The dose effect ofma-l+ andry+ on xanthine dehydrogenase activity inDrosophila melanogaster

Summary Two loci,ma-l ⁺ andry ⁺, necessary for xanthine dehydrogenase activity inDrosophila melanogaster have been studied for dosage effects utilizing deficiencies and duplications induced for this purpose. Comparisons of one, two and three doses ofma-l ⁺ in the female or one and two doses in the male indicate that there is no increase in specific enzyme activity with dose. On the other hand, comparisons of one, two and three doses ofry ⁺ in the male and female reveal an increase in enzyme activity that is roughly proportional to dose. Since dosage ofry ⁺ is limiting, whereas that ofma-l ⁺ is not, the final concentration of xanthine dehydrogenase is shown to depend on the number of doses ofry ⁺.The implications of these findings with respect to the hypothesis of dosage compensation and to the mechanism of control of enzyme and protein concentration are discussed.Operated by Union Carbide Corporation for the U.S. Atomic Energy Commission.     

Observations on the regulation of uricase activity during development of Drosophila melanogaster

To elucidate the mechanisms involved in the regulation of uricase activity in Drosophila melanogaster, a comparative analysis of the patterns of uricase activity during development was undertaken for the wild type, Ore-R, and the mutants ry ² and ma-1. Uricase activity in ry ² and ma-l, unlike that in Ore-R, increased rapidly following emergence of the adult. This study indicates that uricase in Drosophila, in contrast to that in several microorganisms, is not induced by uric acid, since ry ² and ma-l with no detectable uric acid have higher activity than the wild type.This study was supported by grants from NIH, PHS 5-TO 1 GM 0071-13 and 1F02 HD 50,527-01     

Weitere Untersuchungen zu Komplementarit?t und Nicht-Autonomie Deraugenfarb-MutantenMA-L undMA-LBZ vonDrosophila melanogaster

Summary Data found byGlassman (1959, 1960) in compound females of the genotypev bz/v f B x ma-l (wherebz stands forma-l ^bz ) showed complementation between these alleles in both eye color and activity of the enzyme, xanthine dehydrogenase, the latter however being incomplete. These results have been reexamined and extended, using compounds of purebz andma-l stocks as well as various heterozygotes between these stocks and the mutantrosy. Complementation in the red eye pigments (drosopterines), though incomplete in newly emerged compound femalesma-l/bz andbz/ma-l becomes firmly established in older flies, where the red pigments reach the amount present in wildtype eyes. Isoxanthopterin however, as well as enzyme activity do not exceed approximately 10% of the level typical for wildtype. These features of slow and incomplete complementation are very probably due to the interaction ofma-l andbz; they do not occur in a series of other related heterozygotes.Transplantation experiments revealed non-autonomy of bothma-l andbz with regard to the formation of drosopterines in the eyes and isoxanthopterin in the testes.The results are discussed with respect to the allelsm of the two mutants.

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Ausgeführt mit Unterstützung des Schweizerischen Nationalfonds zur Förderung der wissenschaftlichen Forschung. Herrn Prof. Dr.E. Hadorn danke ich herzlich für die Förderung dieser Arbeit, herrn Prof. Dr.U. Leupold für kritische Diskussion.     

Gene expression in Drosophila: post-translational modification of aldehyde oxidase and xanthine dehydrogenase.

Summary Maroon-like homozygotes are completely deficient for xanthine dehydrogenase (XDH) and aldehyde oxidase (AO), however ma-l is not a structural locus for either enzyme. Quantitative immunoelectrophoresis of ma-l and wild type extracts suggests that the ma-l function must be post-translational. To determine whether the ma-l function involves some direct physical changes in XDH and/or AO the enyzmes were characterized with respect to temperature sensitivity and behavior in gel sieving electrophoresis. Since the XDH and AO from complementary ma-l heterozygotes is more thermolabile and different in shape from wild type XDH and AO, we conclude that ma-l is involved in a post-translational modification of these enzymes.     

