Alcohol dehydrogenase polymorphism inDrosophila: Enzyme kinetics of product inhibition

Summary Because 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.     

Regulation of gene function: A comparison of enzyme activity levels in relation to gene dosage in diploids and triploids of Drosophila melanogaster

A study of gene activity in diploid and triploid Drosophila melanogaster females has been performed. Levels of enzyme activity of X-linked glucose 6-phosphate and 6-phosphogluconate dehydrogenases and autosome-linked -glycerophosphate and NADP-dependent isocitrate dehydrogenases were measured and correlated with DNA content, in crude extracts of whole flies or thoraces. The results show that the contribution of each dose of a given gene to the level of enzyme activity is equal in diploid and triploid cells. These findings are discussed in the context of the phenomenon of dosage compensation.This investigation was supported by Research Grant GM-15691 and Genetics Training Grant 2 T1-GM-685 of the National Institutes of Health.Recipient of a Research Career Development Award (K4-GM-13, 277) from the National Institute of General Medical Sciences.     

Regulation of amylase activity in Drosophila melanogaster: Variation in the number of enzyme molecules produced by different amylase genotypes

Purified amylases from high- and low-activity variants of Drosophila melanogaster showed identical specific activities. Immunoelectrophoresis of crude larval homogenates showed severalfold differences between strains in the amounts of cross-reacting material. Control of amylase activity is trans-acting in heterozygotes between high- and low-activity variants. These results suggest the existence of polymorphic regulatory genes affecting the production levels of amylase protein in D. melanogaster.This work was supported by Grant GM-21279 from the Institute of General Medical Science of the NIH to R. C. Lewontin and by an Operating Grant from the Natural Sciences and Engineering Research Council Canada to D. A. Hickey.     

Chromosomal location of isozyme and seed storage protein genes in Dasypyrum villosum (L.) Candargy

Summary The zymogram phenotypes of glucose-phosphate isomerase (GPI), alcohol dehydrogenase-1 (ADH-1), glutamate oxaloacetate transaminase (GOT), superoxide dismutase (SOD), lipoxygenase (LPX), esterase (EST) and the banding patterns of gliadin and glutenin seed storage proteins were determined for Triticum aestivum cv. Chinese Spring (CS), Dasypyrum villosum, the octoploid amphiploid T. aestivum cv. Chinese Spring D. villosum (CS × v) (2n=8x=56; AABBDDVV), and for five CS-D. villosum disomic addition lines. The genes Gpi-V1, Adh-V1, Got-V2, and Sod-V2 coding for GPI-1, ADH-1, GOT-2, and SOD-2 isozymes were located in D. villosum on chromosome 1V, 4V, 6V, and 7V, respectively. Genes coding for gliadin- and glutenin-like subunits are located in D. villosum chromosomes 1V. There are no direct evidence for chromosomal location of genes coding for GOT-3, EST-1 and LPX-2 isozymes. The linkage between genes coding for glutenin-like proteins and GPI-1 isozymes in chromosome 1V is evidence of homoeology between chromosome 1V and the chromosomes of homoeologous group 1 in wheat.Research supported by the National Research Council (Italy) and National Science Foundation (USA). International cooperative project, Grant No. 85.01504.06 (CNR)     

Isolation of Protease-Free Alcohol Dehydrogenase (ADH) from Drosophila simulans and Several Homozygous and Heterozygous Drosophila melanogaster Variants

