Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “UREASE”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 433 records · Page 24Linked to original sources
PubMed1996

Detection of Helicobacter pylori by rapid urease tests: is biopsy size a critical variable?

BACKGROUND: The variables responsible for false-positive and false-negative rapid urease tests are largely unexplored. OBJECTIVES: We compared the results of rapid urease testing with jumbo cup forceps (3.3 mm diameter) and tiny cup forceps (1.8 mm diameter) with two rapid urease tests. METHODS: Antral biopsies were obtained. The order of forceps and rapid urease tests was randomized. Biopsies were also taken for Genta staining. RESULTS: One hundred and two patients were studied; 59 had Helicobacter pylori infection. There were 22 false-negative tests (8 CLOtest, 16 hpfast) and 5 false-positive tests (3 CLOtest, 2 hpfast). All 5 false-positive tests were among those positive only in the second 12 hours. There was no difference in results with the jumbo and tiny cup forceps. Five percent to nine percent (average, 6.6%; 95% confidence interval, 4.4% to 9.6%) of tests with any of the combinations gave an erroneous categorization of H. pylori status. There were no clinical or statistical differences in H. pylori categorizations by CLOtest or hpfast. CONCLUSION: The diagnostic yield for detecting H. pylori infection by rapid urease tests is not adversely affected by small biopsy size, possibly because tiny biopsies obtain superficial tissue where H. pylori reside.

Biopsy↗
PubMed2003

Immobilization of urease onto chemically modified acrylonitrile copolymer membranes.

Poly (acrylonitrile-methylmethacrylate-sodium vinylsulfonate) membranes were subjected to seven different chemical modifications. The amounts of new groups incorporated in the membranes with the modifications were determined. Urease was covalently immobilized on the modified membranes. Both the amount of bound protein and relative activity of immobilized urease were measured. The highest activity was found for urease bound to membranes modified with hydroxylammonium sulfate (68%) and hydrazinium sulfate (67%). Optimum pH of free urease was determined to be 5.8. For positively charged membranes, pH optimum was shifted to higher values, while for negatively charged membranes-to lower pH. The charge of the matrix affected also the rate of the enzyme reaction. The highest rate was measured with urease immobilized on membranes modified with hydroxylammonium sulfate and hydrazinium sulfate. The major part of the immobilized enzyme on different modified membranes remained stable-only ca. 20% of enzyme activity was lost for 4 h at 70 degrees C while the free enzyme was totally inactivated.

Acrylonitrile↗
PubMed1999

Oral immunization with recombinant Helicobacter pylori urease confers long-lasting immunity against Helicobacter felis infection.

Recombinant Helicobacter pylori urease (rUre) has been shown to confer protection against challenge with Helicobacter felis in mice. The purpose of the present study was to examine duration of the immune response and long-term protective efficacy of immunization with rUre. Swiss Webster mice were orally immunized four times at weekly intervals with 100 microg rUre plus 5 microg heat-labile enterotoxin of Escherichia coli (LT) adjuvant, or with LT only. At 4, 10, 20 or 40 weeks post immunization, 25 rUre-immunized mice and control mice were challenged with H. felis and sacrificed at 2 or 10 weeks post-challenge. H. felis infection was assessed by gastric urease assay and by histology. Anti-H. pylori urease specific antibody levels were measured in serum and saliva both pre- and post-challenge. Over the 40 week time period, the infection rates in rUre-immunized mice were significantly lower than those in controls (p < 0.05) as assessed by gastric urease activity. Protection ranged from 79 100% at 2 weeks post-challenge and 63-78% at 10 weeks post-challenge. Gastric bacterial density in rUre-immunized mice was significantly lower than that of controls (p < 0.03) as determined by histologic assessment. Anti-urease antibody levels remained elevated in the serum and mucosal compartments at 39 weeks following immunization. This study shows that immunization with rUre plus LT results in long-lasting protective immunity against challenge with H. felis.

Adjuvants, Immunologic↗
PubMed1991

Urease production by Helicobacter (Campylobacter) pylori.

