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1.
Summary The molybdenum hydroxylases are a ubiquitous class of enzymes which contain molybdenum in association with a low molecular weight cofactor. Genetic evidence suggests that the Drosophila loci, ma-1, cin and lxd are concerned with this cofactor because mutants for any one of these loci simultaneously interrupt activity for two molybdenum hydroxylases, XDH and A0. A third enzyme activity, P0, is also absent in each of the three mutants but evidence classifying P0 as a molybdoenzyme has been lacking. This study utilizes the known tungsten sensitivity of molybdoenzymes to demonstrate directly that pyridoxal oxidase is also a molybdoenzyme. The low molecular weight molybdenum cofactor is found to be severely reduced in extracts of the lxd and cin mutants but ma-1 mutants have high levels of cofactor. A partially purified preparation of XDH crossreacting material from ma-1 was also shown to contain the molybdenum cofactor. These results, considered with data from other workers are taken to indicate that the functions of all three of the loci examined could be concerned with some aspect of cofactor biosynthesis.This work was supported by PHS grant GM 23736 to V. Finnerty  相似文献   

2.
The biochemical effects of several newly induced low xanthine dehydrogenase (lxd) mutations in Drosophila melanogaster were investigated. When homozygous, all lxd alleles simultaneously interrupt each of the molybdoenzyme activities to approximately the same levels: xanthine dehydrogenase, 25%; aldehyde oxidase, 12%; pyridoxal oxidase, 0%; and sulfite oxidase, 2% as compared to the wild type. In order to evaluate potentially small complementation or dosage effects, mutant stains were made coisogenic for 3R. These enzymes require a molybdenum cofactor, and lxd cofactor levels are also reduced to less than 10% of the wild type. These low levels of molybdoenzyme activities and cofactor activity are maintained throughout development from late larval to adult stages. The lxd alleles exhibit a dosage-dependent effect on molybdoenzyme activities, indicating that these mutants are leaky for wild-type function. In addition, cofactor activity is dependent upon the number of lxd + genes present. The lxd mutation results in the production of more thermolabile XDH and AO enzyme activities, but this thermolability is not transferred with the cofactor to a reconstituted Neurospora molybdoenzyme. The lxd gene is localized to salivary region 68 A4-9, 0.1 map unit distal to the superoxide dismutase (Sod) gene.  相似文献   

3.
At least four enzymes contribute to histochemically, electrophoretically, or spectrophotometrically detectable aldehyde oxidase (AO) activity in Drosophila melanogaster. The one we designate AO-1 contributes the majority of activity measured in extracts of whole flies. Pyridoxal oxidase (PO) is also a broad range AO. It is prominent only in midgut and Malpighian tubules, where it apparently accounts for a substantial fraction of total AO activity. The tissue distributions of these enzymes are clearly disparate despite close linkage of their structural loci and parallel dependence on the mal, lxd, and cin loci. A similarly related enzyme, xanthine dehydrogenase (XDH), is detected as an AO only in electrophoretic gels. A fourth broad range AO, not dependent on mal, lxd, and cin, is confined to the ejaculatory bulb. A similar array of AO isozymes is present in phylogenetically distant Drosophila species.This work was supported by NIH Grant 2 RO1 HD 10723.  相似文献   

4.
Summary A reliable method for visualizing the Drosophila enzyme pyridoxal oxidase in polyacrylamide gels is described. Antiserum to pyridoxal oxidase has been produced and used in quantitative immunoelectrophoresis to determine the relative amounts of pyridoxal oxidase cross reacting material (CRM) in several mutants including lpo, lxd, ma-l and cin. The lpo variant did not have CRM for PO, thus further supporting the idea that it represents a structural gene for pyridoxal oxidase in Drosophila. CRM for PO was found in ma-l and lxd indicating that their effects upon the enzyme are probably post-translational. No CRM for PO could be found in the cin mutants.This work was supported by PHS grant GM 23736 to V. Finnerty  相似文献   

