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PubMed1995

Purification and properties of urease from Sporobolomyces roseus.

Urease (EC 3.5.1.5) catalyses the hydrolysis of urea to ammonia and carbon dioxide. The enzyme from Sporobolomyces roseus was enriched 780-fold and purified to apparent homogeneity using heat treatment, ion exchange chromatography on Q-Sepharose fast flow, hydrophobic interaction chromatography on Phenyl-Sepharose, size exclusion chromatography on Sephacryl S 300 HR, and ion exchange chromatography on MonoQ. Analysis of the purified enzyme by SDS-PAGE demonstrated the presence of subunits with a molecular weight of 90 (+/- 4) kDa. The M(r) of the native enzyme was estimated by size exclusion chromatography to be 340 (+/- 30) kDa, suggesting a tetrameric structure different from other ureases isolated so far from both prokaryotes and eukaryotes. The enzyme was heat-stable, showing no loss of activity after incubation at 70 degrees C for 15 min. The highest urease activities were observed after growth on media containing urea as the sole source of nitrogen.

Chelating Agents↗
PubMed1993

Diethylpyrocarbonate reactivity of Klebsiella aerogenes urease: effect of pH and active site ligands on the rate of inactivation.

Reaction of Klebsiella aerogenes urease with diethylpyrocarbonate (DEP) led to a pseudo-first-order loss of enzyme activity by a reaction that exhibited saturation kinetics. The rate of urease inactivation by DEP decreased in the presence of active site ligands (urea, phosphate, and boric acid), consistent with the essential reactive residue being located proximal to the catalytic center. The pH dependence for the rate of inactivation indicated that the reactive residue possessed a pKa of 6.5, identical to that of a group that must be deprotonated for catalysis. Full activity was restored when the inactivated enzyme was treated with hydroxylamine, compatible with histidinyl or tyrosinyl reactivity. Spectrophotometric studies were consistent with DEP derivatization of 12 mol of histidine/mol of native enzyme. In the presence of active site ligands, however, approximately 4 mol of histidine/mol of protein were protected from reaction. Each protein molecule is known to possess two catalytic units; hence, we propose that urease possesses at least one essential histidine per catalytic unit.

Binding Sites↗
PubMed1990

Comparison of four stains and a urease test for rapid detection of Helicobacter pylori in gastric biopsies.

Gastric biopsies were obtained from 125 subjects to compare detection of Helicobacter pylori by culture, a rapid urease test and histopathologic examination using haematoxylin-eosin, Gram, Giemsa, Warthin-Starry silver and acridine orange stains. Helicobacter pylori was isolated from 39 specimens. Acridine orange and Giemsa were the most sensitive stains, detecting 85% and 79% of positive specimens respectively. All stains showed high specificity (97-100%). The sensitivity and specificity of the rapid urease test was 62% and 100% respectively. These stains or the rapid urease test may be useful for rapid detection of Helicobacter pylori in gastric biopsies.

Biopsy↗
PubMed1990

Antigenicity of fractions of Helicobacter pylori prepared by fast protein liquid chromatography and urease captured by monoclonal antibodies.

The antigenicity of Helicobacter pylori protein fractions separated by fast protein liquid chromatography size exclusion was investigated by EIA with sera from patients of well defined Helicobacter pylori status. The antigenic material of Helicobacter pylori was confined to fractions 8 and 14 to 21. Urease containing fractions (14/15) and flagella containing fractions (17/18) were identified. Fraction 8 non-specifically bound human immunoglobulin as demonstrated by the binding of Helicobacter pylori negative sera. The remaining fractions 14 to 21 when used individually as EIA antigens were 91-100% specific, however fractions 16 to 19 showed a reduced sensitivity (78%) compared with the acid extract (95%). The urease fractions were 91% sensitive. Purified urease antigen captured by antiurease monoclonal antibodies was 83% sensitive and 93.3% specific.