Urease activity of 50 Helicobacter pylori (H. pylori) strains was assessed employing a photometric assay. Urea hydrolysis reached a maximum in the late log-phase and during the plateau phase of bacterial growth. The reaction time of H. pylori urease was significantly shorter than that of other urease producing bacteria (P. mirabilis, P. vulgaris, K. pneumoniae, K. oxytoca). Increasing the reaction temperature hardly led to an acceleration of the quick urea hydrolysis of H. pylori, in contrast to the situation with P. mirabilis. Acetohydroxamic acid showed a dose-dependent non-competitive suppression of urease production, whereas 9 antibiotics in subinhibitory concentrations did not influence urease production of H. pylori.

Anti-Bacterial Agents↗
PubMed1993

Influence of omeprazole on urease activity of Helicobacter pylori in vitro.

The influence of omeprazole on urease activity of 13 Helicobacter pylori strains was assessed in vitro employing different inocula of the bacteria and various concentrations of omeprazole. Bacteria were grown in liquid culture supplemented with omeprazole for 48 h. Afterwards, bacterial numbers were assessed and urease activity was measured in a spectrophotometric assay. In 10 strains, omeprazole had no influence on urease activity at concentrations up to 8 mg/l; higher concentrations had a bacteriostatic effect. Three strains were more resistant to omeprazole: These showed a marked diminution of urease activity although bacterial numbers were only slightly reduced. Thus a possible inhibitory effect of omeprazole should be taken into account when urease of Helicobacter pylori is measured for diagnostic purposes.

Helicobacter pylori↗
PubMed2002

Optimization of urease immobilization onto non-porous HEMA incorporated poly(EGDMA) microbeads and estimation of kinetic parameters.

Jack bean urease (urea aminohydrolase, EC 3.5.1.5) was immobilized onto modified non-porous poly(ethylene glycol dimethacrylate/2-hydroxy ethylene methacrylate), (poly(EGDMA/HEMA)), microbeads prepared by suspension copolymerization for the potential use in hemoperfusion columns, not previously reported. The conditions of immobilization; enzyme concentration, medium pH, substrate and ethylene diamine tetra acetic acid (EDTA) presence in the immobilization medium in different concentrations, enzyme loading ratio, processing time and immobilization temperature were investigated for highest apparent activity. Immobilized enzyme retained 73% of its original activity for 75 days of repeated use with a deactivation constant kd = 3.72 x 10(-3) day(-1). A canned non-linear regression program was used to estimate the intrinsic kinetic parameters of immobilized enzyme with a low value of observable Thiele modulus (phi < 0.3) and these parameters were compared with those of free urease. The best-fit kinetic parameters of a Michaelis-Menten model were estimated as Vm = 3.318 x 10(-4) micromol/s mg bound enzyme protein, Km = 15.94 mM for immobilized, and Vm = 1.074 micromol NH3/s mg enzyme protein, Km = 14.49 mM for free urease. The drastic decrease in Vm value was attributed to steric effects, conformational changes in enzyme structure or denaturation of the enzyme during immobilization. Nevertheless, the change in Km value was insignificant for the unchanged affinity of the substrate with immobilization. For higher immobilized urease activity, smaller particle size and concentrated urease with higher specific activity could be used in the immobilization process.

Enzymes, Immobilized↗
PubMed2000

Kinetic and structural characterization of urease active site variants.

