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PubMed1982

Physicochemical properties and states of sulfhydryl groups of uricase from Candida utilis.

Highly purified uricase [urate: oxygen oxidoreductase, EC 1.7.3.3] was obtained from Candida utilis by affinity chromatography with xanthine-agarose conjugate followed by chromatography with Sephadex G-200 in the presence of dithiothreitol. The uricase molecule had a molecular weight of 120,000 and was composed of four identical subunits with a molecular weight of 30,000. The amino acid composition was determined and the N-terminal amino acid was identified as methionine. Other physiochemical properties obtained were as follows; isoelectric point (5,6), alpha-helix content (12%; b0 = -76 +/- 15 degree), specific activity (25.8 units/mg protein) and Km value (1.0 x 10(-5) M) for uric acid at pH 8.5 and 25 degree C. The uricase preparation did not contain detectable amounts of the following metals; Fe, Cu, Mg, Mn, Co, Ni, Zn, and Cd. There were three cysteine residues in the subunit molecule and these were classified into two types by a chemical modification study; one is accessible to a reagent and the remaining two become accessible only after denaturation. The former may be located in the vicinity of the active center of the molecule, although it does not participate directly in the catalysis.

Amino Acids↗
PubMed1997

Expression of a gene for uricase II (nodulin-35) in cotyledons of soybean plants.

A cDNA clone (URcot-35) was isolated from a soybean cotyledonary cDNA library using a cDNA clone (URnod-35) for nodule uricase II as a probe. URcot-35 was a 1,170-bp cDNA with an open reading frame that encoded a protein of putative 309 amino acids with a molecular mass of 35,137 Da. The nucleotide sequence of URcot-35 was identical to that of URnod-35. Expression of the URcot-35 gene in cotyledons was investigated by Northern dot-blot hybridization, by the reverse transcription-polymerase chain reaction with subsequent hybridization assays with the cDNA for nodule uricase II as a probe, and by immunoblotting analysis with a monoclonal antibody that was specific to nodule uricase II. The results suggested that the transcript of URcot-35 was present in developing cotyledons and that uricase II accumulated during the pod-filling stage. This is the first report of the isolation of cDNA for uricase II from non-symbiotic tissue and the results demonstrate that uricase II in soybean cotyledons is identical to that in soybean nodules.

Amino Acid Sequence↗
PubMed2000

Expression of two uricase (Nodulin-35 genes in a non-ureide type legume, Medicago sativa.

Two cDNA clones, MsU2 and MsU9 encoding uricase (EC 1.7.3.3, Nodulin-35) were isolated from a cDNA library prepared from nodule tissues of alfalfa, Medicago sativa, plants. Both MsU2 and MsU9 encoded 308 amino acid polypeptides with a difference of 5 amino acids, and the deduced amino acid sequences shared 98% homology. Between these two cDNA clones and uricase genes of soybean which were designated as Nod-35s, more than 80% identity was observed in nucleotides and deduced amino acid sequences, suggesting that these MsU2 and MsU9 are homologs of Nod-35. Using the reverse transcription-PCR technique, we detected the transcripts of these two genes in almost all tissues of alfalfa. The operation of uricase genes was confirmed by the presence of ureide in the xylem sap and uricase activity in the nodules. In situ hybridization analysis revealed that MsU2 and MsU9 were expressed only in uninfected cells of the infected zone of the nodule tissue. The cell specific-expression of the two uricase genes was observed in an identical manner to that of Nod-35 in soybean nodules.

Amino Acid Sequence↗
PubMed2003

Production of an egg yolk antibody specific to microbial uricase and its inhibitory effects on uricase activity.

Ammonia gas produced from poultry manure can be a potential source of environmental pollution. Microbial uricase in poultry manure is an important target enzyme to reduce ammonia production because ammonia is mainly generated from the microbial decomposition of uric acid in the manure. Thus, the inhibition of microbial uricase is critical in preventing NH3 volatilization. A potential method of inhibiting uricase activity is the use of antibodies specific to microbial uricase. Therefore, this study was designed to evaluate 1) the production of the uricase-specific egg yolk antibody (IgY) from immunized hens and 2) the effect of the uricase-specific IgY on the activity of uricase. A total of 12 Single Comb White Leghorn hens were injected intramuscularly with uricase from Arthrobacter globiformis. The hens were immunized a second and third time at 1 and 2 wk, respectively, after the initial injection. The production of uricase-specific IgY was first detected at 2 wk after the initial immunization, and levels increased more than threefold at 4 wk. The method including water extraction, ammonium sulfate precipitation, and ethanol precipitation showed the most acceptable IgY purity and over 97% uricase-specific IgY recovery. Finally, the effect of the uricase-specific IgY on uricase activity was determined by a uricase assay. The slope ratio showed that the immune-IgY from the uricase-immunized hens and nonimmune IgY from nonimmunized control hens reduced uricase degradation by 58 and 43%, respectively, compared to the uricase treatment. The regression slope indicated that the immune IgY and nonimmune IgY treatments had a significant inhibitory effects on uricase activity compared to the uricase.