5.
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.  相似文献   

6.
Molybdoenzymes are involved in a variety of essential pathways including nitrate assimilation, sulfur and/or purine metabolism and abscisic acid biosynthesis. Most organisms produce several such enzymes requiring a molybdopterin cofactor for catalytic function. Mutations that result in a lack of the molybdopterin cofactor display a pleiotropic loss of molybdoenzyme activities, and this phenotype has been used to identify genes involved in cofactor biosynthesis or utilization. Although several cofactor genes have been analyzed in prokaryotes, much less is known concerning eukaryotic molybdenum cofactor (MoCF) genes. This work is focused on the Drosophila MoCF gene cinnamon (cin) which encodes a multidomain protein, CIN, that shows significant similarity to three proteins encoded by separate prokaryotic MoCF genes. These domains are also present in the product of cnx1, an Arabidopsis MoCF gene, and in GEPHYRIN, a rat protein thought to organize the glycine receptor, GlyR, within the postsynaptic membrane. Since this apparent consolidation of separate prokaryotic genes into a single eukaryotic gene is a feature of other conserved metabolic pathways, we wished to determine whether the protein's function is also conserved. This report shows that the plant gene cnx1 can rescue both enzymatic and physiological defects of Drosophila carrying cin mutations, indicating that the two genes serve similar or identical functions. In addition, we have investigated the relationship between CINNAMON and GEPHYRIN, using immunohistochemical methods to localize the CIN protein in Drosophila embryos. Most of the CIN protein, like GEPHYRIN in the rat CNS, is localized to the cell borders and shows a tissue-specific pattern of expression. In a parallel study, antibody to GEPHYRIN revealed the same tissue-specific expression pattern in fly embryos. Both antibodies show altered staining patterns in cin mutants. Taken together, these results suggest that GEPHYRIN may also carry out a MoCF-related function. Received: 14 September 1998 / Accepted: 8 March 1999  相似文献   

7.
The substrate specificities of aldehyde and pyridoxal oxidases in Drosophila melanogaster have been determined with a variety of aliphatic and aromatic aldehydes. This analysis has led to the discovery that 2,4,5-trimethoxy-benzaldehyde is a specific substrate for pyridoxal oxidase, as based on the histochemical distribution of oxidase activity, the absence of enzymatic activity in the lpo 1strains, and the dosage dependence on the number of lpo +genes present. The tissue-specific localization of aldehyde oxidase (AO) and pyridoxal oxidase (PO) in the larval and adult structures showed that AO was present in all the major internal organs of the larvae and adults, including brain, imaginal discs, Malpighian tubules, digestive system, and reproductive structures. Pyridoxal oxidase is present in many of the same structures which possess AO, but is missing from the cardia, crop, imaginal discs, ovarian follicle cells, paragonia, pericardial cells, and wreath cells. The only structure which possesses PO but lacks AO is the larval salivary gland. These histochemical differences in AO and PO distribution were also confirmed by enzymatic analysis of the activities present in homogenates of ovaries, paragonia, and salivary glands. The general pattern of enzyme expression appears to be established during embryogenesis and maintained throughout the life of the individual.This work was supported by NIH Grants AG01975 and GM27866.This paper is dedicated to Professor Donald F. Poulson, Yale University, a pioneer in Drosophila developmental genetics.  相似文献   

8.
The molybdenum cofactor (MoCo) is a component of aldehyde oxidase (AO EC 1.2.3.1), xanthine dehydrogenase (XDH EC 1.2.1.37) and nitrate reductase (NR, EC 1.6.6.1). The activity of AO, which catalyses the last step of the synthesis of abscisic acid (ABA), was studied in leaves and roots of barley (Hordeum vulgare L.) plants grown on nitrate or ammonia with or without salinity. The activity of AO in roots was enhanced in plants grown with ammonium while nitrate-grown plants exhibited only traces. Root AO in barley was enhanced by salinity in the presence of nitrate or ammonia in the nutrient medium while leaf AO was not significantly affected by the nitrogen source or salinity of the medium.Salinity and ammonium decreased NR activity in roots while increasing the overall MoCo content of the tissue. The highest level of AO in barley roots was observed in plants grown with ammonium and NaCl, treatments that had only a marginal effect on leaf AO. ABA concentration in leaves of plants increased with salinity and ammonium.Keywords: ABA, aldehyde oxidase, ammonium, nitrate, salinity.   相似文献   