Klebsiella aerogenes urease uses a dinuclear nickel active site to catalyze urea hydrolysis at >10(14)-fold the spontaneous rate. To better define the enzyme mechanism, we examined the kinetics and structures for a suite of site-directed variants involving four residues at the active site: His320, His219, Asp221, and Arg336. Compared to wild-type urease, the H320A, H320N, and H320Q variants exhibit similar approximately 10(-)(5)-fold deficiencies in rates, modest K(m) changes, and disorders in the peptide flap covering their active sites. The pH profiles for these mutant enzymes are anomalous with optima near 6 and shoulders that extend to pH 9. H219A urease exhibits 10(3)-fold increased K(m) over that of native enzyme, whereas the increase is less marked ( approximately 10(2)-fold) in the H219N and H219Q variants that retain hydrogen bonding capability. Structures for these variants show clearly resolved active site water molecules covered by well-ordered peptide flaps. Whereas the D221N variant is only moderately affected compared to wild-type enzyme, D221A urease possesses low activity ( approximately 10(-)(3) that of native enzyme), a small increase in K(m), and a pH 5 optimum. The crystal structure for D221A urease is reminiscent of the His320 variants. The R336Q enzyme has a approximately 10(-)(4)-fold decreased catalytic rate with near-normal pH dependence and an unaffected K(m). Phenylglyoxal inactivates the R336Q variant at over half the rate observed for native enzyme, demonstrating that modification of non-active-site arginines can eliminate activity, perhaps by affecting the peptide flap. Our data favor a mechanism in which His219 helps to polarize the substrate carbonyl group, a metal-bound terminal hydroxide or bridging oxo-dianion attacks urea to form a tetrahedral intermediate, and protonation occurs via the general acid His320 with Asp221 and Arg336 orienting and influencing the acidity of this residue. Furthermore, we conclude that the simple bell-shaped pH dependence of k(cat) and k(cat)/K(m) for the native enzyme masks a more complex underlying pH dependence involving at least four pK(a)s.

Aspartic Acid↗
PubMed1996

Structures of the Klebsiella aerogenes urease apoenzyme and two active-site mutants.

Urease from Klebsiella aerogenes [Jabri et al. (1995) Science 268, 998-1004] is an (alpha beta gamma)3 trimer with each alpha-subunit having an (alpha beta)8-barrel domain containing a binickel active center. Here we examine structure-function relations for urease in more detail through structural analysis of the urease apoenzyme at 2.3 A resolution and mutants of two key catalytic residues (H219A and H320A) at 2.5 A resolution. With the exception of the active site, in which a water molecule takes the place of the missing carbamate and nickel atoms, the structure of the apoenzyme is nearly identical to that of the holoenzyme, suggesting a high degree of preorganization which helps explain the tight binding of nickel. In the structure of H219A, the major change involves a conformational shift and ordering of the active site flap, but a small shift in the side chain of Asp alpha 221 could contribute to the lower activity of H219A. In the H320A structure, the catalytic water, primarily a Ni-2 ligand in the holoenzyme, shifts into a bridging position. This shift shows that the nickel ligation is rather sensitive to the environment and the change in ligation may contribute to the 10(5)-fold lower activity of H320A. In addition, these results show that urease is resilient to the loss of nickel ions and mutations. Analysis of the urease tertiary/quaternary structure suggests that the stability of this enzyme may be largely due to its burial of an unusually large fraction of its residues: 50% in the gamma-subunit, 30% in the beta-subunit, and 60% in the alpha-subunit.

Amino Acid Sequence↗
PubMed2000

Fluoride inhibition of Klebsiella aerogenes urease: mechanistic implications of a pseudo-uncompetitive, slow-binding inhibitor.

Klebsiella aerogenes urease uses a dinuclear nickel active site to catalyze the hydrolysis of urea. Here, we describe the steady-state and pre-steady-state kinetics of urease inhibition by fluoride. Urease is slowly inhibited by fluoride in both the presence and absence of substrate. Steady-state rate studies yield parallel double-reciprocal plots; however, we show that fluoride interaction with urease is not compatible with classical uncompetitive inhibition. Rather, we propose that fluoride binds to an enzyme state (E) that is in equilibrium with resting enzyme (E) and produced during catalysis. Fluoride binding rates are directly proportional to inhibitor concentration. Substrate reduces both the rate of fluoride binding to urease and the rate of fluoride dissociation from the complex, consistent with urea binding to E and E.F in addition to E. Fluoride inhibition is pH-dependent due to a protonation event linked to fluoride dissociation. Fluoride binding is pH-independent, suggesting that fluoride anion, not HF, is the actual inhibitor. We assess the kinetic results in terms of the known protein crystal structure and evaluate possible molecular interpretations for the structure of the E state, the site of fluoride binding, and the factors associated with fluoride release. Finally, we note that the apparent uncompetitive inhibition by fluoride as reported for several other metalloenzymes may need to be reinterpreted in terms of fluoride interaction with the corresponding E states.