Animals↗
PubMed2003

Effect of minerals on activity of microbial uricase to reduce ammonia volatilization in poultry manure.

Inhibition of microbial uricase in poultry manure is critical to reduce NH3 volatilization, because hydrolysis of uric acid by microbial uricase is the first step in the production of NH3 gas in poultry manure. Three experiments were conducted to evaluate the effects of minerals on uricase activity and NH3 volatilization from poultry manure. In Experiment 1, an in vitro enzyme assay was used to evaluate the effects of Zn, Cu, Mg, and Mn on the activity of microbial uricase. There were three treatments: uricase, uricase + minerals, and uricase preincubated with minerals. Uric acid concentration was measured at 293 nm with a spectrophotometer. The results indicated that Zn and Cu greatly blocked the activity of microbial uricase (>90% inhibition), whereas Mg and Mn were less inhibitory. Experiment 2 was designed to evaluate the effect of ZnSO4 on the growth of uric acid-utilizing microorganisms by an in vitro assay. There were three treatments: control, ZnSO4 (10 mM), and ZnSO4 (50 mM). The results indicated that ZnSO4 significantly reduced the number of uric acid-utilizing microorganisms compared to the control. In Experiment 3, an NH3-trapping system was used to evaluate the effect of different levels of ZnSO4 on NH3 volatilization and nitrogen retention in poultry manure. Poultry manure (300 g) was mixed with 0, 0.15, 0.3, 1.5, 3, or 6 g ZnSO4 to create manure concentrations of Zn at 0, 0.05, 0.1, 0.5, 1, or 2% (wt/wt), respectively. The 1 and 2% ZnSO4 treatments significantly increased manure uric acid and total nitrogen retention by reducing NH3 volatilization compared to the control during the 3-wk incubation.

Ammonia↗
PubMed1997

Two distinct uricase II (nodulin 35) genes are differentially expressed in soybean plants.

Nodule-specific uricase (uricase II) is a homotetramer of a 33-kDa polypeptide, nodulin 35, and plays a key role in the assimilation of nitrogen fixed by microsymbionts in most legumes that have determinate nodules. We have isolated two distinct genes, UR2 and UR9, that encode for nodulin 35 from a soybean genomic library. Their corresponding cDNAs were also isolated from a nodule cDNA library. UR2 and UR9 both encode for 309 amino acid proteins with 12 amino acid differences. The expression of these two genes in various organs of soybean was examined by reverse transcription-polymerase chain reaction with primers specific to each cDNA sequences. Expression of UR9 was almost specific in root nodules, although it was expressed in roots, primary leaves, and developing seed at very low levels. In contrast, the UR2 transcripts were present in almost all plant organs at low levels, but no enhancement of the expression was observed in nodules. Thus, UR9 behaves as a nodulin gene, whereas UR2 is a nonsymbiotic uricase II gene. The sequences of their potential promoter regions share high homology within regions up to about 400 bp upstream from the translation initiation sites. These results suggest that symbiotic and nonsymbiotic uricase II genes diverged by gene duplication and that relatively small alterations in the promoter sequence enable the nodule-specific expression.

Amino Acid Sequence↗
PubMed2000

Structural and expression analysis of uricase mRNA from Lotus japonicus.

Uricase (nodulin-35) cDNA, LjUr, was isolated from nodules of a model legume, Lotus japonicus. LjUr expression was most abundant in nodules, although it was detected in nonsymbiotic tissues as well, particularly in roots. Expression in nodules was detected in uninfected cells, nodule parenchyma, and, more intensely, in vascular bundles. Phylogenetic analysis of uricase sequences from various legumes indicated that uricases of amide- and ureide-transporting legumes form two distinct clades. LjUr is in the cluster of amide-transport legumes even though L. japonicus bears determinate nodules.

