactin has a direct ADH-like action on the kidney resulting in antidiuresis.