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PubMed1996

Purification and characterization of urease from schizosaccharomyces pombe.

The urease from the ascomycetous fission yeast Schizosaccharomyces pombe was purified about 4000-fold (34% yield) to homogeneity by acetone precipitation, ammonium sulfate precipitation, DEAE-Sepharose ion-exchange column chromatography, and if required, Mono-Q ion-exchange fast protein liquid chromatography. The enzyme was intracellular and only one species of urease was detected by nondenaturing polyacrylamide gel electrophoresis (PAGE). The native enzyme had a M(r) of 212 kDa (Sepharose CL6B-200 gel filtration) and a single subunit was detected with a M(r) of 102 kDa (PAGE with sodium dodecyl sulfate). The subunit stoichiometry was not specifically determined, but the molecular mass estimations indicate that the undissociated enzyme may be a dimer of identical subunits. The specific activity was 700-800 micromols urea.min-1.mg protein-1, the optimum pH for activity was 8.0, and the Km for urea was 1.03 mM. The sequence of the amino terminus was Met-Gln-Pro-Arg-Glu-Leu-His-Lys-Leu-Thr-Leu-His-Gln-Leu-Gly-Ser-Leu-Ala and the sequence of two tryptic peptides of the enzyme were Phe-Ile-Glu-Thr-Asn-Glu-Lys and Leu-Tyr-Ala-Pro-Glu-Asn-Ser-Pro-Gly-Phe-Val-Glu-Val-Leu-Glu-Gly-Glu-Ile- Glu- Leu-Leu-Pro-Asn-Leu-Pro. The N-terminal sequence and physical and kinetic properties indicated that S. pombe urease was more like the plant enzymes than the bacterial ureases.

Amino Acid Sequence↗
PubMed1980

Jack bean urease (EC 3.5.1.5). IV. The molecular size and the mechanism of inhibition by hydroxamic acids. Spectrophotometric titration of enzymes with reversible inhibitors.

Kinetic, spectral, and other studies establish that hydroxamic acids bind reversibly to active-site nickel ion in jack bean urease. Equilibrium ultracentrifugation studies establish that the molecular weight of native urease is 590 000 +/- 30 000 while that of the subunit formed in 6 M guanidinium chloride in the presence of beta-mercaptoethanol is approximately 95 000. Essentially the same subunit molecular weight (approximately 93 000) is found by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, subsequent to denaturation in a guanidinium chloride - beta-mercaptoethanol system at various temperatures. Coupled with an equivalent weight of 96 600 for binding of the inhibitors acetohydroxamic acid and phosphoramidate, these results establish securely that urease is a hexamer with one active site per 96 600-dalton subunit. Consistent values for the equivalent weight are obtained by a routine spectrophotometric titration of the active site of freshly prepared urease with trans-cinnamoylhydroxamic acid. General equations are derived which describe spectrophotometric titrations of binding sites of any enzyme with a reversible inhibitor. These equations allow the evaluation of the difference spectrum of the protein-inhibitor complex even when the binding sites cannot readily be saturated with the inhibitor or vice versa.

Amino Acids↗
PubMed2002

Interactions among the seven Helicobacter pylori proteins encoded by the urease gene cluster.

Survival of Helicobacter pylori in acid depends on intrabacterial urease. This urease is a Ni(2+)-containing oligomeric heterodimer. Regulation of its activity and assembly is important for gastric habitation by this neutralophile. The gene complex encodes catalytic subunits (ureA/B), an acid-gated urea channel (ureI), and accessory assembly proteins (ureE-H). With the use of yeast two-hybrid analysis for determining protein-protein interactions, UreF as bait identified four interacting sequences encoding UreH, whereas UreG as bait detected five UreE sequences. These results were confirmed by coimmunoprecipitation and beta-galactosidase assays. Native PAGE immunoblotting of H. pylori inner membranes showed interaction of UreA/B with UreI, whereas UreI deletion mutants lacked this protein interaction. Deletion of ureE-H did not affect this interaction with UreI. Hence, the accessory proteins UreE/G and UreF/H form dimeric complexes and UreA/B form a membrane complex with UreI, perhaps enabling assembly of the urease apoenzyme at the membrane surface and immediate urea access to intrabacterial urease to allow rapid periplasmic neutralization.

Bacterial Proteins↗
PubMed2001

Cholangiohepatitis and inflammatory bowel disease induced by a novel urease-negative Helicobacter species in A/J and Tac:ICR:HascidfRF mice.