The enzyme alcohol dehydrogenase (ADH) fromseveral naturally occurring ADH variants ofDrosophila melanogaster and Drosophilasimulans was isolated. Affinity chromatography withthe ligand Cibacron Blue and elution with NAD⁺ showed similarbehavior for D. melanogaster ADH-FF, ADH-71k,and D. simulans ADH. Introduction of a secondCibacron Blue affinity chromatography step, withgradient elution with NAD⁺, resulted in pure and stable enzymes. D.melanogaster ADH-SS cannot be eluted from theaffinity chromatography column at a high concentrationof NAD⁺ and required a pH gradient for itspurification, preceded by a wash step with a high concentration ofNAD⁺. Hybrid Drosophila melanogasteralcohol dehydrogenase FS has been isolated fromheterozygous flies, using affinity chromatography withfirst elution at a high concentration NAD⁺, directlyfollowed by affinity chromatography elution with a pHgradient. Incubation of equal amounts of pure homodimersof Drosophila melanogaster ADH-FF and ADH-SS,in the presence of 3 M urea at pH 8.6, for 30 min at roomtemperature, followed by reassociation yielded activeDrosophila melanogaster ADH-FS heterodimers. Noproteolytic degradation was found after incubation ofpurified enzyme preparations in the absence or presenceof SDS, except for some degradation of ADH-SS after verylong incubation times. The thermostabilities of D.melanogaster ADH-71k and ADH-SS were almostidentical and were higher than those of D.melanogaster ADH-FF and D. simulans ADH. Thethermostability of D. melanogaster ADH-FS waslower than those of D. melanogaster ADH-FF andADH-SS. D. melanogaster ADH-FF and ADH-71k have identical inhibition constantswith the ligand Cibacron Blue at pH 8.6, which are twotimes higher at pH 9.5. The K_i values forD. simulans ADH are three times lower at bothpH values. D. melanogaster ADH-SS and ADH-FS havesimilar K_i values, which are lower than thosefor D. melanogaster ADH-FF at pH 8.6. But at pH9.5 the K_i value for ADH-FS is the same as atpH 8.6, while that of ADH-SS is seven times higher. Kinetic parameters ofDrosophila melanogaster ADH-FF, ADH-SS, andADH-71k and Drosophila simulans ADH, at pH 8.6and 9.5, showed little or no variation inK_m ^eth values. TheK_m ^NAD values measured at pH 9.5for Drosophila alcohol dehydrogenases are alllower than those measured at pH 8.6. The rate constants(k_cat) determined for all fourDrosophila alcohol dehydrogenases are higher at pH 9.5 than at pH 8.6. D.melanogaster ADH-FS showed nonlinear kinetics.     

Cloning and sequence analysis of genes involved in erythromycin biosynthesis in Saccharopolyspora erythraea: sequence similarities between EryG and a family of S-adenosylmethionine-dependent methyltransferases.

Summary Treatment of tomato seeds with ethyl methanesulphonate (EMS) followed by allyl alcohol selection of M₂ seeds has led to the identification of one plant (B15-1) heterozygous for an alcohol dehydrogenase (Adh) null mutation. Genetic analysis and expression studies indicated that the mutation corresponded to the structural gene of the Adh-1 locus on chromosome 4. Homozygous Adh-1 null mutants lacked ADH-1 activity in both pollen and seeds. Using an antiserum directed against ADH from Arabidopsis thaliana, which crossreacts with ADH-1 and ADH-2 proteins from tomato, no ADH-1 protein was detected in seeds of the null mutant. Northern blot analysis showed that Adh-1 mRNA was synthesized at wild-type levels in immature seeds of the null mutant, but dropped to 25% in mature seeds. Expression of the Adh-2 gene on chromosome 6 was unaffected. The potential use of the Adh-1 null mutant in selecting rare transposon insertion mutations in a cross with mutable Adh-1 ⁺ tomato lines is discussed.     

The purification and biochemical properties of alcohol dehydrogenase—“Fast (chateau douglas)” from Drosophila melanogaster

Alcohol dehydrogenase has been purified from Drosophila melanogaster lines bearing the Adh ^F, Adh^S, and Adh ^FCh.D. alleles. Biochemical investigations show that the properties of the purified enzymes are very similar to those of crude enzyme extracts except that the pure enzymes are more heat stable. ADH-FCh.D. resembles ADH-S very closely in specific activity, substrate specificity, and a number of kinetic parameters including limiting values for K _m(app.) for ethanol. However, it is considerably more heat stable than either of the two common variants. ADH-F differs from ADH-S and ADH-FCh.D. particularly with regard to the rate of oxidation of secondary alcohols. Atomic absorbtion spectroscopy shows that all three allozymes lack zine or other divalent cations as active-site components. Peptide mapping experiments identify one very active cysteinyl residue; and amide residues in the NAD⁺ binding domain.     

