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At least 19 recordsLinked to original sources
PubMed1996

Potential contribution of optional urease-positive bacteria to idiopathic urinary calcium stone formation. I. Expression of urease activity in bacteria from the urinary tract that are commonly classified as urease-negative.

The aetiology of calcium oxalate stones, which are commonly believed to be sterile, has not yet been fully elucidated. Recent bacteriological studies and investigations using the scanning electron microscope have also shown microorganisms to be present in this type of stone. These microorganisms were assumed not to be able to split urea. To list the most common urease-negative bacteria established in the human urinary system, we isolated apparently urease-negative microorganisms from a consecutive series of 58 urinary stone-forming patients by using standard selecting agars. Pure strains were incubated in an inductive medium lacking all sources of nitrogen except urea. Induction of urease activity was monitored by a test based on the reaction of phenol/hypochlorite with ammonium ions. This test revealed whether the urease negativity of a strain indicated by the selective agar was optional or absolute. All strains we investigated by this method and which were classified by standard methods as urease-negative we found produced urease activity which was clearly measurable, though it was often comparatively small. In the light of these results, the matrix theory of calcium oxalate stone development will need some modifications.

Bacteria↗
PubMed1982

A soybean seed urease-null produces urease in cell culture.

Itachi, a soybean (Glycine max [L.] Merr.) variety with 0.2% normal seed urease activity, was recovered from a screen of 6,000 entries in the United States Department of Agriculture soybean germplasm collection. No urease antigen in Itachi seed extracts was detected by double diffusion or by rocket immunoelectrophoresis. Native gels stained for protein or ureolytic activity revealed no detectable urease holoenzyme. An anti-urease antibody affinity column was used to remove all detectable urease activity and antigen from ;wild type' (cv. Prize) seed extracts. Affinity column effluent and nonchromatographed Itachi extracts both lack a species which comigrates with purified urease subunits in sodium dodecylsulfate polyacrylamide gels. Inability to detect urease antigen or urease protein suggests that during development of Itachi seeds there is no synthesis of urease protein or that, at most, its synthesis is 0.2% of wild type (Prize).No urease activity or only traces of urease activity were detected in cotyledons of developing or germinating Itachi seeds. In contrast, callus cultures induced from cotyledon, shoot tip, root, or root tip tissues of Itachi seedlings exhibited ureolytic activity equivalent to that of Prize cultures. Shoot tip cultures of both Prize and Itachi grew with urea as sole nitrogen source. Most or all of the ureolytic activity in crude extracts of Prize and Itachi suspension culture cells is seed-like urease in thermal stability, recognition by antibodies to the seed enzyme, hydroxyurea sensitivity, and nickel requirement for synthesis. It has been reported previously (Polacco, Havir 1979 J Biol Chem 254: 1707-1715; Polacco, Sparks, Jr, Havir 1979 Genet Eng 1: 241-259) that partially purified cell culture urease is identical to seed urease by immunological and electrophoretic criteria. These results suggest that urease is under different developmental controls in the seed and in cell culture.In both Prize and Itachi cultures, utilization of the ureide allantoin, unlike that of urea, is not dependent on nickel. This suggests that ureide catabolism does not require urease.

Journal Article↗
PubMed2005

Identification of three urease accessory proteins that are required for urease activation in Arabidopsis.

Urease is a nickel-containing urea hydrolase involved in nitrogen recycling from ureide, purine, and arginine catabolism in plants. The process of urease activation by incorporation of nickel into the active site is a prime example of chaperone-mediated metal transfer to an enzyme. Four urease accessory proteins are required for activation in Klebsiella aerogenes. In plants urease accessory proteins have so far been only partially defined. Using reverse genetic tools we identified four genes that are necessary for urease activity in Arabidopsis (Arabidopsis thaliana; ecotypes Columbia and Nössen). Plants bearing T-DNA or Ds element insertions in either the structural gene for urease or in any of the three putative urease accessory genes AtureD, AtureF, and AtureG lacked the corresponding mRNAs and were defective in urease activity. In contrast to wild-type plants, the mutant lines were not able to support growth with urea as the sole nitrogen source. To investigate whether the identified accessory proteins would be sufficient to support eukaryotic urease activation, the corresponding cDNAs were introduced into urease-negative Escherichia coli. In these bacteria, urease activity was observed only when all three plant accessory genes were coexpressed together with the plant urease gene. Remarkably, plant urease activation occurred as well in cell-free E. coli extracts, but only in extracts from cells that had expressed all three accessory proteins. The future molecular dissection of the plant urease activation process may therefore be performed in vitro, providing a powerful tool to further our understanding of the biochemistry of chaperone-mediated metal transfer processes in plants.

