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PubMed2000

Structure of the urease operon of Corynebacterium glutamicum.

Urease activity of Corynebacterium glutamicum results in a rapid pH increase upon addition of urea to the growth medium. The urease operon C. glutamicum was isolated of and sequenced. Seven open reading frames were identified; ureA, ureB, and ureC were homologues of other bacterial urease structural genes, ureE, ureF, ureG, and ureD exhibited homology to urease accessory genes. Disruption of ureC prevented the utilization of urea as a nitrogen source by C. glutamicum. Urease activity was induced by urea and appeared to be independent of the nitrogen regulatory system. Urease activity was not affected by pH. Heterologous expression of a truncated derivative of the urease gene cluster in Escherichia coli showed that ureD was necessary for active expression. Western-blot and primer extension analysis on C. glutamicum grown under different conditions confirmed that the operon was induced by urea. Transcriptional startpoint for the ureA gene was determined.

Amino Acid Sequence↗
PubMed1994

Effect of a urease inhibitor and ceiling fans on ascites in broilers. 2. Blood variables, ascites scores, and body and organ weights.

Restricted ventilation was used to experimentally induce ascites in commercial male broilers. The role of a dietary urease inhibitor (0, 125, and 250 ppm) and ceiling fans to reduce ascites was investigated. At 6 wk of age, birds were bled, euthanatized, weighed, scored for ascites, and heart, liver, and small intestine weights were obtained. Random samples were analyzed for intestinal ammonia. Blood samples were analyzed for blood gases, hemoglobin, red blood cell count, blood urea nitrogen, ammonia, and uric acid. Birds fed 125 and 250 ppm urease inhibitor were significantly (P < .001) lighter at 6 wk, when compared with controls. Urease inhibitor (125 and 250 ppm) significantly decreased large intestine ammonia. Urease inhibitor significantly increased small intestine (250 ppm) and liver weights (125 and 250 ppm), whereas urease inhibitor at 125 ppm decreased right ventricular heart weight. Urease inhibitor had no effect on ascites scores, blood gases, or blood ammonia, but hemoglobin, blood urea nitrogen, red blood cell count, and uric acid were significantly (P < .05) decreased by 125 ppm urease inhibitor.

Animals↗
PubMed1999

Serologic IgG response to urease in Helicobacter pylori-infected persons from Mexico.

Helicobacter pylori urease is required to counteract acidity during colonization of the stomach, and has been suggested as a major immunodominant antigen. The aim of this study was to determine the anti-urease response in a representative national serologic survey in Mexico. The population surveyed included persons 1-90 years of age from all socioeconomic levels and geographic zones of the country. Helicobacter pylori status was determined by ELISA serology. The IgG anti-urease was studied by ELISA using a recombinant apoenzyme. We found that 2,930 of the 7,720 infected patients (38%) were seropositive for IgG urease. The rate of IgG anti-urease positivity increased with age; in children < 10 years old it was < 20% and in persons > 40 years old it was > 50%. Age and a region with a high level of development were risk factors for seropositivity, whereas gender, educational level, crowding, and socioeconomic level were not associated with seropositivity. In conclusion, in natural infection with H. pylori, the response to urease is poor, mainly during the first years of infection. This inconsistent immune response to the enzyme may favor persistence of infection. A vaccine eliciting a consistent anti-urease response might overcome immune evasion and enhance clearance of bacteria after exposure.

Adolescent↗
PubMed2004

Diagnosis of Helicobacter pylori infection in patients with bleeding ulcer disease: rapid urease test and histology.