Bacterial Proteins↗
PubMed2002

Determination of urease activity in soils by carbon dioxide release for ecotoxicological evaluation of contaminated soils.

A method for the quantification of urease enzyme activity has been set up, which is based on the quantification of carbon dioxide set free into the head space of gastight vessels. The method can be applied for ecotoxicological characterisation of contaminated soil samples besides other methods like soil respiration measurements or nitrification inhibition tests. The sieved soil sample can be incubated under nearly natural conditions with an adjusted water content of about 50% of the water holding capacity. Ammonia or urea do not need to be extracted, since carbon dioxide release is correlated to urease activity. Thus carbon dioxide release is a direct result of urease activity which can be measured in the head space using gastight syringes and gaschromatographic equipment. The urease activity is determined by comparing the carbon dioxide release of incubation vessels with and without urea supply. The applicability of this method has been demonstrated by experiments with N-(n-butyl)phosphoric triamide (NBPT), copper ions and zinc ions as known inhibitors of urease activity.

Carbon Dioxide↗
PubMed2000

Suppression of urease levels in Streptococcus salivarius by cysteine, related compounds and by sulfide.

Urease synthesis in Streptococcus salivarius is induced by an acid environment, carbohydrate and a high growth rate. We now report that both cysteine and sulfide above 1 mM strongly suppress S. salivarius urease levels. Close structural relatives of cysteine (cysteamine, ethanedithiol and penicillamine) at 5 mM buffered to pH 7.0 also caused urease suppression, but thiols in general (2-mercaptoethanol, dithiothreitol and glutathione) did not. In cultures buffered below pH 5.9, the cysteine-induced urease suppression was lifted substantially, but the sulfide suppression increased, suggesting involvement of different processes. Urease activity was inhibited 50% by 5 mM mercaptoethanol but unaffected by 5 mM cysteine or sulfide, hence modification of enzyme activity by thiols is not directly related to suppression of their levels in culture. Cysteine, arising primarily through protein hydrolysis which also raises the pH, could be a surrogate pH feedback signal for nearby alkaline conditions, and sulfide may reflect activity of periodontopathic plaques.

Bacterial Proteins↗
PubMed2001

Characterization of gelatin-immobilized pigeonpea urease and preparation of a new urea biosensor.

Urease purified from pigeonpea seeds was immobilized on gelatin beads via cross-linking with glutaraldehyde. The maximum immobilization (75%) was observed at 30 mg/ml gelatin, 0.414 mg of enzyme/bead, 1% (v/v) glutaraldehyde and 4 degrees C. Beads stored in 50 mM Tris/acetate buffer (pH 7.3) at 4 degrees C showed a half-life of 240 days and there was practically no leaching of enzyme (less than 2%) over a period of 30 days. These beads can be reused more than 30 times (with 24 h intervals) without much loss of enzyme activity (i.e. less than 11%). The immobilized urease showed a shift in its optimum pH from 7.3 to 6.5 in Tris/acetate buffer. Optimum temperature also shifted from 47 to 65 degrees C compared with the soluble enzyme. Gelatin-immobilized pigeonpea urease had a higher K(m) (8.3 mM) than that of the soluble enzyme (3.0 mM). The time-dependent temperature inactivation pattern was also found to change from biphasic to monophasic kinetics. The immobilized beads were used for the preparation of a new urea biosensor with a response time of less than 2 min. At least 14 samples of urea can be measured with this biosensor within an hour. The beads, as well as the biosensor, were used to analyse the urea content in clinical samples from the local clinical pathology laboratories. The results obtained with the biosensor were strikingly similar to those obtained with the various commonly employed biochemical/autoanalyzer(R) methods used. These immobilization studies also have a potential role in haemodialysis machines that maintain the urea level in kidney patients and in the construction of a portable/wearable kidney. The easy availability of the pigeonpea urease, the ease of its immobilization on gelatin and a significantly lower cost of the urease described in the present study makes it a suitable product for future applications in therapeutics and diagnostics.