Arabidopsis↗
PubMed1996

Metal ion interaction with urease and UreD-urease apoproteins.

Klebsiella aerogenes urease in a Ni-containing enzyme (two Ni per alpha beta gamma unit) that is purified as an apoprotein from cells grown in Ni-free medium. Partial activation of urease and UreD-urease apoproteins is achieved in vitro by incubation in the presence of Ni(II) and CO2, whereas incubation of these proteins with Ni alone leads to the formation of inactive species [Park, I.-S., & Hausinger, R. P. (1995) Science 267, 1156-1158]. Here we determined the kinetics of these inhibitory reactions and demonstrated the presence of two Ni ions per alpha beta gamma unit in the inactive proteins. Although metal-substituted urease has never been purified from Ni-deprived cell, several other metal ions were shown to bind to the urease apoproteins. Divalent Zn, C, Co, and Mn all inhibited Ni- and Co2-promoted urease activation at concentrations below that of Ni, whereas Mg and Ca ions did not inhibit this process. Ni-inhibited species recovered their ability to be partially activated after EDTA treatment. In contrast, samples that were exposed to Co or Cu ions were irreversibly inactivated, and EDTA treatment of Zn- or Mn-inhibited samples led to reduced levels of activation competence. Mn-substituted urease, generated from urease apoprotein samples in a Mn- and Co2-dependent manner, was shown to be active, whereas other metal-substituted forms if urease lacked activity. The Mn-protein possessed only 2% of the activity of Ni-activated apoprotein [ approximately 8.0 vs approximately 400 mumol min-1 (mg protein)-1], but its KM value was only moderately altered from that of the native enzyme (3.86 +/- 0.15 mM vs 0.2 mM). Unlike the Ni-containing enzyme, Mn-urease was inhibited by EDTA. Given the evidence that urease apoprotein binds numerous metal ions, we speculate on possible roles for the UreD, UreF, and UreG accessory proteins in urease activation.

Apoproteins↗
PubMed1997

Urease from a potentially pathogenic coccoid isolate: purification, characterization, and comparison to other microbial ureases.

Strain SL100 is a gram-positive coccoid isolate prototype with an adhesin specific for gastric mucin and is representative of potentially pathogenic organisms obtained at biopsy from patients with gastric disorders. The urease of this isolate constitutes a significant fraction of the total cell protein, and the outcome of the purification strategy described herein suggests that it is associated with a cell wall fraction. The urease was purified 138-fold to apparent homogeneity, as indicated by gel electrophoresis, to a specific activity of 1,120 U/mg. The urease was unstable during purification in the absence of nickel, which is present in a metallocenter in other microbial ureases. When nickel sulfate was present during growth (5 microM) and in buffers during sonication and purification (100 microM), the urease was completely stable at room temperature during the purification procedure. The native urease was approximately 260 kDa and was composed of three subunits of 65 kDa and three subunits of 21 kDa. The purified urease was relatively stable in acid and retained most of its activity after incubation for 30 min at pH 1.3. The K(m)s for urease measured from whole cells and for the purified enzyme were 0.56 and 1.7 mM, respectively, indicating that some cell wall component(s) affects the affinity of the enzyme for urea. The V(max)s for urea hydrolysis measured from whole cells and for the purified enzyme were 8.1 and 1,120 mol/min/mg of protein, respectively. The kinetic parameters, relative abundance, and subunit composition are more similar to those of the ureases of Helicobacter than to those of the ureases of other microbial species. These similarities are consistent with an adaptation of this organism to colonization of the stomach and indicate that the urease may be a virulence factor during colonization.

Enzyme Stability↗
PubMed2002

Gastric juice urease test and brushing urease test for Helicobacter pylori detection.