Expression of xanthine dehydrogenase activity during embryonic development of Drosophila melanogaster

The contributions of oogenesis and zygotic genome expression to xanthine dehydrogenase activity during embryogenesis were investigated utilizing the mal and ry² mutants. In vitro complementation experiments demonstrated the presence of the mal⁺ complementation factor in the oocyte, suggesting an explanation for the mal maternal effect. The ry⁺ complementation factor synthesized from paternal template was detected at gastrulation. This is the earliest detection of a paternal enzyme during nonmammalian embryonic development.     

Xanthine dehydrogenase AtXDH1 from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> is a potent producer of superoxide anions via its NADH oxidase activity

Xanthine dehydrogenase AtXDH1 from Arabidopsis thaliana is a key enzyme in purine degradation where it oxidizes hypoxanthine to xanthine and xanthine to uric acid. Electrons released from these substrates are either transferred to NAD⁺ or to molecular oxygen, thereby yielding NADH or superoxide, respectively. By an alternative activity, AtXDH1 is capable of oxidizing NADH with concomitant formation of NAD⁺ and superoxide. Here we demonstrate that in comparison to the specific activity with xanthine as substrate, the specific activity of recombinant AtXDH1 with NADH as substrate is about 15-times higher accompanied by a doubling in superoxide production. The observation that NAD⁺ inhibits NADH oxidase activity of AtXDH1 while NADH suppresses NAD⁺-dependent xanthine oxidation indicates that both NAD⁺ and NADH compete for the same binding-site and that both sub-activities are not expressed at the same time. Rather, each sub-activity is determined by specific conditions such as the availability of substrates and co-substrates, which allows regulation of superoxide production by AtXDH1. Since AtXDH1 exhibits the most pronounced NADH oxidase activity among all xanthine dehydrogenase proteins studied thus far, our results imply that in particular by its NADH oxidase activity AtXDH1 is an efficient producer of superoxide also in vivo.     

The isolation and characterization of a mutant allele at a new X-linked locus,mex, affecting NADP+-dependent enzymes inDrosophila melanogaster

The isolation and characterization of mutant alleles in a regulatory gene affecting NADP⁺-dependent enzymes are described. The locus,mex, is at position 26.5 ± 0.74 on the X chromosome ofDrosophila melanogaster. The newly isolated mutant allele,mex ¹, is recessive to either themex allele found in Oregon-R wild-type individuals or that found in thecm v parental stock in which the new mutants were induced. Themex ¹ mutant allele is associated with statistically significant decreases in malic enzyme (ME) specific activity and ME specific immunologically cross-reacting material (ME-CRM) in newly emerged adult males. During this same developmental stage in males, the NADP⁺-dependent isocitrate dehydrogenase specific activity increases to statistically significant levels. Females of themex ¹ mutant strain show statistically significant elevated levels of the pentose phosphate shunt enzymes, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Isoelectric focusing and thermolability comparisons of the active ME from mutant and control organisms indicate that the enzyme is the same. Developmental profiles ofmex ¹ and control strains indicate that this mutant allele differentially modulates the levels of ME enzymatic activity and ME-CRM during development. This work was supported by an Operating Grant from the Natural Sciences and Engineering Research Council of Canada to M.M.B.     

Activity of NAD-dependent glucose-6-phosphate dehydrogenase in yeast-like organisms

Summary From tested yeast-like organisms, onlyGeotrichum candidum showed the same activity of glucose-6-phosphate dehydrogenase with both NAD⁺ and NADP⁺. i. e. 0.017–0.019 mol NADH/min. mg dry weight of cell free extracts. Omission of Mg⁺⁺ in the reaction mixture did not influence the activity of the enzyme in the presence of NAD⁺. Cell free extracts ofEndomyces magnusii showed only low activity of this enzyme and the ratio of its activity in the presence of NAD⁺ and NADP⁺, respectively, varied in individual cultures.Rhodotorula glutinis showed only an NADP⁺-dependent activity.     

