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PubMed2001

In-gel detection of urease with nitroblue tetrazolium and quantification of the enzyme from different crop plants using the indophenol reaction.

Two methods for measurement of urease activity are described and demonstrated on extracts from several crop plants. With an in-gel staining method based on the principle of Fishbein's noninhibitory stain for urease as little as 25 microU of jackbean urease can be detected within 2 h following electrophoresis. A comparison with published in-gel staining methods shows that the sensitivity is improved by at least two orders of magnitude. The second method allows quantification of urease activity from small amounts of plant material without the need for special laboratory equipment. It employs the detection of ammonium by the indophenol reaction. To eliminate reducing agents which are often necessary to maintain urease activity during extraction, but which interfere with ammonium detection, a simple spin-column procedure is used. The quantification of less than 5 mU/ml extract is possible.

Electrophoresis↗
PubMed2001

Marked reduction of Helicobacter pylori-induced gastritis by urease inhibitors, acetohydroxamic acid and flurofamide, in Mongolian gerbils.

Urease has been suggested to be essential for colonization and pathogenesis of Helicobacter pylori infection. In the present study, we evaluated the effects of urease inhibitors [acetohydroxamic acid (AHA) and flurofamide (FFA)] on H. pylori-induced gastritis in Mongolian gerbils. Animals were orally inoculated with H. pylori, and given urease inhibitors in their diet throughout the experimental period of six weeks or four weeks, starting from two weeks after H. pylori inoculation. With the administration of AHA at doses of 100, 500, and 2500 ppm throughout the experimental period, H. pylori-induced gastritis in animals was decreased in a dose-dependent manner, significantly so at 2500 ppm. Suppression of gastric lesions was also evident in animals administered 2500 ppm AHA after the H. pylori infection. Bacterial infection rates were reduced to 40-50% of the control value of 100%, by the highest dose of AHA. The potent urease inhibitor, FFA, also caused marked amelioration of H. pylori-associated gastritis on administration at 100 ppm throughout the six-week experimental period or for four weeks after H. pylori infection. Animals treated with FFA had few visible gastric lesions, and the proportion infected with H. pylori was reduced to less than 10%. Since antibiotic-resistant strains of H. pylori have become a serious problem, nonantibiotic urease inhibitors may be very useful to control H. pylori-associated gastroduodenal disease.

Administration, Oral↗
PubMed1992

The influence of pH and urine composition on urease enzymatic activity in human urine.

It is reasonable to assume that the rate of pH increase in urine induced by urease-producing microorganisms is one of the factors which determine whether crystallisation with subsequent stone formation will occur or not. To evaluate how the time needed to increase urine pH varies between different urine samples and how it depends on urine composition, a standardised amount of urease was added to different human urine samples. The incubations were performed in a pH-stat. This allowed simultaneous study of how urease enzymatic activity depends on urine pH and how it varies between different urines. The enzymatic activity was found to be negatively correlated to urine pH and to vary between different urines. The rate of the pH increase varied markedly between different urines. Small pH increases depended on the native urine pH and urease enzymatic activity. Higher pH increases up to the levels of phosphate crystallisation depended more on urine phosphate, the major urine buffer. The results presented show that urine composition influences the urease-induced pH increase. This might have clinical implications.

Adult↗
PubMed1983

Urease activity in Trichophyton mentagrophytes.

Urease activity was detected in the dermatophyte Trichophyton mentagrophytes cells at early exponential phase of growth. Specific activity of urease decreased with culture age. At exogenous urea concentrations above 2 mM formation of urease was inhibited. The pH optimum lay at 7-7.5, the Km being 14 mM. No urease activity could be detected in cell-free culture fluid of T. mentagrophytes. No endo- or exocellular urease activity could be detected in a T. rubrum strain grown with or without urea.

Ammonia↗
PubMed1998

The effect of urease inhibitors on the encrustation of urethral catheters.