INTRODUCTION: The endoscopic diagnosis of Helicobacter pylori infection in patients with bleeding peptic ulcer is limited by a decreased sensitivity in standard invasive tests, rapid urease test and histology. There is controversy about the convenience of using one, neither, or both diagnostic tests. AIMS: To evaluate the results of simultaneously performed rapid urease test and histology in the diagnosis of Helicobacter pylori infection (H. pylori) in patients with bleeding peptic ulcer. PATIENTS AND METHODS: We included 173 patients, 98 male and 75 female, with an average age of 62 years (18-88), with upper gastrointestinal bleeding secondary to duodenal ulcer (115) or gastric ulcer (58), diagnosed within 24 hours after hospital admission. None of the patients had received treatment for H. pylori, proton pump inhibitors or antibiotics in the two weeks prior to the upper gastrointestinal bleeding episode. H. pylori infection was investigated in all patients by two antral biopsy samples for histological study (hematoxilin-eosin) and one or two antral biopsies for rapid urease test (Jatrox-H.p.-test). In cases with a negative urease test and histology, a 13C urea breath test was performed. Infection was considered present when at least one invasive test or the breath test was positive, whereas both invasive tests and the breath test had to be negative to establish an absent infection. RESULTS: 152 patients (88%) showed H. pylori infection, 104 patients (90%) with duodenal ulcer and 48 patients (83%) with gastric ulcer. In all 119 cases (78%) were diagnosed by the urease test and 112 cases (74%) by histology. Both methods were used to diagnose 134 of 152 cases (88%) (p < 0.05), these being positive in 97 cases and negative in 39 cases. In 18 of these 39 cases, the breath test was positive. CONCLUSIONS: Histology and urease test have similar diagnostic values for the identification of H. pylori in patients with bleeding peptic ulcer. Due to its rapid results, the urease test should be the method of choice. However, additional biopsies should be performed, and, when negative, a histological study should be carried out, since a combination of both methods allows a more precise diagnosis.

Adolescent↗
PubMed2000

Urease activity may contribute to the ability of Actinobacillus pleuropneumoniae to establish infection.

The contribution of urease activity to the pathogenesis of Actinobacillus pleuropneumoniae was investigated using 2 different urease-negative transposon mutants of the virulent serotype 1 strain, CM5 Nalr. One mutant, cbiK::Tn10, is deficient in the uptake of nickel, a cofactor required for urease activity. The other mutant, ureG::Tn10, is unable to produce active urease due to mutation of the urease accessory gene, ureG. In aerosol challenge experiments, pigs developed acute pleuropneumonia following exposure to high doses (10(6) cfu/mL) of the parental strain, CM5 Nalr, and to the cbiK::Tn10 mutant. When low dose (10(3) cfu/mL) challenges were used, neither urease-negative mutant was able to establish infection, whereas the parental strain was able to colonize and cause lesions consistent with acute pleuropneumonia in 8 of the 20 pigs challenged. These findings suggest that urease activity may be needed for A. pleuropneumoniae to establish infection in the respiratory tract of pigs.

Actinobacillus Infections↗
PubMed2001

[Effect of toluene on urease activity of Lou soil].

Urease activity affected by toluene studying results showed as follows: Toluene had little action on Jackbean urease activity, but can remarkably increase soil urease activity in lower toluene concentrations and shorter time, the increasing variation was 1.48-3.96 times than without toluene treatment in seven soil samples tested. The variation was less than 3.98% when sterilized soil samples adsorbed the Jackbean urease. It may be because toluene can kill soil microorganism, release urease within cell and subsequently urease fixed into soil organic-inorganic colloidal. Different soil pH and microbiology can lead to vary differently for urease activity.

Fabaceae↗
PubMed2002

[The study of photochemical immobilization of urease on polyether sulfone film surface].

A new method of using photoactivable ester with azido group was described to immobilize urease on polyether sulfone(PES) film surface. The effects of photoactive enzyme concentration, temperature, pH, irradiation time on the activity of immobilized urease were investigated. Reused times and storage stability were also studied. The results showed that the surface concentration of urease immobilized on PES surface was about 0.33 mg/cm2. When the irradiation time was 5 minutes, the relative activity of immobilized urease was the highest and the activity increased with the increase of the concentration of photoactive urease solution. The optimum pH and temperature of immobilized urease were 7 and 50 degrees C respectively. The relative activity of immobilized urease was stable (50%) after 12 times reused at 50 degrees C.

Enzyme Stability↗
PubMed2000

Plant ureases: roles and regulation.

Both urea and urease were subjects of early scientific investigations. Urea was the first organic molecule to be synthesized and jack bean urease was the first enzyme ever to be crystallized. About 50 years later it was shown to be the first nickel metalloenzyme. Since then, nickel-dependent ureases have been isolated from many bacteria, fungi and higher plants. They have similar structures and mechanisms of catalysis. A urease apoenzyme needs to be activated. This process requires participation of several accessory proteins that incorporate nickel into the urease forming catalytic site. In this review, ureases from various organisms are briefly described and the similarities of their structures discussed. Moreover, the significance of urea recycling in plants is explained and recent literature data about the function and activation of plant ureases are presented.

