Properties of alcohol dehydrogenase isozymes in a strain of Drosophila melanogaster homozygous for the Adh-slow allele

Two isozymes of alcohol dehydrogenase from Drosophila melanogaster homozygous for the Adh-slow allele have been separated by isoelectric focusing. The isozymes differ in their temperature optima, temperature stabilities, specific activities, and at least one of their Michaelis constants. They are immunologically identical. Evidence is presented that NAD may partially convert one isozyme into another. The possible nature of these isozymic differences is discussed.     

Synthesis of Drosophila melanogaster alcohol dehydrogenase in yeast

Expression systems for the heterologous expression of Drosophila melanogaster alcohol dehydrogenase (ADH) in Saccharomyces cerevisiae have been designed, analyzed and compared. Four different yeast/Escherichia coli shuttle vectors were constructed and used to transform four different yeast strains. Expression was detectable in ADH- yeast strains, from either a constitutive promoter, yeast ADH1 promoter (ADCp), or a regulated promoter, yeast GALp. The highest amount of D. melanogaster ADH was obtained from a multicopy plasmid with the D. melanogaster Adh gene expressed constitutively under the control of yeast ADCp promoter. The D. melanogaster enzyme was produced in cell extracts, as assessed by Coomassie blue staining and Western blotting after polyacrylamide-gel electrophoresis and it was fully active and able to complement the yeast ADH deficiency. Results show that D. melanogaster ADH subunits synthesized in yeast are able to assemble into functional dimeric forms. The synthesized D. melanogaster ADH represents up to 3.5% of the total extracted yeast protein.     

Mechanism of action of Drosophila melanogaster alcohol dehydrogenase.

Reported kinetic pH dependence data for alcohol dehydrogenase from Drosophila melanogaster are analyzed with regard to differences in rate behaviour between this non-metallo enzyme and the zinc-containing liver alcohol dehydrogenase present in vertebrates. For the Drosophila enzyme a mechanism of action is proposed according to which catalytic proton release to solution during alcohol oxidation occurs at the binary-complex level as an obligatory step preceding substrate binding. Such proton release involves an ionizing group with a pKa of about 7.6 in the enzyme.NAD+ complex, tentatively identified as a tyrosyl residue. The ionized form of this group is proposed to participate in the binding of alcohol substrates and to act as a nucleophilic catalyst of the subsequent step of hydride ion transfer from the bound alcohol to NAD+. Herein lie fundamental mechanistic differences between the metallo and non-metallo short chain alcohol dehydrogenases.     

The involvement of alcohol dehydrogenase and aldehyde dehydrogenase in alcohol/aldehyde metabolism in Drosophila melanogaster

In this study we have examined the roles of alcohol dehydrogenase, aldehyde oxidase, and aldehyde dehydrogenase in the adaptation of Drosophila melanogaster to alcohol environments. Fifteen strains were characterized for genetic variation at the above loci by protein electrophoresis. Levels of in vitro enzyme activity were also determined. The strains examined showed considerable variation in enzyme activity for all three gene-enzyme systems. Each enzyme was also characterized for coenzyme requirements, effect of inhibitors, subcellular location, and tissue specific expression. A subset of the strains was chosen to assess the physiological role of each gene-enzyme system in alcohol and aldehyde metabolism. These strains were characterized for both the ability to utilize alcohols and aldehydes as carbon sources as well as the capacity to detoxify such substrates. The results of the above analyses demonstrate the importance of both alcohol dehydrogenase and aldehyde dehydrogenase in the in vivo metabolism of alcohols and aldehydes.     

UGA nonsense mutation in the alcohol dehydrogenase gene of Drosophila melanogaster

A mutant gene, which we have designated AdhnB, codes for a defective form of the enzyme alcohol dehydrogenase in Drosophila melanogaster. We show that the polypeptide encoded by AdhnB is approximately 2000 Mr smaller than the protein synthesized under the direction of the wild-type alcohol dehydrogenase gene. In contrast, the alcohol dehydrogenase mRNA produced by both genes is the same size. We cloned and sequenced a portion of the protein-coding region of AdhnB and compared it to the same region in the wild-type gene. We found a single base substitution: a change of the TGG tryptophan codon at amino acid 235 to a TGA termination codon. This nonsense mutation accounts for the observed reduction in size of the alcohol dehydrogenase polypeptide. In further studies, we found that the steady-state levels of alcohol dehydrogenase mRNA in flies carrying the AdhnB gene and the wild-type alcohol dehydrogenase gene were indistinguishable. However, the steady-state level of alcohol dehydrogenase polypeptide was reduced to 1% of wild-type levels in flies with the AdhnB gene. Moreover, the rate of alcohol dehydrogenase synthesis in mutant flies was reduced to 50% of that found in wild type. The aberration in AdhnB thus affects both the rate of synthesis and the rate of degradation of the alcohol dehydrogenase peptide. AdhnB is the first reported nonsense mutant in Drosophila.     

Comparison of alcohol dehydrogenase expression in Drosophila melanogaster and D. simulans

Alcohol dehydrogenase (ADH) gene expression was analyzed in Drosophila melanogaster and its sibling species D. simulans. The levels of ADH activity, ADH-cross-reacting material (CRM), and ADH-mRNA were analyzed for several strains of each species, which derive from diverse geographic locations around the world. There is considerable quantitative variation in ADH activity, CRM level, and RNA level among strains within species at all developmental stages. However, the only consistent differences between the two species are in pupal RNA level and in late-adult activity and CRM level. Late-adult melanogaster flies that are homozygous for the Slow allozyme have approximately twice the level of ADH activity and CRM as do simulans flies. The regression of activity on CRM over strains is highly significant and essentially the same for each species, which means that most, if not all, of the activity difference between the species is due to a difference in concentration of the ADH protein. In contrast, there is no significant regression of CRM level on mRNA level in adults of either species; nor is there a significant difference in RNA level between species. Therefore, the difference in ADH protein concentration is not due to RNA template availability. Thus, the interspecific difference in ADH level in adults must be due either to a difference in the rate of translation of the two RNAs or to a difference in protein stability.     

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

• 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.