An electrophoretically cryptic alcohol dehydrogenase variant in Drosophila melanogaster. I. Activity ratios, thermostability, genetic localization and comparison with two other thermostable variants

The alcohol dehydrogenase (ADH) variant ADH-FCh.D. has a secondary alcohol/primary alcohol activity ratio characteristic of ADH-S although it has an electrophoretic mobility inseparable from ADH-F. ADH-FCh.D. is distinguished from these two common ADH variants by being much more thermostable. Genetic analysis suggests tht ADH-FCh.D. is specified by an allele at the Adh locus. Biochemical comparisons show that ADH-FCh.D. has the same electrophoretic mobility, activity ratio and thermostability as the two other heat-resistant variants which have been reported, ADH-F71K in Europe and ADH-Fr in North America. The geographically widespread distribution of a thermostable ADH variant within the ADH-F electrophoretic class indicates that it should be considered in attempts to explain the Adh polymorphism in natural populations.     

Rapid enzyme kinetic assays of individualDrosophila and comparisons of field-caughtD. melanogaster andD. simulans

Techniques for performing numerous enzyme kinetic assays with minimum time and effort would be valuable to studies of the evolutionary genetics of metabolic control and the quantitative genetics of determinants of kinetic parameters. Microtiter plate readers have been used for a variety of repetitious analytical techniques, and instruments are available that can take repetitive readings with sufficient speed to perform kinetic assays. The ability of these instruments to assay rapidly the kinetic properties of small samples makes them potentially useful for a number of problems in population genetics. While the ability to handle large numbers of samples is very attractive, the small sample volumes and optical imprecision of microtiter plates result in some sacrifice in accuracy. This paper presents methods for performing kinetic assays on individual field-caughtDrosophila, quantifies the precision of these methods, and characterizes differences amongDrosophila melanogaster andD. simulans from samples caught in California and Pennsylvania. Comparisons between field-caught and laboratory rearedD. melanogaster show that most of the characters are very similar, with the exception of GPDH, which has a threefold higher mean activity among field-caught flies. The phenotypic correlations are presented with a brief discussion of their relevance to assessing the evolution of metabolic control of these enzymes.This work was supported by Grant BSR 8717495 from the National Science Foundation and by Grants HD 18379 and HD 00743 from the U.S. Public Health Service.     

Isozyme variability in species of the genus Drosophila. VIII. The alcohol dehydrogenase polymorphism in north carolina populations of D. melanogaster

Adult Drosophila melanogaster flies collected from populations broadly dispersed over ecological and geographic strata of North Carolina, and over a period of 4 years, were analyzed for alcohol dehydrogenase phenotypes by gel electrophoresis. Gene frequencies in spring-summer-fall field collections were remarkably stable over all strata. Two winter collections exhibited contrasting gene frequency changes. In one case the results are interpreted in terms of long-distance migration from Florida, while the other is explicable by assignment of a causal role to environmental factors which accompany the winter season.This investigation was supported in part by NIH Research Grant No. GM11546 from the National Institute of General Medical Sciences and by Contract No. AT-(40-1)-3980 from the United States Atomic Energy Commission.Paper No. 4719 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, North Carolina 27609.     

Characterization of a low-activity allele of NADP+-dependent isocitrate dehydrogenase from Drosophila melanogaster

We have characterized biochemical effects of Idh ^GB1 in Drosophila melanogaster. This is a null-activity allele for NADP⁺-dependent isocitrate dehydrogenase (NADP-IDH) isolated from a natural population. The homozygous mutant strain has 5% of the NADP-IDH specific activity found in controls and less than 24% of the immunologically cross-reacting material (CRM). This mutation maps to 27.2 on the third chromosome, to the right of h. The biochemical phenotype of this mutant strain includes a coordinate reduction in malic enzyme (ME) specific activity and CRM and an increase in specific activity for the pentose-phosphate shunt enzymes, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase. The K _m values for purified NADP-IDH are not different from those found for the purified control enzyme for NADP⁺ or isocitrate. It is suggested that this allele may represent a cis-acting control mutation for one of at least two loci involved in the production of NADP-IDH in D. melanogaster.Research supported by an Alberta Heritage Foundation for Medical Research Establishment Grant to MMB and a Natural Sciences and Engineering Research Council Operating Grant to JHW.     

