Purification and enzyme stability of alcohol dehydrogenase from Drosophila simulans, Drosophila virilis and Drosophila melanogaster adhS.
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ABSTRACT:
SUBMITTER: Juan E
PROVIDER: S-EPMC1161922 | biostudies-other | 1980 Jul
REPOSITORIES: biostudies-other
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Project description:The biochemical properties of the enzyme alcohol dehydrogenase of two different Drosophila species, Drosophila simulans and Drosophila virilis, were studied and compared with those of Drosophila melanogaster Adhs enzyme. All of them consist of two identical subunits of molecular weight 27800 and share significant similarities in function. The substrate specificities of these enzymes were characterized and Km(app.) and Vmax.(app.) values were calculated. All these alcohol dehydrogenases show greater affinity for secondary rather than for primary alcohols. The amino acid compositions of the three enzymes were determined, and there is a close similarity between the D. simulans and the D. melanogaster enzymes, but there are significant differences from the alcohol dehydrogenase of D. virilis. The N-terminal amino acid is blocked and the C-terminal amino acid is the same for all three alcohol dehydrogenases. The enzymes from the three species were carboxymethylated and digested with trypsin. The peptide 'maps' reveal, as expected, more homologies between the enzymes of D. simulans and D. melanogaster than with the enzyme of D. virilis.
| S-EPMC1162855 | biostudies-other
Project description:Drosophila melanogaster and its sibling species, Drosophila simulans, differ in expression of the enzyme alcohol dehydrogenase (ADH). Adult melanogaster flies that are homozygous for the Slow allozyme have approximately twice the level of ADH activity and crossreacting material as simulans adults. There is no corresponding difference in ADH mRNA, however, so this difference in ADH protein level is evidently due to a difference in the rate of translation of the two RNAs and/or to a difference in protein stability. Here we report an interspecific gene-transfer experiment, using P-element transformation, to determine whether this expression difference is due to genetic background differences between the species (trans-acting modifiers) or to cis-acting factors within the Adh gene. When the Adh genes from D. melanogaster and D. simulans are put into the same genetic background, there is no detectable difference in their level of expression. The level is relatively high in the melanogaster background and relatively low in the simulans background. Therefore, the interspecific difference in Adh expression is due entirely to trans-acting modifiers, in spite of the many sequence differences between the Adh genes of the two species, which include two amino acid substitutions.
| S-EPMC55235 | biostudies-other
Project description:The alcohol dehydrogenase of the Drosophila melanogaster adhUF allele (alloenzyme with ultra-fast electrophoretic mobility) was unstable in crude or partially purified preparations. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that inactivation was porbably due to proteolytic degradation, and new method of purification of the enzyme was developed. After three steps, namely salmine sulphate precipitation, hydroxyapatite chromatography and Sephadex G-100 gel filtration, a 10-fold purified preparation was obtained. The enzyme produced was relatively stable compared with alcohol dehydrogenase purified by other methods, and was shown to be proteinase-free. The enzyme had a subunit mol.wt. of 24000 and had a single thiol residue per subunit available for titration with 5,5'-dithiobis-(2-nitrobenzoic acid). The amino acid composition and C-terminal amino acid sequence of the enzyme were determined. The substrate specificity of this alcohol dehydrogenase was also characterized. These results are discussed in relation to experiments on the evolutionary significance of thermostability at the adh locus.
| S-EPMC1164699 | biostudies-other
Project description:The effect of temperature on four purified alleloenzymes of the alcohol dehydrogenase (Adhs, Adhf, AdhD and Adhn-5) of the fruitfly Drosophila melanogaster was investigated in detail. Initial-velocity studies showed that the naturally occurring Adhf and Adhs enzymes differed only in their temperature optima, and evidence of kinetic adaptation to high and low temperature was not apparent. All four alleloenzymes denatured irreversibly on heating purified enzyme solutions at pH 6.0. This technique revealed only small differences in thermostability between Adhf and Adhs, although the two mutant enzymes from AdhD and Adhn-5 were considerably more labile. Electrophoresis of the enzymes though a stable transverse temperature gradient proved to be a discriminating and reproducible technique. Enzymes of different net charge were compared on the same polyacrylamide gel. The Adhf enzyme was shown to be significantly less stable than the Adhs enzyme. Subunit interchange was observed at temperatures below the point at which the unfolding occurred. At pH 4.0, the Adhf/Adhs heterodimer was as stable as the Adhs homodimeric enzyme, and more stable than the Adhf homodimer. Adhn-5 and AdhD alleloenzymes were relatively thermolabile. The stability of the alleloenzymes towards urea denaturation was studied by urea-gradient electrophoresis. Only small differences in stability between the Adhf and Adhs enzymes were observed. The AdhD and Adhn-5 mutants were denatured at the same urea concentration, which was much lower than in the case of the wild-type enzymes. Except at pH 4.0, subunit dissociation could not be distinguished from the unfolding of the monomer.
