Protein catabolism in mosquitoes: ureotely and uricotely in larval and imaginal Aedes aegypti

Catabolism of excess dietary protein by Aedes aegypti was investigated during larval development, during and after metamorphosis. Activity profiles were established for xanthine dehydrogenase (XDH, uricotelic pathway) and arginase (ureotelic pathway). Both enzymes are active at all times during the life-cycle. During the aquatic larval and pupal instars, XDH and arginase activities increase with body size. Maximal activities of these two enzyme systems coincide with the time of metamorphic restructuring.Both enzymes are found in the fatbody tissue: XDH activity is found in 80% of the tissue, while arginase activity is distributed equally between abdominal fatbody and the thorax. This might indicate a role for arginase other than catabolic, such as energy metabolism.Arginase activity is high in the aquatic instars and low in sugar-fed females but increases after blood-feeding. XDH activity, also high in larvae and pupae, increases markedly after a blood meal.Larval excretion is characterized by the ureotelic pathway. The pupae as closed systems excrete neither uric acid nor urea; urate accumulates during larval and pupal periods, is conserved throughout metamorphosis, and is finally voided with the meconium by the teneral imago. This presents a form of transient storage-excretion.     

Regulation of purine hydroxylase and xanthine dehydrogenase from Clostridium purinolyticum in response to purines, selenium, and molybdenum

The discovery that two distinct enzyme catalysts, purine hydroxylase (PH) and xanthine dehydrogenase (XDH), are required for the overall conversion of hypoxanthine to uric acid by Clostridium purinolyticum was unexpected. In this reaction sequence, hypoxanthine is hydroxylated to xanthine by PH and then xanthine is hydroxylated to uric acid by XDH. PH and XDH, which contain a labile selenium cofactor in addition to a molybdenum cofactor, flavin adenine dinucleotide, and FeS centers, were purified and partially characterized as reported previously. In the present study, the activities of these two enzymes were measured in cells grown in media containing various concentrations of selenite, molybdate, and various purine substrates. The levels of PH protein in extracts were determined by immunoblot assay. The amount of PH protein, as well as the specific activities of PH and XDH, increased when either selenite or molybdate was added to the culture medium. PH levels were highest in the cells cultured in the presence of either adenine or purine. XDH activity increased dramatically in cells grown with either xanthine or uric acid. The apparent increases in protein levels and activities of PH and XDH in response to selenium, molybdenum, and purine substrates demonstrate that these enzymes are tightly regulated in response to these nutrients.     

Comprehensive analysis of mechanism underlying hypouricemic effect of glucosyl hesperidin

Hyperuricemia is caused by hepatic overproduction of uric acid and/or underexcretion of urate from the kidneys and small intestine. Although increased intake of citrus fruits, a fructose-rich food, is associated with increased risk of gout in humans, hesperidin, a flavonoid naturally present in citrus fruits, reportedly reduces serum uric acid (SUA) levels by inhibiting xanthine oxidase (XOD) activity in rats. However, the effects of hesperidin on renal and intestinal urate excretion were previously unknown. In this study, we used glucosyl hesperidin (GH), which has greater bioavailability than hesperidin, to clarify comprehensive mechanisms underlying the hypouricemic effects of hesperidin in vivo. GH dose-dependently decreased SUA levels in mice with hyperuricemia induced by potassium oxonate and a fructose-rich diet, and inhibited XOD activity in the liver. GH decreased renal urate excretion without changes in kidney URAT1, ABCG2 or GLUT9 expressions, suggesting that reducing uric acid pool size by inhibiting XOD decreased renal urate excretion. We also found that GH had no effect on intestinal urate excretion or protein expression of ABCG2. Therefore, we concluded that GH exhibits a hypouricemic effect by inhibiting XOD activity in the liver without increasing renal or intestinal urate excretion. Of note, this is the first study to elucidate the effect of a flavonoid on intestinal urate excretion using a mice model, whose findings should prove useful in future food science research in the area of urate metabolism. Taking these findings together, GH may be useful for preventing hyperuricemia, especially in people with the overproduction type.     