Encrustation and blockage of indwelling urethral catheters is primarily brought about by infection of the urinary tract by Proteus mirabilis or other urease-producing species. The bacteria colonise the catheter forming a biofilm community within a polysaccharide matrix. The activity of the urease drives up the urinary pH and causes the crystallisation of calcium and magnesium phosphates in the biofilm. We have used a simple physical model of the catheterised bladder to investigate the ability of urease inhibitors to control encrustation. It was observed that acetohydroxamic acid (1.0 mg/ml) and fluorofamide (1.0 microg/ml) restricted the increase in pH of P. mirabilis-infected urine from 9.1 to 7.6. Significant reductions in the deposition of calcium and magnesium salts were also recorded on the silicone catheters. Electron microscopy confirmed that encrustation and occlusion of the catheter lumen was minimal in the presence of the urease inhibitors. The data from this in vitro study suggests that urease inhibitors, particularly fluorofamide, could have clinical applications in the prevention of catheter encrustation and blockage.

Benzamides↗
PubMed2005

A newly constructed primer pair for the PCR amplification, cloning and sequencing of the flagellin (flaA) gene from isolatesof urease-negative Campylobacter lari.

A newly constructed primer pair (lari-Af/lari-Ar) designed to generate a product of the flagellin (flaA) gene for urease-negative Campylobacter lari produced a PCR amplicon of about 1700 bp for 16 isolates from 7 seagulls, 5 humans, 3 food animals and one mussel in Japan and Northern Ireland. Nucleotide sequencing and alignments of the flaA amplicons from these isolates demonstrated that the deduced amino acid sequences of the possible open reading frame were 564-572 amino acid residues in length with calculated molecular weights of 58,804 to 59,463. The deduced amino acid sequence similarity analysis strongly suggested that the ORF of the flaA from the 16 isolates showed 70-75% sequence similarities to those of Campylobacter jejuni isolates. The approximate Mr of the flagellin purified from some of the isolates of urease-negative C. lari was estimated to range from 59.6 to 61.8 kDa. Thus, flagellin from the isolates of urease-negative C. lari was shown for the first time to have a molecular size similar to those of C. jejuni and Campylobacter coli isolates, but to be different from the shorter flaA and smaller flagellin of urease-positive thermophilic Campylobacter (UPTC) isolates. Flagellins from C. lari spp., consisting of the two representative taxa of urease-negative C. lari and UPTC, thus show genotypic and phenotypic diversity.

Amino Acid Sequence↗
PubMed2006

Inhibition of urease by bismuth(III): implications for the mechanism of action of bismuth drugs.

Bismuth compounds are widely used for the treatment of peptic ulcers and Helicobacter pylori infections. It has been suggested that enzyme inhibition plays an important role in the antibacterial activity of bismuth towards this bacterium. Urease, an enzyme that converts urea into ammonia and carbonic acid, is crucial for colonization of the acidic environment of the stomach by H. pylori. Here, we show that three bismuth complexes exhibit distinct mechanisms of urease inhibition, with some differences dependent on the source of the enzyme. Bi(EDTA) and Bi(Cys)(3) are competitive inhibitors of jack bean urease with K(i) values of 1.74 +/- 0.14 and 1.84 +/- 0.15 mM, while the anti-ulcer drug, ranitidine bismuth citrate (RBC) is a non-competitive inhibitor with a K (i) value of 1.17 +/- 0.09 mM. A (13)C NMR study showed that Bi(Cys)(3) reacts with jack bean urease during a 30 min incubation, releasing free cysteines from the metal complex. Upon incubation with Bi(EDTA) and RBC, the number of accessible cysteine residues in the homohexameric plant enzyme decreased by 5.80 +/- 0.17 and 11.94 +/- 0.13, respectively, after 3 h of reaction with dithiobis(2-nitrobenzoic acid). Kinetic analysis showed that Bi(EDTA) is both a competitive inhibitor and a time-dependent inactivator of the recombinant Klebsiella aerogenes urease. The active C319A mutant of the bacterial enzyme displays a significantly reduced sensitivity toward inactivation by Bi(EDTA) compared with the wild-type enzyme, consistent with binding of Bi(3+) to the active site cysteine (Cys(319)) as the mechanism of enzyme inactivation.

Amino Acid Sequence↗
PubMed1989

Selective silver staining of urease activity in polyacrylamide gels.