Purification and properties of the NAD+-dependent (type D) and O2-dependent (type O) forms of rat liver xanthine dehydrogenase.

The xanthine-oxidizing enzyme of rat liver has been purified as an NAD⁺-dependent dehydrogenase (type D) and as the O₂-dependent oxidase (type O). The purified D and O variants are nearly homogenous as judged by polyacrylamide discontinuous gel electrophoresis and are indistinguishable on sodium dodecyl sulfate-urea gels. The absorption spectrum of the type D enzyme is indistinguishable from that of the type O enzyme and closely resembles the spectra of xanthine-oxidizing enzymes from other sources. The types D and O enzymes have essentially the same cofactor composition. Oxidation of xanthine by type D is stimulated by NAD⁺ with concomitant NADH formation. Type D is able to utilize NADH as well as xanthine as electron donor to various acceptors, in contrast to type O that is unable to oxidize NADH. Arsenite, cyanide and methanol completely abolish xanthine oxidation by the type D enzyme while affecting the activities with NADH to varying extents. In these respects rat liver xanthine dehydrogenase closely resembles chicken liver xanthine dehydrogenase. However, in contrast to the avian enzyme, the purified rat liver enzyme is unstable as a dehydrogenase and is gradually converted to an oxidase. This conversion is accompanied by an increase in the aerobic xanthine → cytochrome c activity. The native type D enzyme in rat liver extracts is precipitable with antibody prepared against purified type O. The K_m for xanthine is not significantly different for the two forms.     

Occurrence of xanthine dehydrogenase in Chlamydomonas reinhardii: A common cofactor shared by xanthine dehydrogenase and nitrate reductase

Wild-type Chlamydomonas reinhardii cells have xanthine dehydrogenase activity when grown with nitrate, nitrite, urea, or amino acid media. Mutant strains 102, 104, and 307 of Chlamydomonas, lacking both xanthine dehydrogenase and nitrate reductase activities, were incapable of restoring the NADPH-nitrate reductase activity of the mutant nit-1 of Neurospora crassa, whereas wild type cells and mutants 203 and 305 had xanthine dehydrogenase and were able to reconstitute the nitrate reductase activity of nit-1 of Neurospora. Therefore, it is concluded that in Chlamydomonas a common cofactor is shared by xanthine dehydrogenase and nitrate reductase. Xanthine dehydrogenase is repressed by ammonia and seems to be inessential for growth of Chlamydomonas.     

Analysis of novel alleles of brother of tout‐velu,the drosophila ortholog of human EXTL3 using a newly developed FRT42D ovoD chromosome

The FLP/FRT system permits rapid phenotypic screening of homozygous lethal mutations in the context of a viable mosaic fly. Combining this system with ovo^D dominant female‐sterile transgenes enables efficient production of embryos derived from mutant germline clones lacking maternal contribution from a gene of interest. Two distinct sets of FRT chromosomes, carrying either the mini‐white (w ^{+ mW.hs}), or rosy (ry⁺) and neomycin (neo^R) transgenes are in common use. Parallel ovo^D lines were developed using w ^{+ mW.hs} FRT insertions on the X and chromosomes 2R and 3L, as well as ry⁺, neo^R FRT insertions on 2L and 3R. Consequently, mutations isolated on the X, 2R and 3L chromosomes in a ry⁺, neo^R FRT background, are not amenable to germline clonal analysis without labor‐intensive recombination onto chromosome arms containing a w ^{+ mW.hs} FRT. Here we report the creation of a new ovo^D line for the ry⁺, neo^R FRT insertion at position FRT42D on chromosome 2R, through induced recombination in males. To establish the developmental relevance of this reagent we characterized the maternal‐effect phenotypes of novel brother of tout‐velu alleles generated on an FRT42D chromosome in a somatic mosaic screen. We find that an apparent null mutation that causes severe defects in somatic tissues has a much milder effect on embryonic patterning, emphasizing the necessity of analyzing mutant phenotypes at multiple developmental stages.     