9.
Sulfite oxidase (sulfite: ferricytochrome c oxidoreductase; EC 1.8.2.1) has been detected in Drosophila melanogaster and some of its properties have been studied. In most respects this enzyme resembles the mammalian sulfite oxidases except for its molecular weight (148,000), which is somewhat higher than that of rat sulfite oxidase (116,000). Cytochrome c, potassium-ferricyanide, and oxygen can serve as electron acceptors in the oxidation of sulfite by the enzyme. Although definite evidence can be obtained only through the analysis of the pure enzyme, experiments involving tungstate feeding suggest that Drosophila sulfite oxidase is most probably a molybdoenzyme. Extracts of mal flies show normal levels of sulfite oxidase, whereas lxd flies have only 5–10% of the activity of wild type, and in cin flies the enzyme is apparently absent. While it is possible that the lxd and cin mutations are at some level responsible for the defective synthesis of a molybdenum-containing cofactor (supposed to be present in most molybdoenzymes), the evidence accumulated so far by several authors and the results of the present investigation argue against the involvement of a Mo cofactor in the multiple enzyme deficiencies observed in mal flies.  相似文献   

10.
Xanthine dehydrogenase (XDH) is the initial enzyme in the purine catabolic pathway ofN. crassa. Secondary nitrogen sources such as purines are metabolized when preferred sources of reduced nitrogen (ammonium or glutamine) are unavailable. XDH synthesis is regulated by glutamine repression and uric acid induction. Thenit-2 locus is believed to encode atrans-acting positive regulator essential for the expression of genes encoding enzymes involved in secondary pathways of nitrogen acquisition, such as XDH and nitrate reductase. However, immunoblot analyses and enzyme assays reveal that XDH protein is synthesized and XDH activity is expressed innit-2 mutants. Nevertheless, XDH responds to nitrogen metabolite repression. The generality thatnit-2 is an obligate control element in nitrogen metabolite repression is questioned. Additionally, mutants defective in XDH activity, namely,xdh-1 and the molybdenum cofactor mutantsnit-1, -7, -8 and -9, are observed to grow on xanthine but not hypoxanthine.This research was supported in part by National Science Foundation Grant DMB 8516203.  相似文献   

11.
Four different genes are known to affect aldehyde oxidase activity (AO) in Drosophila melanogaster. Mutants at each of these loci eliminate AO activity and simultaneously eliminate detectable AO-crossing reacting material (AO-CRM) even though only one is the structural gene for AO (Aldoxn). The other three genes (cin1, lxd and mal) coordinately "control" the levels of activity of AO and two related enzymes, xanthine dehydrogenase (XDH) and pyridoxal oxidase (PO). Contrary to their effects on AO-CRM, neither of these three mutants eliminate XDH-CRM. A model of interaction of these enzymes and genes controlling their activities is discussed.  相似文献   

12.
Drosophila ma-l gene was suggested to encode an enzyme for sulfuration of the desulfo molybdenum cofactor for xanthine dehydrogenase (XDH) and aldehyde oxidase (AO). The human molybdenum cofactor sulfurase (HMCS) gene, the human ma-l homologue, is therefore a candidate gene responsible for classical xanthinuria type II, which involves both XDH and AO deficiencies. However, HMCS has not been identified as yet. In this study, we cloned the HMCS gene from a cDNA library prepared from liver. In two independent patients with classical xanthinuria type II, we identified a C to T base substitution at nucleotide 1255 in the HMCS gene that should cause a CGA (Arg) to TGA (Ter) nonsense substitution at codon 419. A classical xanthinuria type I patient and healthy volunteers lacked this mutation. These results indicate that a functional defect of the HMCS gene is responsible for classical xanthinuria type II, and that HMCS protein functions to provide a sulfur atom for the molybdenum cofactor of XDH and AO.  相似文献   