Nitrogen partitioning and blood meal utilization by Aedes aegypti (Diptera Culicidae)

The utilization of the blood meal by mosquitoes was investigated by first feeding females quantities of blood ranging from 1 to 5 mg, and then analyzing the faeces for the various by-products of protein catabolism that were subsequently eliminated. The nitrogeneous waste products in order of importance were uric acid, histidine, ammonia and arginine. Only traces of the other amino acids were excreted.The total amount of each faecal substance varied linearly with the quantity of blood ingested, however their relative proportions did not change. Regardless of blood meal size the quantily of uric acid and ammonia produced indicates that about 80% of the non-histidine and arginine amino acids are deaminated and utilized for metabolic purposes other than egg protein synthesis.Most of the histidine and about one half of the arginine content of the blood were excreted as free amino acids, but the other amino acids were lost in trace amounts.Nineteen per cent of the total ingested amino acids was incorporated into soluble yolk proteins and this proportion was constant even for small blood meals that result in a reduction in the numbers of eggs produced.The comparative aspects of nitrogen partitioning and blood meal utilization by haematophagous insects, as well as the factors that affect blood meal utilization and fecundity in A. aegypti are discussed.     

Xanthine dehydrogenase expression inNeurospora crassa does not require a functionalnit-2 regulatory gene

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.     

The RNAi-mediated silencing of xanthine dehydrogenase impairs growth and fertility and accelerates leaf senescence in transgenic Arabidopsis plants

Xanthine dehydrogenase (XDH) is a ubiquitous enzyme involved in purine metabolism which catalyzes the oxidation of hypoxanthine and xanthine to uric acid. Although the essential role of XDH is well documented in the nitrogen-fixing nodules of leguminous plants, the physiological importance of this enzyme remains uncertain in non-leguminous species such as Arabidopsis. To evaluate the impact of an XDH deficiency on whole-plant physiology and development in Arabidopsis, RNA interference (RNAi) was used to generate transgenic lines of this species in which AtXDH1 and AtXDH2, the two paralogous genes for XDH in this plant, were silenced simultaneously. The nearly complete reduction in the total XDH protein levels caused by this gene silencing resulted in the dramatic overaccumulation of xanthine and a retarded growth phenotype in which fruit development and seed fertility were also affected. A less severe silencing of XDH did not cause these growth abnormalities. The impaired growth phenotype was mimicked by treating wild-type plants with the XDH inhibitor allopurinol, and was reversed in the RNAi transgenic lines by exogenous supplementation of uric acid. Inactivation of XDH is also associated with precocious senescence in mature leaves displaying accelerated chlorophyll breakdown and by the early induction of senescence-related genes and enzyme markers. In contrast, the XDH protein levels increase with the aging of the wild-type leaves, supporting the physiological relevance of the function of this enzyme in leaf senescence. Our current results thus indicate that XDH functions in various aspects of plant growth and development.     

A deleted portion of one of the two xanthine dehydrogenase genes causes translucent larval skin in the oq mutant of the silkworm (Bombyx mori)

A silkworm mutant, oq, has translucent larval skin because it is deficient in xanthine dehydrogenase (XDH) activity and is unable to synthesize uric acid, which is normally accumulated in the larval epidermis and makes the skin white and opaque. Two XDH bands were found in zymograms of the silkworm fat body: an intense band (XDHalpha) and a faint one (XDHbeta). The oq mutant lacks only XDHalpha, which seemed to be the major source of XDH activity in the fat body. An 8-bp deletion found in BmXDH1, a silkworm XDH gene, generates a premature stop codon. The resulting truncated BmXDH1 protein lacks three molybdenum cofactor-binding domains necessary for enzyme activity. BmXDH2, the other XDH gene, does not show any apparent deficiencies. BmXDH1 expressed in yeast cells yielded an activity band with the same mobility as that of XDHalpha in zymograms. BmXDH1 of the oq mutant did not yield active XDH in yeast, while the activity was restored by filling in the deleted sequence. These results showed that BmXDH1 deletion in the oq mutant is responsible for the absence of significant XDH activity, resulting in the translucent larval skin of the mutant phenotype.     

Is xanthine dehydrogenase involved in response of pea plants (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) to salinity or ammonium treatment?

The effect of salinity and different nitrogen sources on the level of xanthine dehydrogenase (XDH) activity in roots and leaves of pea plants was investigated. Two bands of xanthine dehydrogenase activity (XDH-R2, XDH-R3) were detected in roots after native PAGE and staining with hypoxanthine as substrate. Only one band of XDH activity (XDH-L1) was detected in leaf extracts. Within leaves of three different ages the highest XDH activity was detected in young leaves both under control as well as stress conditions. Salinity did not affect significantly the activity of XDH in pea roots, however, depressed XDH activity in leaves. A significant increase of XDH activity both in roots and leaves was observed only when ammonium was applied as the sole N source. Increased concentration of ureides in the xylem sap of pea plants was observed for both ammonium and high salt treatments, although the higher content of ureides in the xylem sap of 100 mM NaCl treated plants may be rather a result of lower rate of exudation from roots than of increased root ureide biosynthesis. Thus, the changes of root and leaf XDH activity in pea plants seem to be tightly correlated with ureide synthesis that is induced by NH ₄ ⁺ , the product of N fixation, and rather than by salinity. A contribution of pea XDH in increased oxygen species or uric acid production under saline conditions seems to be less than likely.     