Biosensing Techniques↗
PubMed1968

Physical and chemical studies of a low-molecular-weight form of urease.

1. A new form of enzymically active jack-bean [Canavalia ensiformis (L.) DC] urease corresponding to an S(20,w) value of 11.8s and a molecular weight of 260000 was investigated. 2. Conversion of 18s urease (EC 3.5.1.5) into the 12s form depends on both low protein concentration and pH. Above pH5.3 urease exists in the 18s form and below pH4.8 in the 12s form; between these two pH values a 12s-18s rapid-equilibrium process is observed. 3. Comparison of the properties of 18s and 12s urease indicated no major differences. 4. A survey of other good urease sources revealed that the 12s form can also be obtained from soya bean (Glycine soja Sieb. and Zucc. cultivar Biloxi) and the bacterium Bacillus pasteurii (Miguel) Migula, but not from watermelon (Citrullus vulgaris Schrad. cultivar Congo). 5. The 12s forms from jack bean and Bacillus pasteurii did not hybridize.

Bacillus↗
PubMed1999

Helicobacter pylori cadA encodes an essential Cd(II)-Zn(II)-Co(II) resistance factor influencing urease activity.

Inactivation of Helicobacter pylori cadA, encoding a putative transition metal ATPase, was only possible in one of four natural competent H. pylori strains, designated 69A. All tested cadA mutants showed increased growth sensitivity to Cd(II) and Zn(II). In addition, some of them showed both reduced 63Ni accumulation during growth and no or impaired urease activity, which was not due to lack of urease enzyme subunits. Gene complementation experiments with plasmid (pY178)-derived H. pylori cadA failed to correct the deficiencies, whereas resistance to Cd(II) and Zn(II) was restored. Moreover, pY178 conferred increased Co(II) resistance to both the cadA mutants and the wild-type strain 69A. Heterologous expression of H. pylori cadA in an Escherichia coli zntA mutant resulted in an elevated resistance to Cd(II) and Zn(II). Expression of cadA in E. coli SE5000 harbouring H. pylori nixA, which encodes a divalent cation importer along with the H. pylori urease gene cluster, led to about a threefold increase in urease activity compared with E. coli control cells lacking the H. pylori cadA gene. These results suggest that H. pylori CadA is an essential resistance pump with ion specificity towards Cd(II), Zn(II) and Co(II). They also point to a possible role of H. pylori CadA in high-level activity of H. pylori urease, an enzyme sensitive to a variety of metal ions.

Adenosine Triphosphatases↗
PubMed1996

Concrement formation and urease-induced crystallization in urine from patients with continent ileal reservoirs.

OBJECTIVES: To study the relationship between urinary tract infection, urine composition and concrement formation in patients with continent ileal reservoirs for urinary diversion. PATIENTS AND METHODS: The study comprised 27 patients (seven men and 20 women, mean age 47 years, range 23-76) with continent ileal reservoirs who were followed for a mean of 67 months (range 13-146) by annual reservoiroscopy, intravenous urography and urine culture; at the final follow-up, a sample of their morning urine was analysed for a range of compounds and the number and size of any particles present or produced in response to incubation with urease. RESULTS: The presence of urease-producing bacteria was associated with the formation of concrement. However, a few patients in whom an infection with urease-producing organisms was not detected also formed concrement. Urine from those patients forming stones tended to have a high calcium and a low citrate concentration. After incubation with urease, significantly more and larger particles were formed in the urine from stone formers. There was a strong correlation (r = 0.8) between urinary calcium content and urinary pH when the urease-induced precipitation commenced, and between urinary calcium and the size and volume of the crystals developed (r = 0.9) after 4 h of incubation. CONCLUSIONS: There are many factors which might influence the formation of concrement, e.g. outflow conditions, the presence of staples or infection in the reservoir, and the composition of the urine is also important. It thus appears appropriate to determine if measures to reduce urinary calcium and increase urinary citrate can decrease the episodes of stone formation in those patients with continent ileal reservoirs for urinary diversion who frequently form stones.