The aim of the study was to demonstrate the sensitivity, specificity and accuracy of gastric juice urease test and brushing-urease test compared to the biopsy-urease test for Helicobacter pylori (H. pylori) detection. For each patient, two milliliters of gastric juice was collected and one milliliter in the supernatant was tested for rapid urease reactions. One gastric mucus brushing and two biopsies were taken from the body and the antrum. The brushing specimens were tested for rapid urease reaction by shaking the brush into the urea broth. The gold standards for diagnosing of H. pylori are positive H. pylori upon specimen culture or positive identification of H. pylori from polymerase chain reaction (PCR) assay using primer for vac A gene. Forty patients were enrolled in the study including ten patients with gastric ulcer, six patients with duodenal ulcer and twenty four patients with non-ulcer dyspepsia. Brushing-urease test and biopsy-urease test were not different sensitivity (87.50% vs 93.20%), specificity (100% vs 100%) and accuracy (90.25% vs 95.50%). The gastric juice urease test had a sensitivity of 65.25 per cent, specificity of 100 per cent and accuracy of 75 per cent for detecting of H. pylori infection. In conclusion, gastric juice urease test had low sensitivity in the diagnosis of H. pylori infection. Brushing-urease test is as accurate as biopsy-urease test in detecting H. pylori infection. However, the brushing method had lower gastric tissue injury than the biopsy and so should be used for detecting H. pylori infection in patients with coagulopathy.

Adult↗
PubMed1987

Effects of anti-Ureaplasma urease antibody on homologous and heterologous urease activities.

Among organisms in the class Mollicutes only Ureaplasma species possess urease. Antiserum to urease of U. urealyticum strain T960 (CX8) was used to examine the cross-reactivity of urease from other Ureaplasma species, as well as urease of jack bean and several urease-possessing walled bacteria. Immunological cross reactivity was used to establish phylogenetic relationships between various antigens. The ability of monospecific anti-urease antibody to inhibit urease activity was examined. The antiserum inhibited urease activity of the homologous strain the least of any Ureaplasma tested. It is postulated that urease possesses a minimum of two sets of epitopes. Binding of antibody to one epitope causes inhibition of enzyme activity; this epitope is common to urease of all Ureaplasma species. Binding of antibody to the other epitope prevents binding to the inhibition epitope; this epitope is specific to U. urealyticum strain T960 (CX8). No inhibition was observed with urease from jack bean or several walled bacteria.

Animals↗
PubMed1992

Monoclonal antibodies against the native urease of Helicobacter pylori: synergistic inhibition of urease activity by monoclonal antibody combinations.

Monoclonal antibodies (MAbs) against the native urease of Helicobacter pylori NCTC 11637 were found to clearly inhibit the urease activity. Interestingly, synergistic inhibition by two MAbs recognizing different subunits was also observed. Ten MAbs were produced and classified as two isotypes of the immunoglobulin G (IgG) subclass, IgG1, and IgG2a. Western blot (immunoblot) analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that five MAbs recognized the large subunit and the other five recognized the small subunit of the urease. Among the MAbs, L2 and S2, which recognized the large and the small subunits, respectively, were also able to inhibit the urease activity of clinical isolates from H. pylori-infected patients. The combination of L2 and S2 led to augmented synergistic inhibition. L2, but not S2, could also inhibit the urease activity from Helicobacter mustelae; enzyme-linked immunosorbent assay and Western blot analysis showed that L2 cross-reacted with this urease. These results suggested that the epitope recognized by L2 had a structure common to both Helicobacter species and may be involved in the active site of the urease. In contrast to the MAbs, a polyclonal antibody in sera from mice immunized with H. pylori urease did not have the ability to inhibit H. pylori urease activity. However, the polyclonal antibody retained the ability to abolish the inhibitory action of these MAbs. Moreover, other MAbs which could not inhibit H. pylori urease activity also abolished the inhibitory action.

Antibodies, Bacterial↗
PubMed2006

Ureases display biological effects independent of enzymatic activity: is there a connection to diseases caused by urease-producing bacteria?