A selective method for staining urease activity bands in nondenaturing polyacrylamide gels is described. It is based on the deposition of silver at the urease bands after incubation of gels in the presence of urea and photographic developers. Its highly sensitivity (up to 0.015 enzyme units, corresponding to 5 ng of purified urease) is based on both the silver deposition enhancement methodology and the developers used. The selectivity of the procedure is based on the local pH increase catalytically produced by the enzyme in the presence of urea. The densitometric scan of the enzyme bands gives a linear response at least in the range 0.015-0.300 urease units. This selective staining method is about 2.5 times more sensitive than the standard silver staining of proteins, in terms of detectable urease amount.

Densitometry↗
PubMed1976

Photocontrol of urease activity in spiropyran collagen membrane.

1. Collagen fibrils were modified with beta-1-[3,3-dimethyl-6'-nitrospiro-(indoline-2,2'-2H-benzopyran)] propionic anhydride. 2. Urease (urea amidohydrolase, EC 3.5.1.5) was immobilized in spiropyran collagen membrane. The activity of the urease-spiropyran collagen membrane was found to increase in the dark and then decrease with visible light irradiation. 3. The optimum pH of the urease-spiropyran collagen membrane under visible light was lowered in the dark. 4. The apparent Michaelis constant (K'm) of the urease-spiropyran collagen membrane in the dark was almost the same as that under visible light. The apparent maximum velocity was increased in the dark. 5. The diffusion coefficient of urea through the spiropyran collagen membrane in the dark was 1.4 times that under visible light. However, the increase of the diffusion rate was not responsible for the activity increase of the urease-spiropyran collagen membrane.

Collagen↗
PubMed1991

The demonstration of nickel in the urease of Helicobacter pylori by atomic absorption spectroscopy.

Fast protein liquid chromatography and SDS-PAGE have been used to isolate and purify Helicobacter pylori urease. A nickel component of the urease was detected in the purified proteins by atomic absorption spectroscopy. The nickel was present only in the 61 kDa polypeptide and in the ratio of between five and six atoms to one molecule of urease, suggesting a hexameric structure. These results are discussed in relation to other bacterial ureases and urease activity at low pH.

Chromatography, Liquid↗
PubMed1991

Cloning and expression of various staphylococcal genes encoding urease in Staphylococcus carnosus.

The urease genes from Staphylococcus xylosus C2a, Staphylococcus aureus U500, and S. aureus Newman were cloned in Staphylococcus carnosus using the plasmid vectors pCA43 and pCA44. The resulting respective recombinant plasmids pUra 402, pUraUH66, and pUra17 contained chromosomal DNA fragments with sizes of 5.6, 5.8, and 6.8 kb, respectively. Investigations on urease expression of the donor and recombinant strains in media with various nitrogen sources revealed that S. xylosus C2a produced urease constitutively at the highest specific activity. All of the recombinant strains had significantly lower urease activities than their DNA-donor strains. The nickel-dependence of urease was demonstrated in S. aureus U500 by a plate diffusion assay.

Cloning, Molecular↗
PubMed1994

Assessment of four rapid urease test systems for detection of Helicobacter pylori in gastric biopsy specimens.

An in vitro experiment and an in vivo clinical trial were both performed in order to assess the efficacy of four biopsy urease test systems, including one commercial kit, Temmler CUT test (Temmler Pharma, Marburg, Germany), for the rapid detection of Helicobacter pylori. We first evaluated four rapid urease test systems by inoculating bacterial suspensions of different concentrations into urea-containing media and observing the color change at room temperature. We found that the CUT test was superior in vitro to the other three urease test systems. As was expected, the lower the concentration of the inoculum, the slower was the color change and the fewer were the positive results noted. The minimal concentration of H. pylori for a positive urease test at 24 h was 1000-10,000 colony-forming units/ml in 1 drop of bacterial suspension inoculated. We then evaluated four biopsy urease test systems for the rapid diagnosis of H. pylori infection in antral and fundal mucosa biopsy specimens of 37 patients undergoing upper gastrointestinal endoscopy. All four test systems were 100% specific when compared with culture. In 69 culture-positive biopsy specimens, the CUT test was not only more sensitive (72%) than the other three systems (42%, 51%, and 45%, respectively), but also gave the fastest reaction by detecting more culture-positive biopsy specimens after 3 h of incubation at room temperature. The differences were statistically significant.

