Physiological Significance of the Alcohol Dehydrogenase Polymorphism in Larvae of Drosophila

This study deals with biochemical and metabolic-physiological aspects of the relationship between variation in in vivo alcohol dehydrogenase activity and fitness in larvae homozygous for the alleles Adh71k, AdhF, AdhS, of Drosophila melanogaster, and for the common Adh allele of Drosophila simulans. The Adh genotypes differ in the maximum oxidation rates of propan-2-ol into acetone in vivo. There are smaller differences between the Adh genotypes in rates of ethanol elimination. Rates of accumulation of ethanol in vivo are negatively associated with larval-to-adult survival of the Adh genotypes. The rank order of the maximum rates of the ADHs in elimination of propan-2-ol, as well as ethanol, is ADH-71k greater than ADH-F greater than ADH-S greater than simulans-ADH. The ratio of this maximum rate to ADH quantity reveals the rank order of ADH-S greater than ADH-F greater than ADH-71k greater than simulans-ADH, suggesting a compensation for allozymic efficiency by the ADH quantity in D. melanogaster.Our findings show that natural selection may act on the Adh polymorphism in larvae via differences in rates of alcohol metabolism.     

Evolutionary genetics of the Drosophila alcohol dehydrogenase gene-enzyme system

Evolutionary genetics embodies a broad research area that ranges from the DNA level to studies of genetic aspects in populations. In all cases the purpose is to determine the impact of genetic variation on evolutionary change. The broad range of evolutionary genetics requires the involvement of a diverse group of researchers: molecular biologists, (population) geneticists, biochemists, physiologists, ecologists, ethologists and theorists, each of which has its own insights and interests. For example, biochemists are often not concerned with the physiological function of a protein (with respect to pH, substrates, temperature, etc.), while ecologists, in turn, are often not interested in the biochemical-physiological aspects underlying the traits they study. This review deals with several evolutionary aspects of the Drosophila alcohol dehydrogenase gene-enzyme system, and includes my own personal viewpoints. I have tried to condense and integrate the current knowledge in this field as it has developed since the comprehensive review by van Delden (1982). Details on specific issues may be gained from Sofer and Martin (1987), Sullivan, Atkinson and Starmer (1990); Chambers (1988, 1991); Geer, Miller and Heinstra (1991); and Winberg and McKinley-McKee (1992).Dedicated to Professor Billy W. Geer, because of his contributions to knowledge of the biochemical genetics of Drosophila.     

Kinetics and thermodynamics of ethanol oxidation catalyzed by genetic variants of the alcohol dehydrogenase from Drosophila melanogaster and D. simulans

Four naturally occurring variants of the alcohol dehydrogenase enzyme (ADH; EC 1.1.1.1) from Drosophila melanogaster and D. simulans, with different primary structures, have been subjected to kinetic studies of ethanol oxidation at five temperatures. Two amino acid replacements in the N-terminal region which distinguish the ADH of D. simulans from the three ADH allozymes of D. melanogaster generate a significantly different activation enthalpy and entropy, and Gibbs free energy change. The one or two amino acid replacements in the C-terminal region between the ADH allozymes of D. melanogaster do not have such clear-cut effects. All four ADH variants show highly negative activation entropies. Sarcosine oxidation by the ADH-71k variant of D. melanogaster has an activation energy barrier similar to that of ethanol oxidation. Three amino acid differences between the ADH of D. simulans and the ADH-F variant of D. melanogaster influence the kappa cat and kappa cat/Kethm constant by a maximum factor of about 2 and 2.5, respectively, over the whole temperature range. Product inhibition patterns suggest a 'rapid equilibrium random' mechanism of ethanol oxidation by the ADH-71k, and the ADH of D. simulans.     

HERITABLE VARIATION IN ETHANOL TOLERANCE AND ITS ASSOCIATION WITH BIOCHEMICAL TRAITS IN DROSOPHILA MELANOGASTER

To help elucidate mechanisms of larval ethanol tolerance seven isochromosomal lines of Drosophila melanogaster with different second chromosomes were fed a growth-limiting concentration of ethanol (4.5% v/v) and examined for associations between growth traits and biochemical characteristics that had previously been implicated in the determination of tolerance variation. Repeated measures of survival and development time over four generations verified the inherited nature of these traits. Significant variation among the lines were evident for flux from ethanol into lipid, for activity levels of alcohol dehydrogenase and glycerol-3-phosphate oxidase (GPO), and for levels of long chain and unsaturated fatty acids. A high degree of positive association occurred among the variables. A partial correlation analysis controlling for performance of the lines on ethanol-free medium revealed a strong association between the degree of long chain fatty acid content and line survival when ethanol was fed. The correlation between GPO activity and survival in an ethanol environment appeared to depend on the association of GPO activity with long chain fatty acid content. The positive correlations of flux from ethanol into lipid with many of the other variables suggested that the ADH pathway influenced the level of ethanol tolerance. These associations are all consistent with the hypothesis that the lipid content of body tissues, especially the levels of long chain and unsaturated fatty acids in cell membranes, may have an important influence on both spatial and interspecific variation in the ethanol tolerance of larvae.     

Metabolic control analysis and enzyme variation: nutritional manipulation of the flux from ethanol to lipids in Drosophila.

The effect that variation in activities of the enzymes alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) has on the flux from 14C-ethanol to lipids was examined in third-instar larvae of Drosophila melanogaster and D. simulans. The activities of ADH and ALDH were also nutritionally manipulated by the inhibitor, cyanamide. Feeding larvae cyanamide before the flux test eliminated greater than 98% of the ALDH activity but only 40% of the ADH activity. The mean +/- SD flux control coefficient for ADH activity was 0.86 +/- 0.12, and that for ALDH activity was 0.02 +/- 0.07. This suggests that ADH is the major rate-limiting enzyme for the ethanol-to-lipid pathway in Drosophila larvae under the current experimental conditions.     

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.     

The metabolism of ethanol-derived acetaldehyde by alcohol dehydrogenase (EC 1.1.1.1) and aldehyde dehydrogenase (EC 1.2.1.3) in Drosophila melanogaster larvae.

Both aldehyde dehydrogenase (ALDH, EC 1.2.1.3) and the aldehyde dehydrogenase activity of alcohol dehydrogenase (ADH, EC 1.1.1.1) were found to coexist in Drosophila melanogaster larvae. The enzymes, however, showed different inhibition patterns with respect to pyrazole, cyanamide and disulphiram. ALDH-1 and ALDH-2 isoenzymes were detected in larvae by electrophoretic methods. Nonetheless, in tracer studies in vivo, more than 75% of the acetaldehyde converted to acetate by the ADH ethanol-degrading pathway appeared to be also catalysed by the ADH enzyme. The larval fat body probably was the major site of this pathway.     

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

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-l^F1 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.     

Alcohol dehydrogenase controls the flux from ethanol into lipids in Drosophila larvae. A 13C NMR study

The dependence of the flux in the alcohol-degrading pathway on the activity of alcohol dehydrogenase was investigated in Drosophila larvae. Third-instar larvae were supplied with [2-13C]ethanol as a dietary carbon source. Specific carbon enrichments in de novo synthesized fatty acids were determined in vitro by means of 13C nuclear magnetic resonance spectroscopy. Carbon fluxes deduced from these enrichment patterns were correlated with the in vitro alcohol dehydrogenase activities in three different Adh genotypes in seven different strains. The flux control coefficient for alcohol dehydrogenase was shown to be approximately 1.0. This indicates that the alcohol dehydrogenase gene-enzyme system in Drosophila larvae can be a major target of natural selection.