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.     

Effect of salt stress on fatty acid alterations in some strains of Dipodascopsis and Dipodascus spp.

The effect of salt stress (8% w/v NaCl) on fatty acid composition of eight strains of Dipodascus and Dipodascopsis spp. varied from being of slight influence only (Dipodascopsis uninucleata), to decreasing the content of 18:2 (D. reesii, D. tetrasperma and D. australiensis) and to decreasing both 18:1 and 18:2 (D. tothii and D. aggregatus) with a concomitant rise of 14:1 and 16:1. With the exception of D. aggregatus, NaCl inhibited lipid accumulation in all strains. Only trace amounts of fatty acids over C₁₈ in chain length were found.J. ajbidor, M. Lamaka, A. Chrastina and P. Pokreisz are with the Department of Biochemical Technology, Slovak Technical University, Radlinskeho 9, 812 37, Bratislava, Slovak Republic; E. Breirerová is with the Institute of Chemistry, Slovak Academy of Science, Dúbravská cesta 9, Slovak Republic. M. Certík is with the Research Institute of Gerontology, Zámocká ul, P.O. Box 25, 901 01 Malacky, Slovak Republic.     

Alcohol dehydrogenase polymorphism in barrel cactus populations of Drosophila mojavensis

Starch gel electrophoresis revealed that the alcohol dehydrogenase (ADH-2) locus was polymorphic in two populations (from Agua Caliente, California and the Grand Canyon, Arizona) of cactophilic Drosophila mojavensis that utilize barrel cactus (Ferocactus acanthodes) as a host plant. Electromorphs representing products of a slow (S) and a fast (F) allele were found in adult flies. The frequency of the slow allele was 0.448 in flies from Agua Caliente and 0.659 in flies from the Grand Canyon. These frequencies were intermediate to those of the low (Baja California peninsula, Mexico) and high (Sonora, Mexico and southern Arizona) frequency Adh-2^S populations of D. mojavensis that utilize different species of host cacti.     

Linkage analyses using biochemical variants in mice. II. Levulinate dehydratase and autosomal glucose 6-phosphate dehydrogenase

The level of hepatic -aminolevulinate dehydratase varies among inbred strains of mice and is regulated by codominant alleles at the Lv locus. Twenty-two inbred strains have been classified with respect to this locus. Lv is 5±2 recombination units from brown, b, in linkage group VIII. The locus for autosomal glucose 6-phosphate dehydrogenase (Gpd-1) has also been assigned to linkage group VIII and is 32±5 units from brown. The order of the loci is Lv-b-Gpd-1. Incidental note is made of linkage between the malic dehydrogenase (Mdh-1) and dilute (d) loci, linkage group II, with 10±3 % recombination between the two.Supported by the Roche Institute of Molecular Biology, Nutley, New Jersey, and by Public Health Service Research Grant CA-05873 from the National Cancer Institute.     

Purification and characterization of two allelic forms of l-glycerol 3-phosphate dehydrogenase from inbred strains of mice

l-Glycerol 3-phosphate dehydrogenase (E.C. 1.1.1.8) was purified from the muscle of BALB/cJ and C57BL/6J mice. The half-lives of the enzyme at 50 C were 6 and 33 min, respectively, for the BALB/cJ and C57BL/6J strains. Enzyme preparations from the two strains of mice were compared with respect to the following properties and found to be essentially indistinguishable: K _m values for dihydroxyacetone phosphate, NADH, l--glycerophosphate, and NAD⁺; maximum velocity; competitive inhibition by inorganic phosphate; pH optimum; energy of activation; electrophoretic mobility; molecular weight and subunit molecular weight. From these data, it is concluded that the kinetic properties of the purified enzyme are not the factors responsible for the differences in activity found in crude homogenates of mouse tissues.This work was supported by NIH Research Grant HD 06712 from the National Institute of Child Health and Human Development and by an allocation from NIH General Research Support Grant RR-05545 from the Division of Research Resources to The Jackson Laboratory. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Adaptive significance of amylase polymorphism in Drosophila