Bacteriological Techniques↗
PubMedDate not supplied

Cloning of Ureaplasma urealyticum DNA sequences showing genetic homology with urease genes from gram-negative bacteria.

DNA chromosomic sequences from Ureaplasma urealyticum were shown, by DNA hybridization, to possess homology with Providencia stuartii urease genes. The homologous ureaplasma DNA fragment was cloned in Escherichia coli and a DNA probe, designated IC61 probe was specific for only the urease-producing U. urealyticum among the Mollicutes. Genetic homology shown between the IC61 probe and urease genes from Campylobacter pylori, E. coli and P. stuartii suggests that (1) the cloned DNA fragment from U. urealyticum contains urease encoding sequences, and that (2) the urease genes possess conserved sequences among phylogenetically distant prokaryotes.

Cloning, Molecular↗
PubMed2004

Kinetics of novel competitive inhibitors of urease enzymes by a focused library of oxadiazoles/thiadiazoles and triazoles.

Based on structure of the substrate of urease and for the purpose of designing pharmacophore models for urease inhibitors, which could be effective in physiological and pharmacological studies, a series of twenty-five 1,3,4-diazole-2(3H)-thiones-2(3H)-thiones, 1,3,4-diazoles-2(3H)-thiones, and 1,2,4-tri-3-thiones (OSNs) were designed, synthesized, and evaluated for various kinetic parameters of urease inhibition. OSNs inhibited the activity of urease(s) in a concentration dependent fashion. Dixon as well as Lineweaver-Burk plots and their secondary replots indicated that the nature of inhibition was of pure competitive type for all the 25 compounds. 5-[4-(hydroxy)phenyl]-1,3,4-thiadiazole-2(3H)-thione was found to be the most active one with a Ki value of 2 microM. The Ki values were increased with an increase in substrate concentrations. Apparently, OSNs employ a homologous mechanism of action by exploiting a common transition catalysis state and acting as ligand chelators to form octahedral complexes with the urease enzymes in an orientation-specific mode. The inhibition was slightly potentiated by lower pH and not abolished in the presence of NH2OH (a scavenger of histidine residue). Because of their safe profile in the genotoxic assay, they may be pursued in the near future for human testing

Animals↗
PubMed2004

Development of urease and glutamic dehydrogenase amperometric assay for heavy metals screening in polluted samples.

An amperometric assay based on urease inactivation has been developed for the screening of heavy metals in environmental samples. The enzyme urease catalyses the hydrolysis of urea and the formation of NH(4)(+) is determined using a NADH-glutamate dehydrogenase coupled reaction system. NADH consumption is monitored amperometrically using screen-printed three electrode configuration and its oxidation current is then correlated to urease activity. The presence of heavy metals in the samples inhibits the urease activity, resulting in a lower NH(4)(+) production and therefore a decrease in NADH oxidation. The use of metallised carbon electrodes gave a decrease in NADH oxidation potential from +300 mV versus Ag/AgCl compared with > +600 mV for bare carbon electrodes, and thus minimised interferences from oxidizable species present in the samples. Electrodes fouling and possible contamination after reuse and cleaning was also eliminated by using screen-printed disposable electrodes. The linear range obtained for Hg(II) and Cu(II) was 10-100 microgl(-1) with a detection limit of 7.2 microgl(-1) and 8.5 microgl(-1), respectively. Cd(II) and Zn(II) produced enzyme inhibition in the range 1-30 mgl(-1), with limits of detection of 0.3 mgl(-1) for Cd(II) and 0.2 mgl(-1) for Zn(II). Pb(II) did not inactivate the urease enzyme significantly at the studied range (up to 50 mgl(-1)). Coefficients of variation (CV) values were 6-9% in all cases. Application of the assay system to leachate samples gave reliable and accurate toxicity assessments when compared to atomic absorption spectrometry (AAS) and inductively coupled plasma atomic emission spectroscopy (ICP-MS) analysis. This approach provides to be a simple and rapid (15 min, including enzyme inhibition time) method for metal ions detection.