Amino Acid Sequence↗
PubMed2004

Development of ectopic roots from abortive nodule primordia.

The symbiotic phenotype of five Tn5-induced mutants of Rhizobium etli affected in different anabolic pathways (namely, gluconeogenesis and biosynthesis of lysine, purine, or pyrimidine) was analyzed. These mutants induced, on the root of Phaseolus vulgaris, a normal early sequence of morphogenetics events, including root hair deformation and development of nodule primordia. Later on, however, from the resulting root outgrowths, instead of nodules, one or more ectopic roots (spaced closely related and agravitropic) emerged. Therefore, this group of mutant was collectively called "root inducer" (RIND). It was observed that the RIND-induced infection threads aborted early inside the invaded root hair, and that the resulting abortive nodules lack induction of late nodulin genes. Moreover, experiments performed using a conditional mutant (a methionine-requiring invader) revealed that bacterial invasion plays a key role in the maintenance of the program of nodule development and, in particular, in the differentiation of the most specific symbiotic tissue of globose nodules, the central tissue. These data indicate that, in P. vulgaris, the nodule primordium is a root-specified pro-meristematic tissue.

Base Sequence↗
PubMed1988

Uricase protein sequences: conserved during vertebrate evolution but absent in humans.

Uricase is a peroxisomal liver enzyme that catalyzes the oxidation of uric acid to allantoin during purine catabolism. It is present in vertebrates in most species of fish, amphibians, and mammals but its enzymatic activity is absent in hominoids. We have used Western blot analysis in a comparative study to establish a homology among uricases from different species of vertebrates. Using antibodies against denatured rat liver uricase, we have been able to detect for the first time cross-reactivity with the uricase of species ranging in the evolutionary scale from fish to primates (macaque). Our results suggest that these uricases have a common evolutionary origin. Our conclusion is also supported by the fact that uricase from different species exhibits identical tissue, subcellular localization, and similarity of molecular weights. This study was extended to include human liver samples. Using the same approach but with a more sensitive detection system (alkaline phosphatase instead of peroxidase), we did not detect polypeptide species related to rat uricase in human fetal or adult liver samples, which indicates that during hominoid evolution, the mutational event responsible for the loss of uricase activity in humans precluded formation of a translatable uricase mRNA.

Animals↗
PubMed2001

Hyperuricemia exacerbates chronic cyclosporine nephropathy.

BACKGROUND: Hyperuricemia frequently complicates cyclosporine (CSA) therapy. The observation that longstanding hyperuricemia is associated with chronic tubulointerstitial disease and intrarenal vasoconstriction raised the hypothesis that hyperuricemia might contribute to chronic CSA nephropathy. METHODS: CSA nephropathy was induced by the administration of CSA (15 mg/kg/day) for 5 and 7 weeks to rats on a low salt diet (CSA group). The effect of hyperuricemia on CSA nephropathy was determined by blocking the hepatic enzyme uricase with oxonic acid (CSA-OA). Control groups included rats treated with vehicle (VEH) and oxonic acid alone (OA). Histological and functional studies were determined at sacrifice. RESULTS: CSA treated rats developed mild hyperuricemia with arteriolar hyalinosis, tubular injury and striped interstitial fibrosis. CSA-OA treated rats had higher uric acid levels in association with more severe arteriolar hyalinosis and tubulointerstitial damage. Intrarenal urate crystal deposition was absent in all groups. Both CSA and CSA-OA treated rats had increased renin and decreased NOS1 and NOS3 in their kidneys, and these changes are more evident in CSA-OA treated rats. CONCLUSION: An increase in uric acid exacerbates CSA nephropathy in the rat. The mechanism does not involve intrarenal uric acid crystal deposition and appears to involve activation of the renin angiotensin system and inhibition of intrarenal nitric oxide production.

Animals↗
PubMed2002

Pharmacokinetics of 2-chlorodeoxyadenosine in a child undergoing hemofiltration and hemodialysis for acute renal failure.