Thealdox-2 locus ofDrosophila melanogaster also affects sulfite oxidase and molybdenum metabolism

Mutation at thealdox-2 locus inDrosophila melanogaster affects the specific activities of four molybdoenzymes differentially during development. Sulfite oxidase activity is normal during late larval and pupal stages but is reduced during early adult stages inaldox-2 organisms. There was complete concordance among the effects ofaldox-2 on sulfite oxidase, aldehyde oxidase, xanthine dehydrogenase, and pyridoxal oxidase, when 38 stocks were analyzed which were derived from single recombination events betweenc andpx, markers which flankaldox-2. Several different biochemical analyses indicate that the active molybdoenzymes present in thealdox-2 strain are normal with respect to size, shape,pH-activity profile,K _m , and molecular weight. Significant differences were found between thealdox-2 strain and the OR control strain in their responses to dietary Na₂MoO₄ and Na₂WO₄. The mutant strain is much more resistant to the effects of dietary Na₂WO₄ and much more responsive to the administration of Na₂MoO₄ than the OR control strain when these effects are quantitated by measurements of molybdoenzyme specific activities. This evidence suggests that thealdox-2 ⁺ gene product has a molybdenum binding site which can also bind tungsten and that this site is altered in the mutant strain. The hypothesis presented explains the observed effects of thealdox-2 mutation and relates them to the other mutations reported in this gene-enzyme system.This work was supported by an Operating Grant from the Natural Sciences and Engineering Research Council to M.M.B.     

The maternal and zygotic control of development by cinnamon, a new mutant in Drosophila melanogaster

A genetic analysis of the X-linked mutant cinnamon (cin) in Drosophila melanogaster demonstrates that this locus controls a process that is essential for zygote survival. Survival of a zygote is shown to be ensured by either the maternal expression of the cin⁺ allele or by the presence of a cin⁺ allele in the zygotic genome. The cin locus is also shown to affect pteridine pigment metabolism. The presence of a cin⁺ allele in either the mother or zygote is requisite for normal zygotic eye pigmentation. It is shown that cin flies lack xanthine dehydrogenase, an enzyme involved in pteridine metabolism, and accumulate the enzyme's pteridine and purine substrates. The relationship between the effects of cin on viability and xanthine dehydrogenase activity is discussed.     

The enhancement by pervanadate of tyrosine phosphorylation on prostatic proteins occurs through the inhibition of membrane-associated tyrosine phosphatases

The Na⁺–K⁺ ATPase activity and SH group content were decreased whereas malondialdehyde (MDA) content was increased upon treating the porcine cardiac sarcolemma with xanthine plus xanthine oxidase, which is known to generate superoxide and other oxyradicals. Superoxide dismutase either alone or in combination with catalase and mannitol fully prevented changes in SH group content but the xanthine plus xanthine oxidase-induced depression in Na⁺–K⁺ ATPase activity as well as increase in MDA content were prevented partially. The Lineweaver-Burk plot analysis of the data for Na⁺–K⁺ ATPase activity in the presence of different concentrations of MgATP or Na⁺ revealed that the xanthine plus xanthine oxidase-induced depression in the enzyme activity was associated with a decrease in V_max and an increase in K_m for MgATP; however, K_a value for Na⁺ was decreased. Treatment of sarcolemma with H₂O₂ plus Fe²⁺, an hydroxyl and other radical generating system, increased MDA content but decreased both Na⁺–K⁺ ATPase activity and SH group content; mannitol alone or in combination with catalase prevented changes in SH group content fully but the depression in Na⁺–K⁺ ATPase activity and increase in MDA content were prevented partially. The depression in the enzyme activity by H₂O₂ plus Fe²⁺ was associated with a decrease in V_max and an increase in K_m for MgATP. These results indicate that the depressant effect of xanthine plus xanthine oxidase on sarcolemmal Na⁺–K⁺ ATPase may be due to the formation of superoxide, hydroxyl and other radicals. Furthermore, the oxyradical-induced depression in Na⁺–K⁺ ATPase activity may be due to a decrease in the affinity of substrate in the sarcolemmal membrane.     