Biosensing Techniques↗
PubMed2005

Insulator semiconductor structures coated with biodegradable latexes as encapsulation matrix for urease.

A new urea biosensor for clinical applications was obtained by immobilization of urease within different latex polymers functionalized by hydroxy, acetate and lactobionate groups. Responses of these biosensors based on pH-ion-selective field effect insulator-semiconductor (IS) systems to urea additions were evaluated by capacitance measurements. UV-visible spectroscopy was used to check the urease activity in various matrixes. A good retention of the catalytic urease activity in the case of the cationic polymers was observed. In addition, rotating disk electrode experiments were carried out to determine the matrix permeability characteristics. Under optimal conditions, i.e. buffer capacity corresponding to 5 mM phosphate buffer, the urea enzyme insulator semiconductor (ENIS) sensors showed a linear response for urea concentrations in the range 10(-1.5) to 10(-4)M. Furthermore, kinetic parameters for the immobilized urease were obtained from Lineweaver-Burk plot. Clearly, a fast response and a good adhesion for the urease-acetate polymer composite films, prepared without using glutaraldehyde as cross-linking agent was observed.

Absorbable Implants↗
PubMed2005

Urease biogenesis in Streptococcus thermophilus.

Urease biogenesis was monitored in the lactic acid bacterium Streptococcus thermophilus during the growth cycle using in-gel detection and a phenol-hypochloride assay. Zymogram analysis, performed in a non-denaturing polyacrylamide gel, enabled visualization of a complex profile of bands whose number and intensity were dependent on the growth phase and culture conditions. The monitoring of urease biogenesis in batch fermentations revealed the onset of enzyme synthesis starting from the mid-exponential growth phase, with a maximum reached during the late exponential phase. Urease activity strongly increased at acidic pH but to a lesser extent when urea and nickel ions were added to the culture medium. When S. thermophilus cells were cultured with pH maintained at a neutral value, urease activity was detectable only in gel with extremely low signals. Evaluation of beta-glucuronidase activity in strain DSM 20617(T) harboring a transcriptional fusion between a DNA fragment containing the putative urease promoter and the gusA reporter evidenced significant expression at neutral pH that strongly increased in an acidic environment. Further experiments carried out on p(ureI)-gusA recombinant strain revealed that expression of ure genes was not affected by carbohydrates, nickel or urea availability. The presence of consistent expression of ure genes at neutral pH and the absence of induction of expression by carbohydrate availability demonstrated that the transcription of ure genes in S. thermophilus is regulated differently compared with that of the closely related S. salivarius. These differences are discussed taking into consideration the different habitats colonized by the two bacterial species.

Bacterial Proteins↗
PubMed2005

The role of Klebsiella pneumoniae urease in intestinal colonization and resistance to gastrointestinal stress.

The first step in nosocomial infections due to Klebsiella pneumoniae is colonization of the patient's gastrointestinal (GI) tract. In a previous work, signature-tagged mutagenesis was used in a murine model to identify 13 genes required for efficient colonization, two of which were involved in urea metabolism. The role of urease was further investigated by the construction and analysis of an isogenic urease-deficient mutant. The behavior of both the wild-type strain and the urease-deficient mutant was tested under hostile conditions, reproducing stresses encountered in the GI environment. The wild-type strain had an acid tolerance response (ATR) to inorganic acid, was resistant to organic acids (38.5% survival) and was able to survive concentrations of bile encountered in vivo. The absence of urease did not affect the resistance of K. pneumoniae to acid and bile stresses, but the enhanced adhesion response to Int-407 cells after exposure to bile observed with the wild-type strain was no longer detected with the urease mutant. When tested in the murine intestinal colonization model, both strains were mainly recovered in the large intestine parts, and the mutant was impaired in its colonization capacities, but only when tested in competition with the wild-type strain. These findings emphasize the prominent role played by metabolic function in the colonization process of such a complex ecosystem as the host GI tract.

Acids↗