George E.W. Thörig

A dual function of alcohol dehydrogenase in Drosophila

Alcohol dehydrogenase (ADH) of Drosophila not only catalyzes the oxidation of ethanol to acetaldehyde, but additionally catalyzes the conversion of this highly toxic product into acetate. This mechanism is demonstrated by using three different methods. After electrophoresis the oxidation of acetaldehyde is shown in an NAD-dependent reaction revealing bands coinciding with the bands likewise produced by a conventional ADH staining procedure. In spectrophotometric measurements acetaldehyde is oxidized in an NAD-dependent reaction. This activity is effectively inhibited by pyrazole, as specific inhibitor of ADH. By means of gas chromatographic analysis a quick generation of acetate from ethanol could be demonstrated. Our conclusion is further supported by experimental results obtained with either purified ADHF enzyme or genotypes with or without ADH, aldehyde-oxidase, pyridoxal-oxidase and xanthine-dehydrogenase activity. These results are discussed in relation to ethanol tolerance in the living organism in particular with respect to differences found between ADH in Drosophila melanogaster and D. simulans, and in relation to the possible implications for the selective forces acting on ADH-polymorphism.

Dual function of the alcohol dehydrogenase of Drosophila melanogaster: ethanol and acetaldehyde oxidation by two allozymes ADH-71k and ADH-F.

SummaryUntil recently the alcohol dehydrogenase of Drosophila melanogaster was thought to act only in the first step of primary alcohol oxidation, producing an aldehyde. Instead, acetic acid is the main product of a two-step process.A rapid procedure was developed for the isolation and purification of two allozymes. The thermostability of the purified enzymes was found to be very different, t1/2 at 35°C, being 45 min and 130 min for ADH-F and ADH-71k respectively.The kinetic parameters of ethanol oxidation by the two purified allozymes were determined within physiological substrate and coenzyme ranges. The use of artificial electron acceptors has a notable influence on the ethanol oxidation: the apparent Michaelis constants increase: the oxidation rate with ADH-71k increases, whereas it decreases with ADH-F.Purified ADH is shown to be able to catalyze the oxidation of acetaldehyde solely in the presence of NAD+, and PMS and MTT as artificial electron acceptors.From the kinetic data the relative in vivo oxidation rates of ethanol by both ADH allozymes were calculated. ADH-F turned out to be somewhat less effective (30%–40%) than ADH-71k. The physiological consequences of these differences are discussed.

Alcohol dehydrogenase of Drosophila: Conversion and retroconversion of isozyme patterns

Abstract 1. 1. In vitro and in vivo effects involved in the isozyme formation of homodimeric Drosophila alcohol dehydrogenase (ADH) have been studied. 2. 2. Total ADH activity of the three isozymes together as found after electrophoresis and MTT-formazan staining is often a poor predictor of total ADH activity in vivo . 3. 3. When ADH isozymes in zymograms are visualized by NADH fluorescence they do represent their in vivo activities. 4. 4. Various naturally-occurring β-keto metabolites caused conversion patterns of the ADH-isozymes in D. melanogaster and other Drosophila species, but not in two blowfly species. 5. 5. Retroconversion of ADH-isozymes in vivo has been achieved during exposure of flies to ethanol. 6. 6. The significance of these processes is discussed.

Alcohol dehydrogenase polymorphism in Drosophila: enzyme kinetics of product inhibition.

SummaryBecause natural populations ofDrosophila melanogaster are polymorphic for different allozymes of alcohol dehydrogenase (ADH) and becauseD. melanogaster is more tolerant to the toxic effects of ethanol than its sibling speciesD. simulans, information regarding the sensitivities of the different forms of ADH to the products of ethanol degradation are of ecological importance. ADH-F, ADH-S, ADH-71k ofD. melanogaster and the ADH ofD. simulans were inhibited by NADH, but the inhibition was relieved by NAD+. The order of sensitivity of NADH was ADH-F<ADH-71k, ADH-S<ADH-simulans with ADH-F being about four times less sensitive than theD. melanogaster enzymes and 12 times less sensitive than theD. simulans enzyme. Acetaldehyde inhibited the ethanolto-acetaldehyde activity of the ADHs, but at low acetaldehyde concentrations ethanol and NAD+ reduced the inhibition. ADH-71k and ADH-F were more subject to the inhibitory action of acetaldehyde than ADH-S and ADH-simulans, with ADH-71k being seven times more sensitive than ADH-S. The pattern of product inhibition of ADH-71k suggests a rapid equilibrium random mechanism for ethanol oxidation. Thus, although the ADH variants only differ by a few amino acids, these differences exert a far larger impact on their intrinsic properties than previously thought. How differences in product inhibition may be of significance in the evolution of the ADHs is discussed.

Alcohol dehydrogenase of Drosophila melanogaster : metabolic differences mediated through cryptic allozymes

Acetone formation from propan-2-ol, a saturated secondary alcohol, has been analysed in flies of three different Adh-gentypes of D. melanogaster. The in vivo oxidation of propan-2-ol was mainly mediated through ADH activity. It could be demonstrated that flies homozygous for the Adh71k allele produced more acetone than flies homozygous for AdhF. This difference in metabolic flux mediated through the cryptic allozymes under non-saturated ADH-substrate conditions seems to be based on their different kinetic properties in vivo. Product inhibition of ADH monitored by means of ADH-isozymes conversion as observed after electrophoresis was similar for both cryptic allozymes.ADH-71k and ADH-F showed immunological identity, and the in vivo protein levels of ADH-71k were 25–30 per cent higher than ADH-F.The population-genetic implications of our findings have been evaluated.

