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PubMedDate not supplied

Urease activity in gastrointestinal tract of rabbit and electrophoretic behaviour of urease.

Urease activity in the stomach (fundus and antrum), caecal content and soft faeces of rabbit was studied. Significant differences between fundus and antral content (P less than 0.01) and between caecum and soft faeces (P less than 0.05) were observed. The urease zymograms from caecal content and soft faeces of rabbit presented two different bands. The fundus content and the caecal ureolytic Clostridium innocuum bacterium exhibited only one band. Band B of caecal content was not evident at pH 4, whereas band A, also present in the stomach, was observed both at pH 4 and at pH 6. The optimal pH of urease activity of stomach and caecal content was in the range of 4-5 and 5-6, respectively. A comparison of intestinal urease zymograms with those of the single ureolytic bacterial species was suggested in order to clarify their role in urea metabolism.

Animals↗
PubMed1994

Urease-mediated destruction of bacteria is specific for Helicobacter urease and results in total cellular disruption.

The survival of Helicobacter mustelae, Proteus mirobilis, Escherichia coli and Campylobacter jejuni in the presence of urea and citrate at pH 6.0 was examined. H. mustelae, which has urease activity similar to H. pylori, had a markedly reduced survival, median 2.5% (0-78%) (P < 0.001) when incubated under these conditions. Only 7% of the ammonia produced by H. mustelae urease activity was recovered from the buffer, a similar percentage to that previously reported with H. pylori. None of the other organisms, all of which had lower urease activity, had impaired survival under these conditions. Electron microscopical studies demonstrated extensive structural damage to H. pylori following exposure to urea and citrate at pH 6.0. This structural damage to the organisms makes it unlikely that the low recovery of ammonia was due to retention of ammonia within the bacteria and suggests that the ammonia may have been incorporated into glutamate or other amino acids. Incorporation of ammonia into these compounds would deplete the cell of the key metabolic intermediate alpha-ketoglutarate and could thus explain the mechanism of the urease-dependent destruction of the organism.

Ammonia↗
PubMed1998

Urease-positive, acid-sensitive mutants of Helicobacter pylori: urease-independent acid resistance involved in growth at low pH.

Acid resistance is considered an important virulence factor of the human pathogen Helicobacter pylori. The enzyme urease plays an important role in this acid resistance, but there are indications that other systems are present. We set out to establish the relevance of these urease-independent acid-resistance systems for growth at low pH. Four mutants out of a total of 1000 UV-mutants were urease positive, grew identical to wild-type on pH 7 plates, but did not grow on pH 5 plates. Whereas transformation of a mutant with its own chromosomal DNA did not restore growth at pH 5, transformation with wild-type DNA or DNA of one of the other mutants did restore the growth. From these complementation studies, we conclude that in H. pylori a urease-independent acid-resistance system, probably depending on the expression of more than one gene, is involved in the growth at low pH.

Acids↗
PubMed2001

Recombinant urease and urease DNA of Coccidioides immitis elicit an immunoprotective response against coccidioidomycosis in mice.

Coccidioides immitis antigens which stimulate a T helper cell 1 (Th1) pathway of host immune response are considered to be essential components of a vaccine against coccidioidomycosis. Recombinant urease (rURE) and recombinant heat shock protein 60 (rHSP60) of C. immitis were expressed in Escherichia coli and tested as vaccine candidates in BALB/c mice. A synthetic oligodeoxynucleotide which contained unmethylated CpG dinucleotides and was previously shown to enhance a murine Th1 response was used as an immunoadjuvant. T cells isolated from the spleens and lymph nodes of the rURE- and rHSP60-immune mice showed in vitro proliferative responses to the respective recombinant protein, but only those T lymphocytes from rURE-immunized mice revealed markedly elevated levels of expression of selected Th1-type cytokine genes. BALB/c mice immunized subcutaneously with rURE and subsequently challenged by the intraperitoneal (i.p.) route with a lethal inoculum of C. immitis arthroconidia demonstrated a significant reduction in the level of C. immitis infection compared to control animals. rHSP60 was much less effective as a protective antigen. Evaluation of cytokine gene expression in lung tissue and levels of recombinant urease-specific immunoglobulins (immunoglobulin G1 [IgG1] versus IgG2a) in murine sera at 12 days after challenge provided additional evidence that immunization with rURE stimulated a Th1 response to the pathogen. Urease was further evaluated by expression of the URE gene in a mammalian plasmid vector (pSecTag2A.URE) which was used to immunize mice by the intradermal route. In this case, 82% of the vector construct-immunized animals survived more than 40 days after i.p. infection, compared to only 10% of the mice immunized with the vector alone. In addition, 87% of the pSecTag2A.URE-immunized survivors had sterile lungs and spleens. These data support the need for further evaluation of the C. immitis urease as a candidate vaccine against coccidioidomycosis.