| S-EPMC1163060 | biostudies-other
Project description:The Drosophila melanogaster alleloenzymes AdhS and AdhF have been studied with respect to product inhibition by using the two substrate couples propan-2-ol/acetone and ethanol/acetaldehyde together with the coenzyme couple NAD+/NADH. With both substrate couples the reaction was consistent with an ordered Bi Bi mechanism. The substrates added to the enzyme in a compulsory order, with coenzyme as the leading substrate, to give two interconverting ternary complexes. The second ternary complex broke down with release of products in an obligatory order, with the aldehyde/ketone leaving first. Both the acetaldehyde and acetone products formed binary complexes with the enzyme that affected NAD+ binding. However, only an enzyme-acetone complex seemed to affect NADH binding and hence the reverse reaction. The inhibitory pattern with acetaldehyde as product was also affected by the formation of a ternary enzyme-NAD(+)-acetaldehyde complex, which broke down to acetic acid and NADH. The product-inhibition pattern shown in the present work is different from that published for Drosophila Adh previously and this discrepancy can not be explained by the use of different variants of Drosophila Adh.
| S-EPMC1137071 | biostudies-other
Project description:We have determined the nucleotide sequences of eight ethyl methanesulphonate-induced mutants in Drosophila alcohol dehydrogenase (ADH), of which six were previously characterized by Hollocher and Place [(1988) Genetics 116, 253-263 and 265-274]. Four of these ADH mutants contain a single amino acid change: glycine-17 to arginine, glycine-93 to glutamic acid, alanine-159 to threonine, and glycine-184 to aspartic acid. Although these mutants are inactive, three mutants (Gly17Arg, Gly93Glu and Gly184Asp) form stable homodimers, as well as heterodimers with wild-type ADH, in which the wild-type ADH subunit retains full enzyme activity [Hollocher and Place (1988) Genetics 116, 265-274]. Interestingly, the Ala159Thr mutant does not form either stable homodimers or heterodimers with wild-type ADH, suggesting that alanine-159 is important in stabilizing ADH dimers. The mutations were analysed in terms of a three-dimensional model of ADH using bacterial 20 beta-hydroxysteroid dehydrogenase and rat dihydropteridine reductase as templates. The model indicates that mutations in glycine-17 and glycine-93 affect the binding of NAD+. It also shows that alanine-159 is part of a hydrophobic anchor on the dimer interface of ADH. Replacement of alanine-159 with threonine, which has a larger side chain and can hydrogen bond with water, is likely to reduce the strength of the hydrophobic interaction. The three-dimensional model shows that glycine-184 is close to the substrate binding site. Replacement of glycine-184 with aspartic acid is likely to alter the position of threonine-186, which we propose hydrogen bonds to the carboxamide moiety of NAD+. Also, the negative charge on the aspartic acid side chain may interact with the substrate and/or residues in the substrate binding site. These mutations provide information about ADH catalysis and the stability of dimers, which may also be useful in understanding homologous dehydrogenases, which include the human 17 beta-hydroxysteroid, 11 beta-hydroxysteroid and 15-hydroxyprostaglandin dehydrogenases.
| S-EPMC1136942 | biostudies-other
Project description:Examples of clinal variation in phenotypes and genotypes across latitudinal transects have served as important models for understanding how spatially varying selection and demographic forces shape variation within species. Here, we examine the selective and demographic contributions to latitudinal variation through the largest comparative genomic study to date of Drosophila simulans and Drosophila melanogaster, with genomic sequence data from 382 individual fruit flies, collected across a spatial transect of 19 degrees latitude and at multiple time points over 2 years. Consistent with phenotypic studies, we find less clinal variation in D. simulans than D. melanogaster, particularly for the autosomes. Moreover, we find that clinally varying loci in D. simulans are less stable over multiple years than comparable clines in D. melanogaster. D. simulans shows a significantly weaker pattern of isolation by distance than D. melanogaster and we find evidence for a stronger contribution of migration to D. simulans population genetic structure. While population bottlenecks and migration can plausibly explain the differences in stability of clinal variation between the two species, we also observe a significant enrichment of shared clinal genes, suggesting that the selective forces associated with climate are acting on the same genes and phenotypes in D. simulans and D. melanogaster.