The clearance of 2-chlorodeoxyadenosine (2-CdA) in patients with renal insufficiency has not been characterized previously. The authors describe the clinical course and the pharmacokinetics of 2-CdA in a child with acute monoblastic leukemia who experienced acute renal failure during treatment with cytarabine and 2-CdA. 2-CdA (9 mg/m per day) was infused over 30 minutes daily for 5 days. Plasma and dialysate concentrations of 2-CdA were measured by high-performance liquid chromatography. The rate of this patient's 2-CdA clearance was lower than the rates reported for children with normal renal function. The average clearance rate, reflecting systemic clearance and clearance by continuous venovenous hemofiltration and hemodialysis, was 12.4 L/hour per m for the first 3 days of 2-CdA therapy. He did not experience untoward hematologic toxicity. Because high 2-CdA plasma concentrations were observed in this patient, clinicians are advised to exercise caution when using this drug in patients with renal dysfunction. More experience in the administration of 2-CdA to patients with renal insufficiency will be necessary to determine the need for dosage adjustment.

Acute Kidney Injury↗
PubMedDate not supplied

Degradation of low molecular weight uremic solutes by oral delivery of encapsulated enzymes.

An alginate microcapsule was developed that contains three enzymes (urease, uricase, and creatininase) capable of effectively degrading urea, uric acid, and creatinine, which are elevated to pathologic levels in patients with kidney failure. The capsules were evaluated in vitro and in vivo in a rodent model and evidenced considerable potential as a possible adjunctive therapy in the treatment of ESRD. In vitro, 5 mL of the capsules incorporating a quantity of enzymes in the mg range effectively degraded all the uric acid, 97% of the urea, and 70% of the creatinine within 24 hours in a 100 mL test solution simulating the concentration of these solutes in uremic plasma. Enzyme degradation of urea followed Michaelis-Menten kinetics, and the Lineweaver-Burk plots for both encapsulated enzymes and unencapsulated control animals were superimposable, indicating that mass transfer through the capsules was not rate limiting in the degradation process. A chemically induced acute renal failure model in the rat was used to evaluate the ability of encapsulated enzymes, along with an oral sorbent (ion exchange resin), to degrade uremic toxins in vivo. Encapsulated enzyme therapy decreased the severity of azotemia by as much as 70%. Preliminary scale up calculations indicated that oral delivery to humans would involve a practical and manageable quantity of enzymes. This is the first study using a combination of enzymes in a single delivery vehicle to degrade multiple uremic toxins.

Acute Kidney Injury↗
PubMed1985

The all2 gene is required for the induction of the purine deamination pathway in Schizosaccharomyces pombe.

Five mutants were isolated at the all2 gene on the basis of their inability to utilize hypoxanthine as a sole source of nitrogen. These mutants failed to utilize the purines adenine, hypoxanthine, xanthine, uric acid, allantoin and allantoic acid, although they could utilize urea and ammonium. The all2 mutants appeared to be defective in purine induction of uricase, allantoinase, allantoicase and ureidoglycollase activities but retained wild-type activity of the constitutively synthesized urease. The all2 mutations were recessive.

Amidine-Lyases↗
PubMed2002

Effect of short-term N(2) deficiency on expression of the ureide pathway in cowpea root nodules.

Root systems of 28-d-old cowpea (Vigna unguiculata L. Walp cv Vita 3: Bradyrhizobium sp. strain CB756) plants bearing nitrogen-fixing nodules in sand culture were exposed to an atmosphere of Ar:O(2) (80:20, v/v) for 48 h and then returned to air. Root systems of control plants were maintained in air throughout. Nodules were harvested at the same times in control and Ar:O(2)-treated root systems. Activities of two enzymes of de novo purine synthesis, glycinamide ribonucleotide transformylase (GART; EC 2.1.2.2), aminoimidazole ribonucleotide synthetase (AIRS; EC 6.3.3.1), uricase (EC 1.7.3.3), and phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) were measured together with the protein level of each using immune-specific polyclonal antibodies. AIRS activity and protein both declined to very low levels within 6 h in Ar:O(2) together with a decline in transcript level of pur5, the encoding gene. GART activity, protein, and transcript (pur3) levels were relatively stable. Uricase activity declined in Ar:O(2) as rapidly as AIRS activity but the protein was stable. PEPC activity showed evidence of increased sensitivity to inhibition by malate but the protein level was stable. The data indicate that the flux of fixed N from bacteroids (N(2)-fixing nodule bacteria) is in some way associated with transcriptional control over pur5 and possibly also catabolism of AIRS protein. In contrast, there is limited posttranslational control over GART and PEPC and close posttranslational control over uricase activity. The significance of these different levels of regulation is discussed in relation to the overall control of enhanced expression of plant enzymes in the cowpea symbiosis.

Argon↗