Ureases are enzymes from plants, fungi and bacteria that catalyze the hydrolysis of urea to form ammonia and carbon dioxide. While fungal and plant ureases are homo-oligomers of 90-kDa subunits, bacterial ureases are multimers of two or three subunit complexes. We showed that some isoforms of jack bean urease, canatoxin and the classical urease, bind to glycoconjugates and induce platelet aggregation. Canatoxin also promotes release of histamine from mast cells, insulin from pancreatic cells and neurotransmitters from brain synaptosomes. In vivo it induces rat paw edema and neutrophil chemotaxis. These effects are independent of ureolytic activity and require activation of eicosanoid metabolism and calcium channels. Helicobacter pylori, a Gram-negative bacterium that colonizes the human stomach mucosa, causes gastric ulcers and cancer by a mechanism that is not understood. H. pylori produces factors that damage gastric epithelial cells, such as the vacuolating cytotoxin VacA, the cytotoxin-associated protein CagA, and a urease (up to 10% of bacterial protein) that neutralizes the acidic medium permitting its survival in the stomach. H. pylori whole cells or extracts of its water-soluble proteins promote inflammation, activate neutrophils and induce the release of cytokines. In this paper we review data from the literature suggesting that H. pylori urease displays many of the biological activities observed for jack bean ureases and show that bacterial ureases have a secretagogue effect modulated by eicosanoid metabolites through lipoxygenase pathways. These findings could be relevant to the elucidation of the role of urease in the pathogenesis of the gastrointestinal disease caused by H. pylori.

Animals↗
PubMed2006

Cysteine based novel noncompetitive inhibitors of urease(s)--distinctive inhibition susceptibility of microbial and plant ureases.

Based on the catalysis mechanism of urease, a homologous series of 10 cysteine derivatives (CysDs) was designed and synthesized, and their inhibitory activities were evaluated for microbial ureases (Bacillus pasteurii, BPU, and Proteus mirabilis, PMU) and for a plant urease [jack bean (Cavavalia ensiformis), JBU]. As already described, thiol-compounds might inhibit urease activity by chelating the nickel atoms involved in the catalysis process. In contrast to cysteine, which has been reported to be a very weak urease inhibitor, we verified a potential inhibitory activity of these CysDs. The kinetic data demonstrate that thiol derivatives are more effective than the respective thioether derivatives. Besides, thiol-CysDs had a reduced activity in acidic pH (5.0). Lineweaver-Burk plots indicated that the nature of inhibition was of noncompetitive type for all 10 compounds, with the minimum Ki value of 2 microM for N,N-dimethyl L-cysteine. It is proposed that these classes of compounds are more potent inhibitors of the bacterial ureases, compared with the plant-originated urease. Since microbial urease is directly involved in the infection process of many pathological organisms, this work demonstrates that thiol-CysDs represent a class of new potential urease inhibitors.

Bacteria↗
PubMed1998

Affinity purification of Helicobacter pylori urease. Relevance to gastric mucin adherence by urease protein.

A simple, reproducible and high yield method of Helicobacter pylori urease enzyme purification was developed using a heparinoid (Cellufine sulfate) affinity gel. The purification method involved two sequential steps using the same gel that takes advantage of the differential affinity of urease to the heparinoid at two levels of hydrogen ion concentration. SDS-polyacrylamide gel electrophoresis analysis of affinity-purified urease revealed two major protein bands with about 62- and 30-kDa molecular mass. When whole cell lysates of clinical and laboratory strains of H. pylori were probed by Western blot, anti-urease hyperimmune serum produced by affinity-purified urease in rabbit recognized only the two bands corresponding to the urease A and B subunits. To probe the molecular relevance of affinity gel adherence to mucin adherence, the purified urease was derivatized with N-hydroxysuccinimidobiotin and used in adherence assays. Competitive inhibition tests revealed commonality of urease receptors among gastric mucin, heparin, and heparinoid. Composite data on adherence kinetics modulated by pH, salt, incubation time, and concentration of urease or mucin were indicative of conformation-dependent ligand-receptor interaction.

Animals↗
PubMed1993

Cloning of Bordetella bronchiseptica urease genes and analysis of colonization by a urease-negative mutant strain in a guinea-pig model.

The genes encoding urease were cloned from Bordetella bronchiseptica and the 5.2 kb of DNA essential for expression analysed in a T7 RNA polymerase transcription-translation system. At least four polypeptides with predicted molecular weights of 69,000, 26,000, 12,200 and 11,000 were found. Partial DNA sequence of the gene encoding the 69,000 Da polypeptide revealed high amino acid identity to the alpha-subunit of Proteus mirabilis urease, UreC and jack bean urease. A stable, unmarked deletion was constructed in this gene to create a urease-negative mutant of B. bronchiseptica. To assess colonization in a guinea-pig model, the urease-negative strain was inoculated with the urease-positive parental strain in a mixed infection. The urease-negative strain out competed the urease-positive strain in the trachea, lungs and caecum. We demonstrate that urease is not essential for B. bronchiseptica colonization of the guinea-pig respiratory and digestive tracts.