In vitro and in vivo activity of soluble cross-linked uricase-albumin polymers: A model for enzyme therapy

The possibility of using soluble cross-linked enzyme-albumin polymers as a means of enzyme therapy for the treatment of certain enzyme deficiency diseases is investigated. The hyperuricemic Dalmatian coach hound is used as an experimental animal and the enzyme uricase (urate oxidase) as the administered enzyme. Chemically cross-linking uricase with an excess of canine albumin yields a soluble enzyme polymer that is significantly more heat stable and resistant to proteolytic activity than the native enzyme. Intravenous administration of similar amounts of enzyme in the native or polymeric form indicated that the “solubilized” enzyme survived in the circulation for a longer period of time (clearance half-time of 26 hours as opposed to 4 hours for the native enzyme) and was more effective in lowering plasma uric acid levels for longer periods. In vivo administration of the native enzyme lowered uric acid levels by about 35% with a return to normal levels with a half-time of about 24 hours. Subsequent injections of native uricase proved less effective and produced a severe hypersensitivity reaction following the third injection. No such adverse reactions or decreased activity of the administered “solubilized” uricase-albumin polymers were observed. The plasma uric acid levels were decreased by about 40% and only after 48 hours did the substrate levels begin to rise towards their resting levels.     

The association between serum ferritin and uric acid in humans

OBJECTIVE: Urate forms a coordination complex with Fe(3+) which does not support electron transport. The only enzymatic source of urate is xanthine oxidoreductase. If a major purpose of xanthine oxidoreductase is the production of urate to function as an iron chelator and antioxidant, a system for coupling the activity of this enzyme to the availability of catalytically-active metal would be required. We tested the hypothesis that there is an association between iron availability and urate production in healthy humans by correlating serum concentrations of ferritin with uric acid levels. MATERIALS AND METHODS: The study population included 4932 females and 4794 males in the National Health and Nutrition Examination Survey III. They were 20 years of age or older and in good health. RESULTS: Serum concentrations of ferritin correlated positively with uric acid levels in healthy individuals (R(2) = 0.41, p<0.001). This association was independent of an effect of gender, age, race/ethnic group, body mass, and alcohol consumption. CONCLUSIONS: The relationship between serum ferritin and uric acid predicts hyperuricemia and gout in groups with iron accumulation. This elevation in the production of uric acid with increased concentrations of iron could possibly reflect a response of the host to diminish the oxidative stress presented by available metal as the uric acid assumes the empty or loosely bound coordination sites of the iron to diminish electron transport and subsequent oxidant generation.     

Purification and properties of milk xanthine dehydrogenase.

Milk xanthine oxidase (XO) has been prepared in a dehydrogenase form (XDH) by purifying the enzyme in the presence of 2.5 mM dithiothreitol. Unlike XO, which reacts rapidly only with oxygen and not with NAD, the XDH form of the enzyme reacts rapidly with NAD. XDH has a turnover number for the NAD-dependent conversion of xanthine to urate of 380 mol/min/mol at pH 7.5, 25 degrees C, with a Km = < or = 1 microM for xanthine and a Km = 7 microM for NAD, but has very little O2-dependent activity. There is evidence that the two forms of the enzyme have different flavin environments: XDH stabilizes the neutral form of the flavin semiquinone and XO does not. Further, XDH binds the artificial flavin 8-mercapto-FAD in its neutral form, shifting the pK of this flavin by 5 pH units, while XO binds 8-mercapto-FAD in its benzoquinoid anionic form. XDH can be converted back to the XO form by the addition of three to four equivalents of the disulfide-forming reagent 4,4'-dithiodipyridine, suggesting that, in the XDH form of the enzyme, disulfide bonds are broken; this may cause a conformational change which creates a binding site for NAD and changes the protein structure near the flavin.     