Animals↗
PubMed2005

Effect of the urease accessory genes on activation of the Helicobacter pylori urease apoprotein.

The roles that accessory gene products play in activating the Helicobacter pylori urease apoprotein were examined. The activity of the urease apoprotein increased in the following order when it was expressed with the accessory genes: ureG<ureGH<ureFGH<ureEFGH<ureIEFGH. Moreover, stepwise additions of ureE and ureI to ureFGH significantly increased urease activity. Urease apoproteins coexpressed with ureFGH, ureEFGH, and ureIEFGH had similar low chymotrypsin susceptibilities. In vivo and in vitro activation studies showed that the cooperative effect of the accessory proteins involved processes in which the UreFGH complex, UreE, and UreI were implicated. Thus, the UreFGH complex may serve to alter the conformation of the apoprotein into one that is more competent to assemble a stable metallocenter, and that facilitates cooperative effects.

Apoproteins↗
PubMed1996

[A comparison of purified urease antigen and whole cell antigen of Helicobacter pylori by ELISA test--study on the application and serum diagnoses of Helicobacter pylori urease diagnostic reagent].

A useful assay for epidemiological survey of H. pylori infection was reported, using the urease antigen of H. pylori to detect the anti-urease antibody in sera from 676 patients suffered from gastropathy with ELISA technique, and compared with whole cell antigen. Results showed that the purified urease antigen was better than whole cell antigen. The partially purified urease antigens rapid diagnostic reagent of H. pylori was examined in hospital/institution and compared with whole-cell antigens. Results of sera from 676 H. pylori-positive gastritis and non-ulcer dyspepsia patients being tested showed that specificity and sensitivity of ELISA were 96% and 98% respectively. It seemed to be very useful for epidemiological studies on H. pylori infection. The use of ELISA in the detection of IgG antibodies against H. pylori was also sensitive, specific and rapid in assessing the improvement of both acute and chronic inflammation, cleaning of bacteria and the antibody titers after treatment, so as recognized an ideal diagnostic method.

Adolescent↗
PubMed1994

A 4.6 kb DNA region of Rhizobium meliloti involved in determining urease and hydrogenase activities carries the structural genes for urease (ureA, ureB, ureC) interrupted by other open reading frames.

A 4.6 kb DNA region of the Rhizobium meliloti strain AK631 was found to contain seven open reading frames (ORFs), all oriented in the same direction. The putative gene products of four of these ORFs were highly homologous to UreA, UreB and UreC of Klebsiella aerogenes, Proteus mirabilis, Proteus vulgaris and Canavalia ensiformis. The overall organisation of the DNA region analysed was ORF1, ureA (ORF2), ORF3, ureB (ORF4), ORF5, ORF6 and ureC (ORF7), indicating that the organisation of the urease structural genes in R. meliloti differs from that of other urease genes so far characterized. ORF1 was incomplete; only the 3' end of the coding region was present. The six complete ORFs coded for polypeptides of 11.1 (UreA), 8.9 (ORF3), 10.8 (UreB), 15.0 (ORF5), 13.8 (ORF6) and 60.7 kDa (UreC). No sequence homology to known polypeptides could be detected for the gene products of ORF1, ORF3, ORF5 and ORF6. Using a lacZ fusion and insertional mutagenesis it was shown that the seven ORFs identified were all located in the same transcription unit. For mutational analysis a resistance gene cassette was introduced into each of the complete ORFs resulting in apolar mutations. Mutations in ureA, ureB and ureC, but not in ORF3, ORF5 and ORF6, abolished urease activity in R. meliloti. The determination of hydrogen uptake in these R. meliloti mutants revealed that only ORF6 and ureB are necessary for hydrogen uptake.