Xanthine dehydrogenase processes retinol to retinoic acid in human mammary epithelial cells

Retinoic acid is considered to be the active metabolite of retinol, able to control differentiation and proliferation of epithelia. Retinoic acid biosynthesis has been widely described with the implication of multiple enzymatic activities. However, our understanding of the cell biological function and regulation of this process is limited. In a recent study we evidenced that milk xanthine oxidase (E.C. 1.17.3.2.) is capable to oxidize all-trans-retinol bound to CRBP (holo-CRBP) to all-trans-retinaldehyde and then to all-trans-retinoic acid. To get further knowledge regarding this process we have evaluated the biosynthetic pathway of retinoic acid in a human mammary epithelial cell line (HMEC) in which xanthine dehydrogenase (E.C. 1.17.1.4.), the native form of xanthine oxidase, is expressed. Here we report the demonstration of a novel retinol oxidation pathway that in the HMEC cytoplasm directly conduces to retinoic acid. After isolation and immunoassay of the cytosolic protein showing retinol oxidizing activity we identified it with the well-known enzyme xanthine dehydrogenase. The NAD⁺ dependent retinol oxidation catalyzed by xanthine dehydrogenase is strictly dependent on cellular retinol binding proteins and is inhibited by oxypurinol. In this work, a new insight into the biological role of xanthine dehydrogenase is given.     

Selenium requirement for active xanthine dehydrogenase from Clostridium acidiurici and Clostridium cylindrosporum

The xanthine dehydrogenase of Clostridium acidiurici and C. cylindrosporum was assayed with methyl viologen as acceptor. In C. acidiurici the basal activity level was about 0.3 mol/min x mg of protein. Cells grown on uric acid in the presence of 10^-7 M selenite showed a 14-fold increase in xanthine dehydrogenase activity, which decreased with higher selenite concentrations (10^-5 M). The supplementation with 10^-7 M molybdate or tungstate was without effect. High concentrations of tungstate decreased the xanthine dehydrogenase if selenite was also present. In comparison, high concentrations of molybdate affected only a small decrease in activity level at the optimal concentration for selenite and relieved to some degree the inhibitory effect of 10^-5 M selenite. With hypoxanthine and xanthine as substrates for growth again only the addition of selenite was necessary to show a similar increase in xanthine dehydrogenase activity. C. acidiurici could be grown in a mineral medium. Both xanthine dehydrogenase and formate dehydrogenase exhibited the highest level of activity if selenite and tungstate were present in that medium.In C. cylindrosporum the basal activity level of xanthine dehydrogenase was about 0.95 mol/min x mg of protein. The addition of 10^-7 M selenite to the growth medium increased the activity level about 3-fold, but the highest level (3.7 U/mg) was reached if 10^-7 M molybdate was also added. The presence of tungstate resulted in a decreased enzyme activity.     

Molybdoenzymes in Drosophila. IV. Further characterization of the cinnamon phenotype

Summary Mutations at the cin gene display drastically lowered levels of the molybdoenzymes, xanthine dehydrogenase (XDH) and aldehyde oxidase (AO), and lack pyridoxal oxidase (PO) and sulfite oxidase (SO) activities. Certain mutations at cin also display varying degrees of female sterility, which is maternally affected. Here we characterize five new cin alleles with respect to the molybdoenzyme activities as well as the molybdenum cofactor, commonly required for molybdoenzyme activity. In complementing cin heterozygotes we find that, in addition to the previously reported unusually high levels of XDH and AO activities, there are unusually elevated levels of SO activity, as well as complementation for PO activity. The levels of immunologically crossreacting material in such heterozygotes indicate that the elevated levels of molybdoenzyme activities cannot be due to increases in the number of enzyme molecules. Measurements of the level of molybdenum cofactor activity normally present in XDH, AO, PO, and SO point to the possibility that a larger fraction of the enzyme molecules are active in these heterozygotes. The possible role of SO with respect to cinnamon's female sterility is also discussed.     