Association between intronic SNP in urate-anion exchanger gene, SLC22A12, and serum uric acid levels in Japanese

Serum uric acid levels are maintained by urate synthesis and excretion. URAT1 (coded by SLC22CA12) was recently proposed to be the major absorptive urate transporter protein in the kidney regulating blood urate levels. Because genetic background is known to affect serum urate levels, we hypothesized that genetic variations in SLC22A12 may predispose humans to hyperuricemia and gout. We investigated rs893006 polymorphism (GG, GT and TT) in SLC22A12 in a total of 326 Japanese subjects. Differences in clinical characteristics among the genotype groups were tested by the analysis of variance (ANOVA). In male subjects, mean serum uric acid levels were significantly different among the three genotypes. Levels in the GG genotype subjects were the highest, followed by those with the GT and TT genotypes. However, no differences between the groups were seen in the distributions of creatinine, Fasting plasma glucose (FPG), HbA(1c), total cholesterol, triglyceride, HDL cholesterol levels or BMI. A single nucleotide polymorphism (SNP) in the urate transporter gene SLC22CA12 was found to be associated with elevated serum uric acid levels among Japanese subjects. This SNP may be an independent genetic marker for predicting hyperuricemia.     

The function of subcellular fractions in the oxidation of glutathione in rat liver homogenate

1. The aerobic loss of GSH added to the supernatant fraction from rat liver is much increased by including the microsome fraction, which both inhibits the concurrent reduction of the GSSG formed and also augments the net oxidation rate. 2. Oxidation occurs with a mixture of dialysed supernatant and a protein-free filtrate; the latter is replaceable by hypoxanthine and the former by xanthine oxidase, whereas fractions lacking this enzyme give no oxidation. 3. In all these instances augmentation occurs with microsomes, with fractions having urate oxidase activity and with the purified enzyme; uric acid and microsomes alone also support the oxidation. 4. Evidence implicating additional protein factors is discussed. 5. It is suggested that GSH oxidation by homogenate is linked through glutathione peroxidase to the reaction of endogenous substrate with supernatant xanthine oxidase and of the uric acid formed with peroxisomal urate oxidase.     

Uric acid synthesis by avian exocrine pancreas

1. Chicken pancreas has been shown to synthesize and secrete uric acid. Uric acid synthesis from xanthine in vitro by isolated pancreatic acinii is saturable and dependent on the activity of xanthine dehydrogenase. 2. Chicken pancreas is unable to synthesize uric acid de novo but the variety of substrates which support urate synthesis suggests that it occurs by the purine degradation pathway.     

Crystal structures of the active and alloxanthine-inhibited forms of xanthine dehydrogenase from Rhodobacter capsulatus.

Xanthine dehydrogenase (XDH), a complex molybdo/iron-sulfur/flavoprotein, catalyzes the oxidation of hypoxanthine to xanthine followed by oxidation of xanthine to uric acid with concomitant reduction of NAD+. The 2.7 A resolution structure of Rhodobacter capsulatus XDH reveals that the bacterial and bovine XDH have highly similar folds despite differences in subunit composition. The NAD+ binding pocket of the bacterial XDH resembles that of the dehydrogenase form of the bovine enzyme rather than that of the oxidase form, which reduces O(2) instead of NAD+. The drug allopurinol is used to treat XDH-catalyzed uric acid build-up occurring in gout or during cancer chemotherapy. As a hypoxanthine analog, it is oxidized to alloxanthine, which cannot be further oxidized but acts as a tight binding inhibitor of XDH. The 3.0 A resolution structure of the XDH-alloxanthine complex shows direct coordination of alloxanthine to the molybdenum via a nitrogen atom. These results provide a starting point for the rational design of new XDH inhibitors.     

Isolation and characterization of the Xanthine dehydrogenase gene of the Mediterranean fruit fly, Ceratitis capitata

Xanthine dehydrogenase (XDH) is a member of the molybdenum hydroxylase family of enzymes catalyzing the oxidation of hypoxanthine and xanthine to uric acid. The enzyme is also required for the production of one of the major Drosophila eye pigments, drosopterin. The XDH gene has been isolated in many species representing a broad cross section of the major groups of living organisms, including the cDNA encoding XDH from the Mediterranean fruit fly Ceratitis capitata (CcXDH) described here. CcXDH is closely related to other insect XDHs and is able to rescue the phenotype of the Drosophila melanogaster XDH mutant, rosy, in germline transformation experiments. A previously identified medfly mutant, termed rosy, whose phenotype is suggestive of a disruption in XDH function, has been examined for possible mutations in the XDH gene. However, we find no direct evidence that a mutation in the CcXDH gene or that a reduction in the CcXDH enzyme activity is present in rosy medflies. Conclusive studies of the nature of the medfly rosy mutant will require rescue by germline transformation of mutant medflies.     