Amino Acid Sequence↗
PubMed1996

Potential contribution of optional urease-positive bacteria to idiopathic urinary calcium stone formation. II. Microlith formation kinetics in a fermenter model of the urinary tract infected by optional urease-positive microorganisms.

We investigated the effects of weak to moderate urease hydrolysis by optional urease-positive microorganisms in an artificial urine model enriched with calcium phosphate and calcium oxalate in respect of calcium stone formation. The incubation experiments were performed using a discontinuously running fermenter device to simulate the urinary system. The kinetics of cell division rates, pH and ammonium ion production were measured and correlated to crystallite appearance in the incubation medium. Qualitative analyses of the sediments revealed apatite. Investigations using light microscopy and scanning electron microscopy (SEM) confirmed the matrix effect of bacterial glycoproteins. It was shown that initiation of calcium oxalate stone formation is in all probability equally determined by matrix effects and by heteronuclear crystallization if the urinary tract is infected by optional urease-positive bacteria. When urinary inorganic phosphate is present, calcium phosphate nidi are always initially formed, and may subsequently be coated by calcium oxalate.

Calcium Oxalate↗
PubMed1994

The urease structural gene ureA in Rhizobium meliloti is preceded by an open reading frame necessary for urease activity.

An open reading frame (ORF1) located upstream of the urease structural gene ureA in Rhizobium meliloti strain AK631 was cloned and characterized by DNA sequencing. Comparison of the amino acid sequence revealed partial homology with the urease accessory gene ureD of Klebsiella aerogenes and Proteus mirabilis. Mutational analysis of ORF1 showed that the gene is necessary for urease activity. Its function is still unknown.

Amino Acid Sequence↗
PubMed2006

Genetic heterogeneity of urease gene loci in urease-positive thermophilic Campylobacter (UPTC).

Degenerate PCR primers in silico based on the two urease structural genes, ureA and ureB, were designed for urease-positive thermophilic Campylobacter (UPTC). Resultant PCR amplification employing these primers generated an amplicon of approximately 2kb, which was cloned and sequenced in UPTC (n=12) isolated from various parts of Europe and Japan. Overall, sequence similarities were shown to be 96.7 to 99.9%. Following sequence alignment analysis, the approximate 1.96kb regions were deduced to consist of parts of ureA (about 570bps) and ureB (about 1390bps) with an overlapping region between the ureA and ureB gene loci. Although a total of 144 heterogeneous sites of all substitutions were located throughout this region, the substitution ratio was higher in the ureA region (1/Omega10bases) than in the ureB region (1/Omega15bases). A resulting dendrogram was constructed, which was based on the nucleotide sequence data of 12 UPTC isolates and demonstrated that the UPTC were genetically variable. They formed a major cluster with Helicobacter, separate from the other urease-producing bacteria examined, suggesting a shared ancestry between UPTC and Helicobacter.

Animals↗
PubMed1999

Urease and hexadecylamine-urease films at the air-water interface: an x-ray reflection and grazing incidence x-ray diffraction study.

We report the results of surface x-ray scattering measurements performed on urease and hexadecylamine-urease films at the air-aqueous solution interface. It is demonstrated that although hexadecylamine does not form a stable monolayer on the pure aqueous surface, it does self-assemble into a stable, well-organized structure when spread on top of a urease film at the air-water interface. It is also likely that protein and hexadecylamine domains coexist at the interface.

Air↗
PubMed1997

Urease-independent chemotactic responses of Helicobacter pylori to urea, urease inhibitors, and sodium bicarbonate.

Helicobacter pylori CPY3401 and an isogenic urease-negative mutant, HPT73, showed chemotactic responses to urea, flurofamide (a potent urease inhibitor), and sodium bicarbonate. Since urea and sodium bicarbonate are secreted through the gastric epithelial surface and hydrolysis of urea by urease on the bacterial surface is essential for colonization, the chemotactic response of H. pylori may be crucial for its colonization and persistence in the stomach.

Chemotaxis↗
PubMed2005

Biosynthesis of active Bacillus subtilis urease in the absence of known urease accessory proteins.