Metabolism of secondary alcohols in Drosophila melanogaster: Effects on alcohol dehydrogenase

Abstract 1. 1. In vivo metabolism of a secondary alcohol in Drosophila melanogaster and its effects on alcohol dehydrogenase (ADH) have been studied. 2. 2. ADH-mediated breakdown of the secondary alcohol, propan-2-ol, was the main source of the acetone produced. 3. 3. Acetone formation declined and stopped ultimately, suggesting inhibition of ADH activity in vivo which has been confirmed in in vitro studies. 4. 4. A powerful ketone-trapping agent, semicarbazide, did not restore the ADH activity in vitro , whereas aldehyde substrates of ADH did restore activity. 5. 5. The final formation of a dead-end ADH:NAD-acetone ternary complex has been proposed and its consequences discussed.

Evidence for a multiple function of the alcohol dehydrogenase allozyme ADH71k of Drosophila melanogaster

Alcohol dehydrogenase of Drosophila melanogaster catalyzes the oxidation of many primary and secondary alcohols. We show that sarcosine, choline and dihydroorotate are substrates of ADH in vitro. The first two substrates are regular substrates of the choline shunt, and the latter of the de novo pyrimidine synthesis. Differences in oxidative ability towards sarcosine and dihydroorotate between two ADH allozymes, ADH71k and ADHF, are observed. The catalytic activity of ADH71k towards sarcosine and dihydroorotate might be responsible for its allelic fixation in Notch8 mutant stocks, in which Notch females have a decreased level of the regular enzymes for these substrates. Their oxidation by ADH71k might act as a bypass, which restores at least part of the decreased activity of enzymes encoded by the Notch locus.

Multiple function of pteridines in Drosophila: The fluorescence of the ejaculatory bulb in Drosophila melanogaster

Abstract It is demonstrated that the strong fluorescence of the ejaculatory bulb of Drosophila melanogaster males is caused by the presence of pteridines. The pteridine composition in the bulb is affected by the mutations ry2 and ma-lF1 in which isoxanthopterin has also been detected. Our results show that the bulbs of wild-type and white-eyed mutant males possess the same pteridines. Some data suggest that the bulbal pteridines originate from the testis region. Partly on the basis of former histochemical findings it is suggested that in the bulbal cavity the pH is high favouring the fluorescent dihydro-states of the pteridines present. All these and additional literature data on the ejaculatory bulb are discussed in connection with various biological processes. Some internal larval structures in which pteridines play or might play a functional role were found to present autofluorescence.

Induction of notch-like phenocopies by methoxyacetate dependent on alcohol dehydrogenase allozymes of Drosophila melanogaster

Abstract 1. 1. Third instar larvae of two wild type strains of Drosophila melanogaster, homozygous for Adh71k and AdhF respectively were exposed to methoxyacetate, an inhibitor of sarcosine dehydrogenase. The two strains showed a different Notch-like phenocopy frequency. This is explained as a consequence of the different oxidation rates of sarcosine by the two alcohol dehydrogenase allozymes, ADH71k and ADHF. 2. 2. Inhibition of ADH activity in vivo by acetone, before the administration of methoxyacetate, enhanced in both strains differentially the frequency of wing-notches and mortality. Then the phenocopy frequency in the Adh71k strain almost equalled that of the AdhF-strain. 3. 3. The activity of sarcosine dehydrogenase is controlled by the Notch locus. Activity of sarcosine dehydrogenase is lowered by Notch mutations and methoxyacetate, whereas at the same time they do not affect the activity of alcohol dehydrogenase. 4. 4. Therefore we suppose that ADH71k forms a bypass for sarcosine oxidation, when in vivo sarcosine dehydrogenase activity is reduced either by artificial in vivo inhibition or by a mutation. 5. 5. This explains also the fixation of the Adh71k allele in stocks with Notch mutants of Drosophila melanogaster.

The Effects of Recessive Lethal Notch Mutations of Drosophila melanogaster on Flavoprotein Enzyme Activities Whose Inhibitions Cause Notch-like Phenocopies

The biochemical action of the Notch locus whose mutants cause morphological aberrations in flies, viz., notches of wings and bristle multiplication, has been analyzed (1) by the addition to the food medium of enzyme inhibitors causing phenocopies of Notch and (2) by comparison of enzyme activity patterns of Notch mutants with different degrees of phenotypic expression. Notch phenocopies were induced by inhibitors of enzyme activities in two biochemical pathways: (1) the de novo pyrimidine synthesis by 5-methylorotate (inhibitor of dihydroorotate dehydrogenase) and (2) the choline shunt by amobarbital (inhibits choline dehydrogenase) and methoxyacetate (inhibits sarcosine dehydrogenase). The inhibition of de novo pyrimidine synthesis prevents the production of deoxyuridine-5-phosphate, the substrate for the synthesis of thymidine-5-phosphate via thymidylate synthase, whereas the inhibition of the choline shunt prevents the production of HCHO groups and glycine, both of which are involved in the synthesis of 5,10-methylenetetrahydrofolate, which is a cofactor of thymidylate synthase. It was already known that the inhibition of the latter enzyme in vivo induces Notch phenocopies. Notch mutants with a strong morphological expression show low enzyme activities for dihydroorotate dehydrogenase and choline dehydrogenase. Both are flavoprotein enzymes linked to the respiratory chain. The correspondence between the low enzyme activities in Notch mutants with a strong morphological expression and the phenocopying effect of antimetabolites on these enzymes in the two biochemical pathways involved strongly suggests that the morphological effects of Notch on flies are a consequence of lowered activities of choline dehydrogenase and dihydroorotate dehydrogenase.