Enzyme Activation↗
PubMed2004

[Immunological rapid urease test].

Immunological rapid urease test for detection of Helicobacter pylori infection, composed of a solid-phase tip coated with monoclonal antibody against H. pylori's urease and ion-sensitive field transistor (ISFT)-based pH sensor system, was developed. The monoclonal antibody against H. pylori's urease was useful to avoid the contamination of urease activity in other bacteria. Because ISFTT had high ability to detect pH change, the sensitivity and specificity of immunological rapid urease test was significantly improved comparing with that of conventional rapid urease test. The utility of immunological rapid urease test was evaluated in some clinical studies.

Antibodies, Monoclonal↗
PubMed2005

Urease-induced alkalinization of extracellular pH and its antitumor activity in human breast and lung cancers.

Jack bean urease catalyzes the decomposition of urea into ammonia, which in turn increases the pH of the surrounding medium. Based on these two properties, we have investigated the antitumor effects of urease in vitro and in vivo on human lung and breast cancer cell lines either by the enzyme itself or in combination with other chemotherapeutic drugs. First, through the generation of toxic ammonia, urease exerted direct cytotoxicity on A549 and MDA-MB-231 tumor cells with LC50 of 0.22 and 0.45 U/ml, respectively. The cytotoxic effects could effectively be blocked using the reversible urease inhibitor acetohydroxamic acid. Complete protection was observed at dose > or = 2 mM. In addition, nude mouse xenograft models demonstrated that intratumoral urease injections (1 - 10 U/dose) inhibited A549 and MCF-7 tumor growth in vivo. Second, when combined with weak-base anticancer drugs, urease provided indirect antitumor effects via pH augmentation. Alkalinization of extracellular pH by urease (2 U/ml) and urea (> or = 2 mM) was found to enhance the antitumor efficacy of doxorubicin (50 microM) and vinblastine (100 microM) significantly.

Ammonia↗
PubMed1989

A procedure for purifying jack bean urease for clinical use.

Urease with a purity meeting the requirements of analytical use was purified from jack bean meal through steps consisting of 20% acetone extraction, heat treatment, acid precipitation, and lyophilization. For extraction of urease, one part of bean meal was mixed with 5 parts of 20% acetone containing 1 mM EDTA and 1 mM 2-mercaptoethanol, and stirred at 20 degrees C for 5 min. Milky substances in the extract were removed by heat treatment. Urease in the clear yellow supernatant was precipitated by adjusting the pH of the solution to 5.4 with citric acid. The acid precipitated urease was neutralized by dissolving in 0.015 M phosphate buffer, pH 8.5 (final pH 6.8 to 7.0) and then lyophilized. By this procedure, the purity of the enzyme was increase 14.7 fold, the recovery of activity was 63%, and the yield was 6.75 g from 1 kg of bean seeds. The specific activity of the preparation was 411 units/mg protein (240 units/mg solid), and the free ammonia content was less than 0.01 microgram per unit. Some other proteins were present in the urease preparation as examined by gel filtration and gradient polyacrylamide gel electrophoresis. The molecular weight of the enzyme estimated by gel filtration was 480,000. However, two urease activity bands with molecular weight of 230,000 and 480,000 were observed in the polyacrylamide gel electrophoregram. From the result of determination of blood urea nitrogen (BUN), this simple purification procedure could be used for practical preparation of urease from jack bean meal for clinical analysis.

Acetone↗
PubMed1987

Purification and characterization of the nickel-containing multicomponent urease from Klebsiella aerogenes.