Helicobacter bilis and H. hepaticus, both urease-positive intestinal helicobacters of mice, have been shown experimentally to induce proliferative typhlocolitis in scid mice. We recently isolated a urease-negative Helicobacter sp. (H. sp.) that also induced proliferative typhlocolitis in pilot studies in scid mice. To determine the pathogenic potential of H. sp. in immunocompromised and immunocompetent mice, 5-week old male A/J or Tac:Icr:Ha(ICR)-scidfRF mice were inoculated by intraperitoneal (IP) injection with approximately 3 x 10(7) colony-forming units (CFU) of H. sp. Mice were necropsied at various time points postinoculation (PI). Sham-inoculated mice had no clinical, gross, or histopathological lesions. In contrast, scid mice inoculated IP with H. sp. had severe hemorrhagic diarrhea and decreased weight gain at 2, 7, and 18 weeks postinoculation (PI), with severe proliferative typhlocolitis, phlebothrombosis, and hepatitis. A/J mice had no clinical signs, but had mild to moderate proliferative typhlocolitis and moderate to marked cholangiohepatitis at 7 and 24 weeks PI. A/J mice infected with H. sp. developed robust immune responses of a predominant Th1 type. This report demonstrates that infection with a urease-negative helicobacter can cause inflammatory bowel disease (IBD) and hepatitis in scid and immunocompetent A/J mice. These results provide a new model of IBD and cholangio-hepatitis associated with a specific urease-negative, novel H. species.

Animals↗
PubMed2005

Role of rapid urease test and histopathology in the diagnosis of Helicobacter pylori infection in a developing country.

BACKGROUND: The aim of this study was to determine the effect of commonly self-prescribed proton pump inhibitors (PPI) on the results of rapid urease test and histology for the diagnosis of H. pylori infection. METHODS: One hundred-nine consecutive patients with dyspeptic symptoms attending the endoscopy suite were enrolled in this study. Antrum biopsy specimens were collected at endoscopy for the rapid urease test (Pronto Dry, Medical Instrument Corp, France) and histopathology. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and like-hood ratio of a positive and negative of Pronto Dry test were compared against histology. The gold standard test for the diagnosis of H. pylori infection was histopathology. RESULTS: Sixty-one percent (66/109) patients were males with mean age of 43 +/- 14.1 years and age range 17-80 years. Fifty-two percent (57/109) were not on any medications while 48% (52/109) used PPI before presentation to the outpatients. Pronto Dry was positive in 40% (44/109) and negative in 60% (65/109). Histopathology was positive for H. pylori in 57% (62/109) and negative in 43% (47/109). The sensitivity, specificity, PPV, NPV and like-hood ratio of a positive and negative Pronto Dry test with and without PPI were 43.3%, 86.4%, 81.3%, 3.18, 0.656 and 52.8% vs 71.9%, 80%, 82.1%, 69%, 3.59 and 0.35. CONCLUSION: This study shows that the sensitivity, specificity, NPV and PPV of rapid urease test was reduced in patients who are on PPI. The exclusive use of the rapid urease test for the diagnosis of Helicobacter pylori cannot be recommended in patients with prior PPI use.

Adolescent↗
PubMed1992

Gas phase derivatization of ammonia with 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole and its application to urease assay.

An ammonia-specific and rapid fluorometric method for determination of ammonia and urease activity was developed. The method is designed to assay ammonia levels or urease activity for the rapid diagnosis of Helicobacter pylori infection. 4-Fluoro-7-nitrobenzo-2-oxa-1,3-diazole was used to derivatize ammonia and 4-amino-7-nitrobenzo-2-oxa-1,3-diazole was analysed by high performance liquid chromatography at an excitation wavelength of 455 nm and an emission wavelength of 520 nm. Derivatization was designed to react with ammonia gas produced in a strong alkaline pH sample. The fluorescent intensity was linear in the range of 0.1-10 mM ammonia per tube when the reaction was carried out for 15 min at 37 degrees C. Urease activity, judged as the amount of ammonia production from urea, could be measured at 25 ng per tube (S/N = 1.5) with Jack bean meal urease. Because of its rapidity, this assay is potentially superior to the current standard method in use in clinical settings.

4-Chloro-7-nitrobenzofurazan↗
PubMed1992

Inhibition of urease activity by dipeptidyl hydroxamic acids.