Laboratory populations of D. busckii flies were kept for one generation on media containing different carbohydrate sources (maltose and rice, potato or maize starch). The flies maintained on standard potato medium served as a control. Progeny were analyzed for -amylase activity and Amy-electromorph frequencies.Spectrophotometrically assayed amylase activity was highest in the flies cultured on potato starch medium and lowest in specimens kept on maltose. Carbohydrate source in some substrates affected both frequencies of Amy-alleles and Amy-genotypes. Phenotypic differences at a biochemical level, i.e. in -amylase activity, might be connected to Amy-structural gene polymorphism in the examined Drosophila species.     

The association between malathion resistance and acetylcholinesterase in Drosophila melanogaster

The relationship between the 50% survival time for flies feeding on a malathion-containing medium and the activity of acetylcholinesterase (AChE) was determined for 15 isofemale lines of Drosophila melanogaster. A significant correlation was found (r=0.28, P<0.05), with more resistant lines tending to have a lower level of AChE activity. An association between AChE and malathion resistance was also observed in a selection experiment. The AChE activity decreased in two of two populations selected for malathion resistance. AChE from these populations was altered in kinetic parameters (measured in crude head extracts) and electrophoretic mobility. Although the resistant AChE had a lower activity (V _m) on either a per milligram protein or a per individual basis, its apparent K _m for acetylthiocholine was lower than that of susceptible AChE. Recombination mapping of both low activity and fast electrophoretic mobility localized these traits to the region of the structural locus (Ace) on the third chromosome. The AChE activity of flies heterozygous for a variety of Ace lesions (kindly provided by Dr. W. M. Gelbart) was consistent with this location. The changes in AChE were suggested to have been caused by selection of alleles at the Ace locus.This work was supported by NSERC Grants A5857, G0183, and A0629.     

Adh n5: A temperature-sensitive mutant at the Adh locus in Drosophila

Individuals of an alcohol dehydrogenase-negative strain of Drosophila melanogaster designated Adh ⁿ⁵ are resistant to ethanol poisoning at low but not at high temperatures. The basis for this behavior is that Adh ⁿ⁵ flies contain a mutant form of alcohol dehydrogenase which is less heat stable than that of wild-type flies. The mutation in Adh ⁿ⁵ maps at, or very close to, the presumptive structural locus and is not complemented by any of 11 other alcohol dehydrogenase-null mutants.This research was supported by Grant No. GM 18254 from the National Institutes of Health and Grant No. M55.2217 from the National Cancer Institute.Publication No. 768 from the Department of Biology, Johns Hopkins University.     

Staphylococcus aureus alpha-toxin-induced pores: Channel-like behavior in lipid bilayers and patch clamped cells

The conductance of pores induced by Staphylococcus aureus -toxin in Lettre cells has been compared to that in bilayers composed of synthetic lipids or Lettre cell membrane constituents. Previously described characteristics of toxin-induced conductance changes in lipid bilayers, namely rectification, voltage-dependent closure, and closure at low pH or in the presence of divalent cations (Menestrina, 1986) are displayed also in bilayers prepared from Lettre cell membranes and in patch clamped Lettre cells. It is concluded that endogenous proteins do not affect the properties of -toxininduced channels significantly and that the relative lack of ion channels in Lettre cells makes them ideal for studies of pore-forming toxins by the patch clamp technique.Dr. Sviderskaya is on leave of absence from the Physiology Institute, University of St. Petersburg, RussiaWe are grateful to Dr. J.P. Arbuthnott and Dr. K. Hungerer for gifts of S. aureus -toxin, to Dr. T.B. Bolton for collaboration with patch clamped cells and to Dr. J.M. Graham for help with the preparation of Lettre cell plasma membranes. This study was supported by the Cell Surface Research Fund, Medical Research Council, Science and Engineering Research Council, UNESCO (Molecular and Cell Biology Network) and The Wellcome Trust.