Klebsiella aerogenes urease was purified 1,070-fold with a 25% yield by a simple procedure involving DEAE-Sepharose, phenyl-Sepharose, Mono Q, and Superose 6 chromatographies. The enzyme preparation was comprised of three polypeptides with estimated Mr = 72,000, 11,000, and 9,000 in a alpha 2 beta 4 gamma 4 quaternary structure. The three components remained associated during native gel electrophoresis, Mono Q chromatography, and Superose 6 chromatography despite the presence of thiols, glycols, detergents, and varied buffer conditions. The apparent compositional complexity of K. aerogenes urease contrasts with the simple well-characterized homohexameric structure for jack bean urease (Dixon, N. E., Hinds, J. A., Fihelly, A. K., Gazzola, C., Winzor, D. J., Blakeley, R. L., and Zerner, B. (1980) Can. J. Biochem. 58, 1323-1334); however, heteromeric subunit compositions were also observed for the enzymes from Proteus mirabilis, Sporosarcina ureae, and Selemonomas ruminantium. K. aerogenes urease exhibited a Km for urea of 2.8 +/- 0.6 mM and a Vmax of 2,800 +/- 200 mumol of urea min-1 mg-1 at 37 degrees C in 25 mM N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid, 5.0 mM EDTA buffer, pH 7.75. The enzyme activity was stable in 1% sodium dodecyl sulfate, 5% Triton X-100, 1 M KCl, and over a pH range from 5 to 10.5, with maximum activity observed at pH 7.75. Two active site groups were defined by their pKa values of 6.55 and 8.85. The amino acid composition of K. aerogenes urease more closely resembled that for the enzyme from Brevibacter ammoniagenes (Nakano, H., Takenishi, S., and Watanabe, Y. (1984) Agric. Biol. Chem. 48, 1495-1502) than those for plant ureases. Atomic absorption analysis was used to establish the presence of 2.1 +/- 0.3 mol of nickel per mol of 72,000-dalton subunit in K. aerogenes urease.

Amino Acids↗
PubMed1985

[Activity of urease in microcapsules].

The activity of urease in microcapsules formed by a symplex membrane from cellulose sulphate and polydimethyldiallyl ammonium chloride is studied and compared with that of free urease in solution. Both free and encapsulated urease exhibits a similar pH optimum. The interaction of urease with cellulose sulphate causes a decrease of the activity down to 35 to 38%. In spite of its lower absolute activity the encapsulated urease shows a higher long time stability than the free urease. The presented immobilization method for urease could be of importance for an active detoxification in uremia.

Capsules↗
PubMed1995

In situ mapping of urease-positive areas in porcine gastric mucosa.

Urease activity is a feature of gastric helicobacters, and its abundant production provides an indirect means of detecting their colonization. A method for mapping urease-positive areas directly on the gastric mucosa was developed, and 57.8% of pigs had evidence of Helicobacter colonization based on urease assay. Moreover, 89.2% of urease-positive pigs had gastritis, confirming that the known association found in man between Helicobacter and gastritis was found also in pigs. The proposed urease assay allowed detection of all urease-positive areas on the gastric mucosa, thus overcoming the biopsy sampling problems derived from the patchy distribution of helicobacters. In this way, gastric mucosa specimens from urease-positive areas could be usefully utilized for culture attempts and for microscopic examination.

Animals↗
PubMed1995

Survey of urease activity in ruminal bacteria isolated from domestic and wild ruminants.

A total of 909 strains, including Selenomonas ruminantium, Lactobacillus sp., Enterococcus sp. and Staphylococcus sp., from the rumen of 104 domestic and wild ruminants was used in tests for urease activity. Tests showed that 56.7% of S. ruminantium strains and 18.5% of lactobacilli manifested medium urease activity with mean values of 14.4 +/- 2.5 and 13.85 +/- 0.25 nkat ml-1, respectively. Most of the Enterococcus faecium (62.2%) and all of the E. faecalis isolates expressed urease activity with mean values of 3.9 +/- 0.05 and 4.1 +/- 0.06 nkat ml-1. E. malodoratus, E. solitarius, E. mundtii, Streptococcus bovis and S. uberis did not produce any urease. All the staphylococci screened were urease-producing strains, mostly with medium or low urease activity. The highest level of urease was measured in the rumen epithelial wall of isolates SE30 (19.7 +/- 2.71 nkat ml-1 of rumen content), SCU32 (29.3 +/- 1.6 nkat ml-1) and also in the rumen content isolates EF35 (19.9 +/- 0.94 nkat ml-1) and S. ruminantium 77 (25.35 nkat ml-1). This survey contributes new data to existing information relating to the ureolytic microbial ecosystem in ruminants.

Animals↗
PubMed1999

Recycling of urea associated with the host plant urease in the silkworm larvae, Bombyx mori.