Bacillus subtilis contains urease structural genes but lacks the accessory genes typically required for GTP-dependent incorporation of nickel. Nevertheless, B. subtilis was shown to possess a functional urease, and the recombinant enzyme conferred low levels of nickel-dependent activity to Escherichia coli. Additional investigations of the system lead to the suggestion that B. subtilis may use unidentified accessory proteins for in vivo urease activation.

Bacillus subtilis↗
PubMed1980

Jack bean urease (EC 3.5.1.5). V. On the mechanism of action of urease on urea, formamide, acetamide, N-methylurea, and related compounds.

Acetamide and N-methylurea have been shown for the first time to be substrates for jack bean urease. In the enzymatic hydrolysis of urea, formamide, acetamide, and N-methylurea at pH 7.0 and 38 degrees C, kcat has the values 5870, 85, 0.55, and 0.075 s-1, respectively. The urease-catalyzed hydrolysis of all these substrates involves the active-site nickel ion(s). Enzymatic hydrolysis of the following compounds could not be detected: phenyl formate, p-nitroformanilide, trifluoroacetamide, p-nitrophenyl carbamate, thiourea, and O-methylisouronium ion. In the enzymatic hydrolysis of urea, the pH dependence of kcat between pH 3.4 and 7.8 indicates that at least two prototropic forms are active. Enzymatic hydrolysis of urea in the presence of methanol gave no detectable methyl carbamate. A mechanism of action for urease is proposed which involves initially an O-bonded complex between urea and an active-site Ni2+ ion and subsequently an O-bonded carbamato-enzyme intermediate.

Acetamides↗
PubMed1993

[Helicobacter pylori and urease activity--comparative study between urease positive and negative mutant strains].

The ammonia concentration in the gastric juice from H. pylori positive cases was significantly higher than that from negative cases, and there was positive correlation between number of H. pylori, gastritis score and ammonia concentration in gastric juice. It is suggested that ammonia in gastric juice which is produced by powerful urease activity of H. pylori is one of the pathogenic factors in gastritis. We succeeded in producing a urease-negative mutant strain of H. pylori. This strain did not colonize gastric mucosa of Cynomolgus monkeys. H. pylori survival time in vitro was increased at pH 2.0 in the presence of urea. These results suggest that urease facilitates colonization of gastric mucosa by H. pylori, and prolongs the survival of H. pylori in an acidic environment.

Ammonia↗
PubMed1995

Essential role of urease in vitro and in vivo Helicobacter pylori colonization study using a wild-type and isogenic urease mutant strain.

To determine the role of urease in colonization of Helicobacter pylori, we constructed a Ure- mutant by allelic exchange mutagenesis and examined the characteristics of the Ure- mutant and its parental strain. We also evaluated whether both strains would be able to colonize the stomach of nude mice. Kmr transformants obtained showed the Ure- phenotype, and one transformant, HPT73, was established as a knock-out mutant of the ureB gene by Southern analysis. Except for the null activity of the urease, HPT73 showed no difference from its parental strain, CPY3401, in growth rate in liquid medium, production of cytotoxin, and motility. However, CPY3401 had more acid resistance than HPT73. Moreover, CPY3401, but not HPT73, was detected in the nude mouse stomach. In the presence of HRA, both CPY3401 and HPT73 colonized the stomachs of nude mice. In summary, H. pylori requires urease for colonization in vivo and its survival under the acidic conditions of the stomach.

Alleles↗
PubMed1989

Algal partner regulates fungal urease in the lichen Evernia prunastri by producing a protein which inhibits urease synthesis.

Occurrence of a protein controlling urease synthesis (PIUS) at the transcriptional level in the lichen Evernia prunastri has been previously reported (Perez-Urria & Vicente, Physiol Plant 65: 433-438, 1985; id. Endocyt C Res 3: 311-316, 1986). In this work it was found that 0.1 mM cycloheximide seems to inhibit PIUS synthesis when lichen thalli are incubated on PIUS inducer, L-arginine. PIUS has been purified and characterized by PAGE, electrofocusing and amino acid analysis. It is a glycoprotein containing a homopolymer of fructose bound to the protein. PIUS has been located in whole thallus and lichenized mycobiont but remains undetectable in cultured fungi. PIUS is only detected in photobiont cells when they are axenically cultured on arginine. Thus, it is postulated that PIUS could be synthesized by lichenized photobionts from which it moves to mycobionts where it inhibits the production of fungal urease.

Amino Acids↗