Adult↗
PubMed2001

Cell lysis is responsible for the appearance of extracellular urease in Helicobacter pylori.

BACKGROUND: Helicobacter pylori is a neutralophilic bacterium that colonizes the acidic human gastric surface using the neutralizing capacity of a constitutively produced urease. Urease is present both in the cytoplasm and bound to the outside surface of the bacteria. The origin of the surface urease continues to be controversial. This study provides additional evidence that the origin of surface urease is cell lysis, not secretion. METHODS: H. Pylori was transformed with a plasmid encoding green fluorescent protein (GFP), a non-native cytoplasmic protein. Cultures supplemented with beta-cyclodextrin or horse serum were collected over various time periods and spun through a ficoll cushion to gently separate whole bacteria from released protein. The pellet and supernatant fractions were analyzed by fluorimetry, SDS-PAGE and Coomassie blue or Western analysis. RESULTS: GFP fluorescence and antigenic reactivity in the supernatant increased at each time point. GFP, the non-native cytoplasmic protein, and UreB, a native cytoplasmic protein, increased over time in the supernatant and both proteins were always present in the pellet fraction. UreI, an inner membrane protein, was only present in the pellet fraction. beta-galactosidase, a protein not found in H. pylori, was used as a negative control. CONCLUSIONS: Since it is unlikely that there is an intrinsic secretion system for GFP, a non-native protein, its increasing presence over time in the supernate fraction along with UreB, and retention of UreI in the pellet fraction implies that cell lysis accounts for the presence of urease on the surface of H. pylori.

Bacteriolysis↗
PubMed1999

GTP-dependent activation of urease apoprotein in complex with the UreD, UreF, and UreG accessory proteins.

Syntheses of metal-containing enzymes often require the participation of accessory proteins. The roles played by many of these accessory proteins are poorly characterized. Klebsiella aerogenes urease, a nickel-containing enzyme, provides an ideal system to study metallocenter assembly. Here, we describe a method for isolating a complex containing urease apoprotein and the UreD, UreF, and UreG accessory proteins. We demonstrate that urease apoprotein in this complex is activated to near wild-type enzyme levels when incubated with nickel ions and high (approximately 100 mM) concentrations of bicarbonate. Significantly, we also observed nickel-dependent activation at physiologically relevant (approximately 100 microM) bicarbonate levels, but only in the presence of GTP. Based on studies involving a nonhydrolyzable analog of GTP, we conclude that nucleotide hydrolysis, not just binding, is required for this process. The critical nucleotide-binding site was localized to UreG on the basis of experiments using a variant complex. These studies highlight the relevance of the UreD-UreF-UreG-urease apoprotein complex to nickel metallocenter assembly and explain the previously identified in vivo energy requirement for urease activation.

Apoenzymes↗
PubMed1999

A molecular mechanical analysis of the active site of urease with a special emphasis on determining the binding conformations available to oxygen-bound urea.

In order to model the active site of urease which contains two nickel ions with differing coordination geometries new parameters were derived for the AMBER* force field. These parameters were obtained by structure based optimization and use a single set of parameters with points on a sphere approach to model nickel(II) high-spin in all its coordination geometries. The force field was successfully used to model the active site of urease and to predict that a bridging water between the two nickel ions in urease was missing from the solid state structure of urease. A thorough conformational search was undertaken to find the conformations available to urea within urease. All the low energy conformations found were used to determine a consensus urea binding model.

Binding Sites↗