Urea concentration and urease activity in the midgut content were compared between larvae of the silkworm, Bombyx mori fed an artificial diet and those fed fresh mulberry leaves. A considerable amount of urea was found in the midgut content of the both larvae, however it was significantly lower in the larvae fed fresh mulberry leaves than in the larvae fed the artificial diet; average urea concentrations in the midgut content of the larvae fed fresh mulberry leaves and the artificial diet were 2.9 and 4.6 &mgr;mol/g, respectively. Urea in the midgut content seems to be secreted from the insect itself since the amount of urea in both diets were negligibly small. Urease activity was detected only in the midgut content of the larvae fed fresh mulberry leaves but not in other tissues of the larvae. On the other hand, no urease activity was detected in the midgut content of the larvae fed the artificial diet. Subsequently, to elucidate the role of mulberry leaf urease in the midgut lumen, larvae that had been reared on the artificial diet were switched to fresh mulberry leaves. The diet switch caused a rapid decrease in urea concentration in the midgut content and an increase in ammonia concentration in the midgut content, suggesting that secreted urea could be hydrolyzed to ammonia by mulberry leaf urease in the midgut lumen. Furthermore, to investigate the physiological significance of mulberry leaf urease on urea metabolism of the silkworm, (15)N-urea was injected into the hemocoel, and after 12 h the larvae were dissected for (15)N analysis. A considerable amount of (15)N was found to be incorporated into the silk-protein of the larvae fed fresh mulberry leaves, but there was little incorporation of (15)N into the silk-protein of the larvae fed the artificial diet. These data indicate that urea is converted into ammonia by the action of mulberry leaf urease in the midgut lumen and used as a nitrogen source in larvae fed mulberry leaves.

Journal Article↗
PubMed1989

Potential phytotoxicity associated with the use of soil urease inhibitors.

Recent work in our laboratory showed that the adverse effect of urea fertilizer on seed germination and seedling growth in soil is due to ammonia produced through hydrolysis of urea by soil urease (NH(2)CONH(2) + H(2)O --> 2NH(3) + CO(2)) and can be eliminated by amending the fertilizer with a small amount of a urease inhibitor such as N-(n-butyl)thiophosphoric triamide or phenylphosphorodiamidate. Continuation of this work showed that these inhibitors can induce leaf-tip necrosis in plants. Research to account for this phytotoxicity indicated that it resulted from an accumulation of toxic amounts of urea in plants through inhibition of urease activity by N-(n-butyl)thiophosphoric triamide and phenylphosphorodiamidate. Support for this conclusion was provided by experiments showing that these urease inhibitors increased both leaf-tip necrosis and urea concentrations in wheat (Triticum aestivum L.) and sorghum [Sorghum bicolor(L.) Moench] plants grown in soils treated with urea and that the necrotic areas of such plants had a much higher concentration of urea than did the nonnecrotic areas. The potential of urease inhibitors for inducing phytotoxicity should not preclude their use to eliminate the adverse effects of urea fertilizers on seed germination and seedling growth in soil because the ammonia produced through hydrolysis of urea fertilizer by urease is much more detrimental to plant growth than is the urea accumulation induced by urease inhibitors.

Journal Article↗
PubMed1992

Urease-null and hydrogenase-null phenotypes of a phylloplane bacterium reveal altered nickel metabolism in two soybean mutants.

Mutation at either of two genetic loci (Eu2 or Eu3) in soybean (Glycine max [L.] Merr.) results in a pleiotropic elimination of the activity of both major urease isozymes. Surprisingly, the phenotype of a phylloplane bacterium, Methylobacterium mesophilicum, living on the leaves of eu2/eu2 or eu3-e1/eu3-e1 mutants is also affected by these plant mutations. The bacteria isolated from leaves of these soybean mutants have transient urease- and hydrogenase-deficient phenotypes that can be corrected by the addition of nickel to free-living cultures. The same bacterium growing on wild-type soybeans or on urease mutants eu1-sun/eu1-sun or eu4/eu4, each deficient in only one urease isozyme, are urease-positive. These results suggest that the bacterium living on the eu2/eu2 or eu3-e1/eu3-e1 mutant is unable to produce an active urease or hydrogenase because it is effectively starved for nickel. We infer that mutations at Eu2 or Eu3 result in defects in nickel metabolism but not in Ni(2+) uptake or transport, because eu2/eu2 and eu3-e1/eu3-e1 mutants exhibit normal uptake of (63)NiCl(2). Moreover, wild-type plants grafted on mutant rootstocks produce seeds with fully active urease, indicating unimpeded transport of nickel through mutant roots and stems.

Journal Article↗