A series of dipeptidyl hydroxamic acids (H-X-Gly-NHOH: X = amino acid residues) was synthesized, and the inhibitory activity against Jack bean and Proteus mirabilis ureases [EC 3.5.1.5] was examined. A number of H-X-Gly-NHOH inhibited Jack bean urease with an I50 of the order of 10(-6) M and inhibited Proteus mirabilis urease with an I50 of the order of 10(-5) M. The inhibition against Jack bean urease was more potent than that with the corresponding aminoacyl hydroxamic acids (H-X-NHOH).

Dipeptides↗
PubMed1998

Screening system for urease inhibitors using 13C-NMR.

Urease inhibitors are candidate drugs to treat infection with the human pathogen, Helicobacter pylori, which produces a potent urease [urea amidohydrolase; EC 3.5.1.5]. We developed a screening system based on 13C-NMR measurement of the time course of decrease in the signal of 13C-urea in the presence of urease. The effect on urease activity of known inhibitors, hydroxamic acids, L-ascorbic acid, 2,2'-dipyridyl disulfide and ninhydrin, was speedily and conveniently measured by this method.

Binding, Competitive↗
PubMed1988

Effect of ammonia on the gastric mucosa in rats: pathophysiological importance of urease in gastric ulcer disease.

We examined the pathophysiological roles of the urea-urease-ammonia system in gastric ulcer disease using rats. Exposure of the stomach to ammonia (0.01-1.0%) decreased the transmucosal potential difference (PD) and histological injury in a concentration-dependent manner. Exposure of the stomach for 20 min to urea (0.025-0.2%) together with urease (100 IU) produced a decrease in PD and microscopic injury similarly, and the lesion was closely associated with the amount of ammonia produced. Urea and urease alone had no effect on the gastric mucosa. These results suggested the pathophysiological importance of urea, urease and ammonia in gastric ulcer disease.

Ammonia↗
PubMed1999

Effect of thermosensitive matrix-phase transition on urease-catalyzed urea hydrolysis.

Temperature dependencies of kinetic and equilibrium parameters of urea hydrolysis catalyzed by native urease and the urease immobilized in a thermosensitive poly-N-isopropylacrylamide gel have been studied. The swelling ratio of the collapsed urease-containing gel is shown to increase in the presence of urea. Below a lower critical solution temperature (LCST) of the polymer, the immobilized urease actually has the same catalytic properties as the native enzyme. At temperatures above LCST, the observed catalytic activity of the immobilized enzyme depends chiefly not only on the thermoreversible matrix state, but also on gel water content.

Diffusion↗
PubMedDate not supplied

Diagnosis of Helicobacter pylori: comparison of an urease test, histological visualization of curved bacteria and culture.

Helicobacter pylori was investigated in 189 patients for culture, microscopic visualization of campylobacter-like organisms (CLO) and a ten minute urease test. In 136 (72%) the bacteria was isolated, and in 98 of them CLO were histologically detected. Specificity, sensitivity, positive and negative predictive values of microscopic visualization of CLO were: 0.77, 0.73, 0.97 and 0.51, respectively; 98 culture-positive patients were urease test positive. Specificity, sensitivity, positive and negative predictive values of the urease test were: 0.83, 0.72, 0.92 and 0.54, respectively. Comparing the urease test with culture of H. pylori combined with microscopic visualization of CLO, its specificity, sensitivity, positive and negative predictive values were: 0.95, 0.71, 0.98 and 0.48, respectively. Probably, these values are not real, since bacteria different from H. pylori could be misclassified as CLO.

Clinical Enzyme Tests↗
PubMed2000

Influence of the novel urease inhibitor N-(n-butyl) thiophosphoric triamide on ruminant nitrogen metabolism: II. Ruminal nitrogen metabolism, diet digestibility, and nitrogen balance in lambs.