Amino Acid Sequence↗
PubMed1993

Immunological specificity of Helicobacter pylori urease and identification by immunological detection of its specific urease.

Helicobacter pylori urease was recovered as a single peak by DEAE-Sepharose column chromatography and Sephacryl S-200 gel filtration. The purified urease was obtained by fast protein liquid chromatography using a Mono Q column. The purified urease preparation gave a single band in polyacrylamide gel disc electrophoresis. Latex particles were sensitized with anti-urease immunoglobulin. The sensitized latex particles were agglutinated with the purified urease and by cell sonicates obtained from 55 strains of H. pylori which were isolated from the gastric mucosa of patients with gastric and duodenal disorders, while they did not react with those obtained from related bacteria known to be urease producers, such as Helicobacter mustelae and urease- positive "Campylobacter lari variants", or by urease of some strains of Enterobacteriae. We have developed a specific and sensitive method for detecting the urease by using the reversed passive latex agglutination technique, in order to identify of the organism.

Antigens, Bacterial↗
PubMed1992

Kinetic properties of Helicobacter pylori urease compared with jack bean urease.

The urease proteins of the jack bean (Canavalia ensiformis) and Helicobacter pylori are similar in molecular mass when separated by non-denaturing gradient polyacrylamide gel electrophoresis, both having three main forms. The molecular mass of their major protein form is within the range 440-480 kDa with the other two lesser forms at 230-260 kDa and 660-740 kDa. These forms are all urease active; however, significant kinetic differences exist between the H. pylori and jack bean ureases. Jack bean urease has a single pH optimum at 7.4, whereas H. pylori urease has two pH optima of 4.6 and 8.2 in barbitone and phosphate buffers that were capable of spanning the pH range 3 to 10. The H. pylori Km was 0.6 mM at pH 4.6 and 1.0 mM at pH 8.2 in barbitone buffer, greater than 10.0 mM, and 1.1 mM respectively in phosphate buffer and also greater than 10.0 mM in Tris.HCl at pH 8.2. By comparison, the jack bean urease had a Km of 1.3 mM in Tris.HCl under our experimental conditions. The findings show that the urease activity of H. pylori was inhibited at the pH optimum of 4.6 in the phosphate buffer, but not in the barbitone buffer. This was shown to be due to competitive inhibition by the sodium and potassium ions in the phosphate buffer, not the phosphate ions as suggested earlier. Jack bean urease activity was similarly inhibited by phosphate buffer but again due to the effect of sodium and potassium ions.

Fabaceae↗
PubMed2002

Leaf urea metabolism in potato. Urease activity profile and patterns of recovery and distribution of (15)N after foliar urea application in wild-type and urease-antisense transgenics.

The influence of urease activity on N distribution and losses after foliar urea application was investigated using wild-type and transgenic potato (Solanum tuberosum cv Désirée) plants in which urease activity was down-regulated. A good correlation between urease activity and (15)N urea metabolism (NH(3) accumulation) was found. The general accumulation of ammonium in leaves treated with urea indicated that urease activity is not rate limiting, at least initially, for the assimilation of urea N by the plant. It is surprising that there was no effect of urease activity on either N losses or (15)N distribution in the plants after foliar urea application. Experiments with wild-type plants in the field using foliar-applied (15)N urea demonstrated an initial rapid export of N from urea-treated leaves to the tubers within 48 h, followed by a more gradual redistribution during the subsequent days. Only 10% to 18% of urea N applied was lost (presumably because of NH(3) volatilization) in contrast to far greater losses reported in several other studies. The pattern of urease activity in the canopy was investigated during plant development. The activity per unit protein increased up to 10-fold with leaf and plant age, suggesting a correlation with increased N recycling in senescing tissues. Whereas several reports have claimed that plant urease is inducible by urea, no evidence for urease induction could be found in potato.

Enzyme Induction↗