13.
Two nitrate reductase (NaR)-deficient mutants of pea (Pisum sativum L.), E1 and A300, both disturbed in the molybdenum cofactor function and isolated, respectively, from cv Rondo and cv Juneau, were tested for allelism and were compared in biochemical and growth characteristics. The F1 plants of the cross E1 × A300 possessed NaR and xanthine dehydrogenase (XDH) activities comparable to those of the wild types, indicating that these mutants belong to different complementation groups, representing two different loci. Therefore, mutant E1 represents, besides mutant A300 and the allelic mutants A317 and A334, a third locus governing NaR and is assigned the gene destignation nar 3. In comparison with the wild types, cytochrome c reductase activity was increased in both mutants. The mutants had different cytochrome c reductase distribution patterns, indicating that mutant A300 could be disturbed in the ability to dimerize NaR apoprotein monomers, and mutant E1 in the catalytic function of the molybdenum cofactor. In growth characteristics studied, A300 did not differ from the wild types, whereas fully grown leaves of mutant E1 became necrotic in soil and in liquid media containing nitrate.  相似文献   

14.
Molybdoenzymes are involved in a variety of essential pathways including nitrate assimilation, sulfur and/or purine metabolism and abscisic acid biosynthesis. Most organisms produce several such enzymes requiring a molybdopterin cofactor for catalytic function. Mutations that result in a lack of the molybdopterin cofactor display a pleiotropic loss of molybdoenzyme activities, and this phenotype has been used to identify genes involved in cofactor biosynthesis or utilization. Although several cofactor genes have been analyzed in prokaryotes, much less is known concerning eukaryotic molybdenum cofactor (MoCF) genes. This work is focused on the Drosophila MoCF gene cinnamon (cin) which encodes a multidomain protein, CIN, that shows significant similarity to three proteins encoded by separate prokaryotic MoCF genes. These domains are also present in the product of cnx1, an Arabidopsis MoCF gene, and in GEPHYRIN, a rat protein thought to organize the glycine receptor, GlyR, within the postsynaptic membrane. Since this apparent consolidation of separate prokaryotic genes into a single eukaryotic gene is a feature of other conserved metabolic pathways, we wished to determine whether the protein's function is also conserved. This report shows that the plant gene cnx1 can rescue both enzymatic and physiological defects of Drosophila carrying cin mutations, indicating that the two genes serve similar or identical functions. In addition, we have investigated the relationship between CINNAMON and GEPHYRIN, using immunohistochemical methods to localize the CIN protein in Drosophila embryos. Most of the CIN protein, like GEPHYRIN in the rat CNS, is localized to the cell borders and shows a tissue-specific pattern of expression. In a parallel study, antibody to GEPHYRIN revealed the same tissue-specific expression pattern in fly embryos. Both antibodies show altered staining patterns in cin mutants. Taken together, these results suggest that GEPHYRIN may also carry out a MoCF-related function.  相似文献   

15.
Rocket immunoelectrophoresis was used to estimate xanthine dehydrogenase cross-reacting material (XDH-CRM) in strains containing the cin 1 and cin 9 mutant genes, which are deficient in XDH enzymatic activity. CRM levels were determined as percentages of CRM in the Oregon-R wild-type strain. The mutant strains contain 72 and 76% of Oregon-R CRM, respectively. CRM levels in strains containing the XDH-deficient mutant genes lxd and mal are 93 and 105%, respectively. The high levels of CRM in these four mutant strains indicate that the primary effects of the mutant genes are on the function of XDH protein rather than its accumulation.This research was supported by a grant from the Natural Sciences and Engineering Research Council of Canada to L. W. B.  相似文献   