Three lamb metabolism experiments were conducted to investigate the effects of chronic administration of the novel urease inhibitor N (n-butyl) thiophosphoric triamide (NBPT) on ruminal N metabolism, fermentation, and N balance. In Exp. 1, ruminally cannulated wethers (n = 28; 45.0 +/- .9 kg) were administered one of seven doses of NBPT (0 [control], .125, .25, .5, 1, 2, or 4 g of NBPT daily) and fed a common cracked corn/cottonseed hull-based diet twice daily containing 2% urea at 2.5% of initial BW for the duration of the 15-d experiment. Overall, NBPT decreased (linear P < .0001; quadratic P < .001) ruminal urease activity, resulting in linear increases (P < .0001) in ruminal urea and decreases in ruminal NH3 N concentrations. However, the detection of an NBPT x day interaction (d 2 vs 15; P < .01) indicated that this depression in urea degradation diminished as the experiment progressed. Increasing NBPT linearly decreased (P < .01) total VFA concentrations on d 2 of the experiment, but it had no effect (P > .10) on d 15. Increasing NBPT had no effect (P > .10) on DM or ADF digestibilities, but it linearly decreased (P < .01) N digestibility. Supplementing NBPT produced a linear increase (P < .05) in urinary N excretion and a linear decrease (P < .01) in N retention. In Exp. 2, ruminally cannulated wethers (n = 30; 46.8 +/- .6 kg) were fed one of two basal diets (2.0 vs 1.1% dietary urea) at 2.5% of initial BW and dosed with either 0 (control), .25, or 2 g of NBPT daily for the duration of the 15-d experiment. There were no NBPT x dietary urea interactions (P > .10) for Exp. 2. Increasing NBPT depressed (linear and quadratic P < .0001) ruminal urease activity, producing linear (P < .0001) increases in urea N and linear decreases in NH3 N in the rumen. As in Exp. 1, an NBPT x day interaction (P < .05) was noted for urea, NH3 N, and total VFA concentrations; the maximum response to NBPT occurred on d 2 but diminished by d 15 of the experiment. Administration of NBPT did not influence (P > .10) DM, ADF, or N digestibilities in Exp. 2. In Exp. 3, wether lambs (n = 30; 26.4 +/- .7 kg) were subjected to the same treatment regimen as in Exp. 2 for a 14-d N balance experiment. Although several NBPT x dietary urea interactions (P < .05) were noted, increasing NBPT did not affect (P > .10) N digestibility. Administration of NBPT quadratically increased (P < .10) urinary N excretion, producing a linear decrease (P < .05) in N retention. These results suggest that although NBPT is capable of inhibiting ruminal urease short-term, the ruminal microflora may be capable of adapting to chronic NBPT administration, thereby limiting its practical use in improving the utilization of dietary urea.

Ammonia↗
PubMed1993

Influence of Escherichia coli on urease-induced crystallisation in human urine.

Urease was added to urines inoculated with Escherichia coli 24 hours earlier and to control urines not inoculated with E. coli. The inoculation did not change the concentration of the measured urine components. The urease-induced ammonium ion production and pH increase was reduced in E. coli-inoculated urines compared to control urines. This suggests that E. coli can inhibit urease. The precipitation of both phosphate and magnesium on glass rods inserted in the urine was reduced with 40-50% in the E. coli-inoculated urines. The results demonstrate that E. coli can influence urease-induced crystallisation.

Adult↗
PubMed1996

Faecal microflora and urease activity during the first six months of infancy.

Gastrointestinal degradation of urea might, according to a new hypothesis, have consequences for the regulation of acid-base balance as well as control of breathing during infancy. Thirteen infants were investigated from their first few days of life to the age of 6 months by collecting faecal samples at the age of 3 days, 2, 3, and 6 months, respectively. The faecal microflora was determined after aerobic and anaerobic cultivation and the faecal urease activity was assessed after 36 h aerobic and anaerobic preincubation. The infants were mostly breast fed and had a faecal microflora containing anaerobic bacteria such as Bifidobacteria, Bacterioides and Lactobacilli but also aerobics such as Escherichia coli, Enterococci and sometimes Klebsiella. The faecal pH increased from approximately 5.30 to 5.90, the pH after anaerobic preincubation being on an average 0.2 pH units lower than after aerobic preincubation. Simultaneously the nitric oxide production of the faecal specimens increased approximately 10-fold and the urease activity decreased by a factor of 3 to 5. We also found an inhibitory action of nitrate, nitrite (in mumolar concentration) and nitric oxide (in parts per million concentration) on the faecal urease activity. Hence, the present results warrant further research in order to determine more precisely the action of different concentrations of various nitrous oxides on individual bacterial species, and furthermore, to assay the faecal urease activity in victims of sudden infant death syndrome as well as in infants dead due to other causes.

Bacteria, Aerobic↗
PubMed1988

The ecology and pathogenicity of urease-producing bacteria in the urinary tract.