Enzymes of ammonium assimilation inStreptomyces avermitilis

Glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH), alanine dehydrogenase (ADH) and alanine aminotransferase (GPT) were detected in the cell-free homogenate ofStreptomyces avermitilis grown in a defined medium containing ammonium sulfate as the only nitrogen source. At an initial NH₄ ⁺ concentration of 7.5 mmol/L, high activities of GS, GOGAT and GDH were found while that of ADH was low. The ADH activity was markedly increased at initially millimolar NH₄ ⁺ concentrations. In some characteristics of its NH₄ ⁺-assimilating system (e.g. control of some enzyme activities, the NADPH specificity of GOGAT, the presence of alanine aminotransferase),S. avermitilis differs from other known streptomycetes.     

The tricarboxylic acid cycle in L₃ Teladorsagia circumcincta: metabolism of acetyl CoA to succinyl CoA

Nematodes, like other species, derive much of the energy for cellular processes from mitochondrial pathways including the TCA cycle. Previously, we have shown L₃Teladorsagia circumcincta consume oxygen and so may utilise a full TCA cycle for aerobic energy metabolism. We have assessed the relative activity levels and substrate affinities of citrate synthase, aconitase, isocitrate dehydrogenase (both NAD⁺ and NADP⁺ specific) and α-ketoglutarate dehydrogenase in homogenates of L₃T. circumcincta. All of these enzymes were present in homogenates. Compared with citrate synthase, low levels of enzyme activity and low catalytic efficiency was observed for NAD⁺ isocitrate dehydrogenase and especially α-ketoglutarate dehydrogenase. Therefore, it is likely that the activity of these to enzymes regulate overall metabolite flow through the TCA cycle, especially when [NAD⁺] limits enzyme activity. Of the enzymes tested, only citrate synthase had substrate affinities which were markedly different from values obtained from mammalian species. Overall, the results are consistent with the suggestion that a full TCA cycle exists within L₃T. circumcincta. While there may subtle variations in enzyme properties, particularly for citrate synthase, the control points for the TCA cycle in L₃T. circumcincta are probably similar to those in the tissues of their host species.     

Xanthine dehydrogenase from Pseudomonas putida 86: specificity,oxidation–reduction potentials of its redox-active centers,and first EPR characterization

Xanthine dehydrogenase (XDH) from Pseudomonas putida 86, which was induced 65-fold by growth on hypoxanthine, was purified to homogeneity. It catalyzes the oxidation of hypoxanthine, xanthine, purine, and some aromatic aldehydes, using NAD⁺ as the preferred electron acceptor. In the hypoxanthine:NAD⁺ assay, the specific activity of purified XDH was 26.7 U (mg protein)⁻¹. Its activity with ferricyanide and dioxygen was 58% and 4%, respectively, relative to the activity observed with NAD⁺. XDH from P. putida 86 consists of 91.0 kDa and 46.2 kDa subunits presumably forming an α₄β₄ structure and contains the same set of redox-active centers as eukaryotic XDHs. After reduction of the enzyme with xanthine, electron paramagnetic resonance (EPR) signals of the neutral FAD semiquinone radical and the Mo(V) rapid signal were observed at 77 K. Resonances from FeSI and FeSII were detected at 15 K. Whereas the observable g factors for FeSII resemble those of other molybdenum hydroxylases, the FeSI center in contrast to most other known FeSI centers has nearly axial symmetry. The EPR features of the redox-active centers of P. putida XDH are very similar to those of eukaryotic XDHs/xanthine oxidases, suggesting that the environment of each center and their functionality are analogous in these enzymes. The midpoint potentials determined for the molybdenum, FeSI and FAD redox couples are close to each other and resemble those of the corresponding centers in eukaryotic XDHs.