| S-EPMC5089931 | biostudies-literature
Project description:Neighboring sequences of a gene can influence its expression. In the phenomenon known as transcriptional interference, transcription at one region in the genome can repress transcription at a nearby region in cis Transcriptional interference occurs at a number of eukaryotic loci, including the alcohol dehydrogenase (Adh) gene in Drosophila melanogaster Adh is regulated by two promoters, which are distinct in their developmental timing of activation. It has been shown using transgene insertion that when the promoter distal from the Adh start codon is deleted, transcription from the proximal promoter becomes de-regulated. As a result, the Adh proximal promoter, which is normally active only during the early larval stages, becomes abnormally activated in adults. Whether this type of regulation occurs in the endogenous Adh context, however, remains unclear. Here, we employed the CRISPR/Cas9 system to edit the endogenous Adh locus and found that removal of the distal promoter also resulted in the untimely expression of the proximal promoter-driven mRNA isoform in adults, albeit at lower levels than previously reported. Importantly, transcription from the distal promoter was sufficient to repress proximal transcription in larvae, and the degree of this repression was dependent on the degree of distal promoter activity. Finally, upregulation of the distal Adh transcript led to the enrichment of histone 3 lysine 36 trimethylation over the Adh proximal promoter. We conclude that the endogenous Adh locus is developmentally regulated by transcriptional interference in a tunable manner.
| S-EPMC7202008 | biostudies-literature
Project description:When females of Drosophila melanogaster and males of Drosophila simulans are mated, the male progeny are inviable, whereas the female progeny display manifold malformations and are sterile. These abnormalities result from genetic incompatibilities accumulated since the time the lineages of the species diverged, and may have their origin in aberrant gene transcription. Because compensatory changes within species may obscure differences at the regulatory level in conventional comparisons of the expression profile between species, we have compared the gene-expression profile of hybrid females with those of females of the parental species in order to identify regulatory incompatibilities. In the hybrid females, we find abnormal levels of messenger RNA for a large fraction of the Drosophila transcriptome. These include a gross underexpression of genes preferentially expressed in females, accompanying gonadal atrophy. The hybrid females also show significant overexpression of male-biased genes, which we attribute to incompatibilities in the regulatory mechanisms that normally act to control the expression of these genes in females. The net result of the multiple incompatibilities is that the gene-expression profiles of the parental females are more similar to each other than either is to that of the hybrid.
| S-EPMC353219 | biostudies-literature
Project description:A group of small ventrolateral neurons (s-LN(v)'s) are the principal pacemaker for circadian locomotor rhythmicity of Drosophila melanogaster, and the pigment-dispersing factor (Pdf) neuropeptide plays an essential role as a clock messenger within these neurons. In our comparative studies on Pdf-associated circadian rhythms, we found that daily locomotor activity patterns of D. virilis were significantly different from those of D. melanogaster. Activities of D. virilis adults were mainly restricted to the photophase under light:dark cycles and subsequently became arrhythmic or weakly rhythmic in constant conditions. Such activity patterns resemble those of Pdf(01) mutant of D. melanogaster. Intriguingly, endogenous D. virilis Pdf (DvPdf) expression was not detected in the s-LN(v)-like neurons in the adult brains, implying that the Pdf(01)-like behavioral phenotypes of D. virilis are attributed in part to the lack of DvPdf in the s-LN(v)-like neurons. Heterologous transgenic analysis showed that cis-regulatory elements of the DvPdf transgene are capable of directing their expression in all endogenous Pdf neurons including s-LN(v)'s, as well as in non-Pdf clock neurons (LN(d)'s and fifth s-LN(v)) in a D. melanogaster host. Together these findings suggest a significant difference in the regulatory mechanisms of Pdf transcription between the two species and such a difference is causally associated with species-specific establishment of daily locomotor activity patterns.
| S-EPMC2651067 | biostudies-literature