16.
The molybdenum cofactor is shared by nitrate reductase (NR), xanthine dehydrogenase (XDH), and abscisic acid (ABA) aldehyde oxidase in higher plants (M. Walker-Simmons, D.A. Kudrna, R.L. Warner [1989] Plant Physiol 90:728-733). In agreement with this, cnx mutants are simultaneously deficient for these three enzyme activities and have physiological characteristics of ABA-deficient plants. In this report we show that aba1 mutants, initially characterized as ABA-deficient mutants, are impaired in both ABA aldehyde oxidase and XDH activity but overexpress NR. These characteristics suggest that aba1 is in fact involved in the last step of molybdenum cofactor biosynthesis specific to XDH and ABA aldehyde oxidase; aba1 probably has the same function as hxB in Aspergillus. The significance of NR overexpression in aba1 mutants is discussed.  相似文献   

17.
The effects of five new mal alleles on aldehyde oxidase (AO) and xanthine dehydrogenase (XDH) activities and CRM levels in Drosophila melanogaster are described. These alleles were isolated by taking full advantage of the pleiotropic phenotype exhibited by all previously described mal alleles and represent at least three unique examples of mal function. Al least one of these alleles is a representative of a new complementation group. Two other alleles exhibit a wild-type eye color in homozygous stock and one of these is "leaky", exhibiting some 50% of the XDH activity normally found in Oregon-R control flies and some 12% of the AO activity. CRM and activity levels have been quantitated for both enzymes in all allelic heterozygotes. XDH-CRM levels vary only slightly around wild-type levels while AO-CRM levels appear much more sensitive to mutational alterations.  相似文献   

18.
【目的】微小杆菌属(Exiguobacterium)细菌广泛分布于海洋及非海洋环境中,具有多种代谢途径以适应复杂多样的生境。本研究从能量代谢途径角度出发,探究该属菌株对不同生境的适应能力。【方法】从美国国家生物科技数据中心(National Center for Biotechnology Information, NCBI)数据库中获取146个Exiguobacterium属菌株的基因组,查找并统计光营养、厌氧呼吸和底物代谢等多种能量代谢途径的关键蛋白或关键酶基因在各菌株基因组中的分布,包括光营养型的视紫红质基因、厌氧呼吸营养型的钼辅因子合成蛋白基因,以及底物代谢营养型中乙醛酸分流途径的异柠檬酸裂解酶及苹果酸合酶基因等。根据对应的氨基酸序列构建视紫红质、MoaC和异柠檬酸裂解酶的系统发育树,分析不同能量代谢途径在该属菌株进化过程中的保守性,推测其对于该属菌株的重要性。【结果】Exiguobacterium属中50%的种具有视紫红质基因,其中分离自非海洋生境的菌株更趋向于含有视紫红质基因。本研究所统计的全部非海洋生境菌株中,含有视紫红质基因的菌株占比约为70%,而在海洋生境菌株中该比例...  相似文献   

19.
Phenoloxidase (PO) activity in the albumen gland (AG) and egg masses (EM) ofBiomphalaria glabrata was assessed using high-performance liquid chromatography combined with electrochemical detection and colorimetric techniques. Both AG and EM extracts catalyzed the hydroxylation ofl-tyrosine (monophenol oxidase activity, MPO) and oxidation ofl-dopa (diphenol oxidase activity, DPO). However, no PO activity was found in the ovotestis. Both MPO and DPO activities in AG and EM were significantly inhibited by 1-phenyl-2-thiourea and inactivated by boiling. Approximately 35% of MPO and 44% of DPO activities were detected in the soluble fraction of homogenized EM, in contrast to that of homogenized AG, which contained about 5% and 12%, respectively, of MPO and DPO activities. N-acetyl-dopamine, a diphenolic compound, enhanced the hydroxylation of tyrosine by the PO. The presence of both MPO and DPO activities also was confirmed by the accelerated accumulation of dopachrome during incubation of EM extracts withl-tyrosine in the absence of ascorbate. Temperature and pH optima for this enzyme were 30°C and 7.5, respectively. The potential roles of PO in egg formation inB. glabrata are discussed.  相似文献   

20.
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.  相似文献   

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