Urease activity is a physiological function of many bacteria that enables these organisms to utilize urea as a source of nitrogen. The association of ureolytic bacteria with human or animal hosts varies widely from a commensal relationship as demonstrated with skin microflora, a symbiotic relationship in the gastrointestinal tract, to a pathogenic relationship in the urinary tract. Since similar or identical species of bacteria such as Staphylococcus aureus are found in all three environments, the effect of urease activity on the host must be solely a function of the environment of these organisms. In this review, the importance of urease to bacteria is discussed, identifying the gastrointestinal tract as a major reservoir of ureolytic bacteria and investigating the urinary tract environment and the infectious struvite stone production that often accompanies urease-producing bacteria there. Finally, an infection model is presented which explains the development and growth of these urinary calculi and their remarkable persistence in spite of modern urological treatments.

Animals↗
PubMed2003

Analysis of serotype AD strains from F1 progenies between urease-positive- and negative-strains of Cryptococcus neoformans.

Cryptococcus neoformans is a pathogenic basidiomycete with a defined sexual cycle involving mating between haploid yeast cells with a transient diploid state. We examined F1 progeny from a crossing between the urease-negative strain (environmental isolate, serotype A, mating type alfa, haploid) and a tester strain (B 3502 from NIH of USA; urease-positive, serotype D, mating type a, haploid) for serotype, mating type, ploidy and urease activity, and performed partial sequencing of the urease gene. Phenotypes of the F1 progeny and results of SSCP analyses suggested that the serotype AD strain of the F1 progeny is a hybrid of the parental serotype A and D strains.

Cryptococcus neoformans↗
PubMed1998

[Studies on infection urolithiasis--urease induced crystallization in synthetic urine].

BACKGROUND: In the present study, urease induced crystallization in synthetic urine was studied by an aggregometer technique. MATERIALS AND METHODS: The synthetic urine was made by the method by Griffith et al (1976). The synthetic urine of 200 microliters portions were stirred constantly at 37 degrees C, and then 10 microliters of urease solution (1000 U/ml) was added. An aggregometer were recorded as turbidity curves on a chart during the incubation for the crystallization simultaneously. RESULTS: A two-phase turbidity curve was obtained from the reaction of synthetic urine and urease. Firstly the mild turbidity appeared gradually up until to approximately 13 min, and then the turbidity increased rapidly. The mild turbidity was called "early crystallization" and the rapid turbidity was called "late crystallization". The early crystallization appeared gradually at pH 7.3 and 0.05 M of anmmonia concentration, and this amount was completely depended on the concentration of calcium in the synthetic urine. The late crystallization occurred rapidly at pH 8.5 and 0.06-0.08 M of anmmonia concentration and this amount was completely depended on the concentration of magnesium in the synthetic urine. We confirmed the early crystallization as calcium phosphate and the late crystallization as magnesium ammonium phosphate by the observations of polarized light microscopy as well as the estimations using an infrared spectrometer. CONCLUSION: Urease induced crystallizations in the synthetic urine had a two-phase turbidity. Firstly the mild turbidity of calcium phosphate appeared gradually, and then rapid turbidity of magnesium ammonium phosphate occurred. Therefore, it is suggested that various crystallizations may be developed in the infected human urine according to the constituents of calcium and magnesium.

Calcium↗
PubMed2002

[Validation of a rapid urease test for the diagnosis of Helicobacter pylori infection].

OBJECTIVE: Several non-commercial rapid urease tests have been designed with the objective of reducing the cost of Helicobacter Pylori infection diagnosis. The objective of the present prospective trial is to assess one of these tests, using the histologic evaluation for the presence of Helicobacter Pylori, as the standard reference of diagnosing this infection. METHODS: Patients undergoing upper endoscopy for various reasons were prospectively enrolled. Three endoscopic biopsies of the antrum and three from the corpus of the stomach were taken in every patient enrolled. The specimens were evaluated by the rapid urease test on an individual basis, comparing the results with the histology assessment of the Helicobacter Pylori status, which was considered as the standard reference for the diagnosis of the infection. RESULTS: One hundred and four patients were enrolled, of which 94 were eligible. Fifty-five patients (60.43%) were infected with Helicobacter Pylori. The sensibility and specificity of the urease test evaluated at 4 hours was 65.45% and 100% respectively. The evaluation at 24 hours of the sensibility and specificity was 83% and 94% respectively. CONCLUSIONS: The non-commercial rapid urease test is a practical, fast, and cost effective method for the detection of Helicobacter Pylori infection, and its diagnostic utility is similar to the commercial test available.

Adult↗