Biogenesis and metabolic significance of microbodies in urate-utilizing yeasts

Growth of Candida famata and Trichosporon cutaneum on uric acid as the sole source of carbon and nitrogen was associated with the development of a number of microbodies in the cells. Cytochemical staining experiments showed that the organelles contained urate oxidase, a key enzyme of uric acid metabolism, and catalase. Transfer of cells, precultured on glucose or glycerol, into uric acid-containing media indicated that these microbodies originated from the organelles, originally present in the inoculum cells, by growth and division. In urate-grown C. famata the microbodies were frequently observed in large clusters; in both organisms they existed in close association with mitochondria and strands of ER. The organelles lacked crystalline inclusions. In freeze-fractured cells their surrounding membranes showed smooth fracture faces.Exposure of urate-grown cells to glucose-excess conditions led to a rapid inactivation of urate oxidase activity but catalase was only slightly inactivated. Glucose-induced enzyme inactivation was not associated with the degradation of the microbodies present in the cells. Similarly, repression of urate oxidase synthesis by ammonium ions also did not lead to the degradation of peroxisomes.     

Gene silencing in phlebotomine sand flies: Xanthine dehydrogenase knock down by dsRNA microinjections

Lutzomyia longipalpis are vectors of medically important visceral leishmaniasis in South America. Blood-fed adult females digest large amounts of protein, and xanthine dehydrogenase is thought to be a key enzyme involved in protein catabolism through the production of urate. Large amounts of heme are also released during digestion with potentially damaging consequences, as heme can generate oxygen radicals that damage lipids, proteins and nucleic acids. However, urate is an antioxidant that may prevent such oxidative damage produced by heme. We investigated xanthine dehydrogenase by developing the RNAi technique for sand flies and used this technique to knock down the Lu. longipalpis xanthine dehydrogenase gene to evaluate its role in survival of adult females after blood feeding. The gene sequence of Lu. longipalpis xanthine dehydrogenase is described together with expression in different life cycle stages and RNAi knock down. Semi-quantitative RT-PCR of xanthine dehydrogenase expression showed a significant increase in expression after bloodmeal ingestion. Microinjection of dsRNA via the thorax of 1-day-old adult female sand flies resulted in approximately 40% reduction of xanthine dehydrogenase gene expression in comparison to flies injected with a control dsRNA. A significant reduction of urate in the whole body and excretions of Lu. longipalpis was observed after dsRNA xanthine dehydrogenase microinjection and feeding 96h later on rabbit blood. Sand flies injected with XDH dsRNA also exhibit significantly reduced life span in comparison with the mock-injected group when fed on sucrose or when rabbit blood fed, showing that urate could be indeed an important free radical scavenger in Lu. longipalpis. The demonstration of xanthine dehydrogenase knock down by dsRNA microinjection, low mortality of microinjected insects and the successful bloodfeeding of injected insects demonstrated the utility of RNAi as a tool for functional analysis of genes in phlebotomine sand flies.     

Uric acid as electronic acceptor of chicken liver's XDH (author's transl)]

Uric acid seems to act as an electronic acceptor in the dehydrogenation of hypoxanthine catalyzed by chicken liver's xanthinedehydrogenase (XDH). Oxidation was observed in crude homogenates under anaerobic conditions, although dialyzed homogenates or purified hepatic XDH also induce a similar action either in aerobic or anaerobic conditions. The reaction pH optimum is about 6.0. Xanthine appears to be the only inhibited product of the reaction when its concentration is greater than 1 X 10(-4) M. When hypoxanthine and uric acid concentrations exceed 2 X 10(-3) M and 1 X 10(-4) M, respectively, they induce inhibition by substrate. Purine is a fairly good substrate of XDH when uric acid acts as acceptor. Allopurinol inhibits hypoxanthine oxidation by uric acid in the presence of XDH. XDH also catalyzes the dismutation of xanthine to hypoxanthine and uric acid.     

Relevance of purine catabolism to hypoxia and recovery in euryoxic and stenoxic marine invertebrates,particularly bivalve molluscs

1. Activities of xanthine dehydrogenase (XDH) and xanthine oxidase (XOD) were measured in a variety of euryoxic and stenoxic marine molluscs.2. Euryoxic bivalves contain only XDH activity which, unlike the mammalian enzyme, is not converted to XOD during anoxic exposure.3. XOD activity was detected predominantly in stenoxic bivalves such as Pecten maximus, Placopeclen magellanicus, and in the cephalopod Loligo opalescens. Although extremely variable, XOD activity increased 4-fold in Cardium edule and 13-fold in Pecten maximus during anoxic exposures of 56 hr and 0.5 hr respectively.4. The data suggest that euryoxic species may tolerate anoxic-normoxic transitions in part by possessing a form of XDH that resists conversion to XOD (a source of Superoxide radicals responsible for ischemia-reperfusion tissue injury in mammals).5. XDH activities in Carcinus maenas digestive gland are sufficient to account fully for the urate reported to accumulate during hypoxia.