Adult↗
PubMed1994

A clinico-epidemiological analysis of Helicobacter pylori (H. pylori) by Southern blotting with A urease gene probe.

Helicobacter pylori (H. pylori) is a gram-negative bacillus thought to be involved in such diseases of the upper gastrointestinal tract as gastritis, peptic ulcers, and gastric cancer. Urease is regarded as the factor responsible for the pathogenic nature of this bacterium. Therefore, in our examination of the genetic polymorphism of H. pylori, by means of Southern blotting, we used the urease gene as a probe. The Southern blot patterns of H. pylori isolated from different patients differed greatly, the inter-individual variation being so marked that it allowed approximate distinction between individual patients. The Southern blot patterns of individual strains of H. pylori did not change, even when they were stored and passed from generation to generation in our laboratory. These results suggest that DNA fingerprints with a urease gene probe will be useful in epidemiologically tracing H. pylori infection. Almost all strains of H. pylori isolated from different sites in the stomach of a patient on different occasions showed the same pattern, allowing us to confirm that only one strain of H. pylori was responsible for H. pylori infection in individual patients.

Blotting, Southern↗
PubMed1994

Kinetic studies of the urease-catalyzed hydrolysis of urea in a buffer-free system.

The kinetics of urea hydrolysis catalyzed by urease, mainly in the absence of buffers by use of the self-buffer effect of the products, was investigated. The effect of pH, temperature, and concentration of enzyme, substrate, product, salt ions, and buffers on the kinetic behavior of urease was examined. A kinetic model of a modified Michaelis-Menten form, incorporating substrate and product inhibition, pH dependence, and temperature effect, was developed to describe the reaction rate. Experimental data indicated that urease in a buffer-free solution was less susceptible to the inhibition of substrate product. The Michaelis constant keeps almost constant with the variation of pH and temperature, and increases with the addition of buffers and salts. The data also suggested that the noncompetitive pattern of the product inhibition, which is not significantly affected by temperature, increases gently with increasing pH. A Monod form rate expression was proposed to analyze the pH effect on the maximum rate. The proposed kinetic model was also examined by the long-time experiments in which pH, substrate, and product concentration varied obviously during the reaction course.

Ammonia↗
PubMed1984

Effects of urease-induced hyperammonemia in mouse liver. Ultrastructural, stereologic and biochemical study.

Intraperitoneal injections of urease induced a marked and sustained hyperammonemia in mice. Ultrastructural and stereologic analysis of hepatocytes from urease-treated mice showed striking changes in the mitochondria, rough and smooth endoplasmic reticulum and lysosomes. Thus, mitochondria became larger and rounder, and contained a less electron-dense matrix although their volume density remained similar to that of control cells. In addition, increases in the smooth and rough reticulum and the lysosomal compartment, were observed. Biochemical analysis of the livers from urease-treated mice revealed a significant increase in the intracellular content of water and lipids. Although the mechanism by which ammonia induces these changes remains unclear, the possible relationship between these findings and those described in the liver of humans and experimental animals in conditions of sustained hyperammonemia is discussed.

Ammonia↗
PubMedDate not supplied

Kinetics and activity distribution of urease coencapsulated with hemoglobin within polyamide membranes.

A 91.5% mass yield of urease and hemoglobin (Hb), co-encapsulated within polyamide membranes, was determined spectrophotometrically. The specific activity yield of microencapsulation was 84%, twofold higher than values previously reported, as a result of optimization of encapsulation conditions. The kinetic parameters and pH activity profiles of intracapsular urease were determined to be similar to those corresponding to the free enzyme. Similar activities were also observed for intact and microcapsule homogenate, indicating minimal mass transfer and diffusional limitation. The active configuration of the enzyme appears to remain intact upon microencapsulation. The application of a kinetic model for encapsulated urease further indicated that the kinetics were reaction-controlled with minimal mass transfer restrictions.

Capsules↗
PubMed1997

Identification of the ure1+ gene encoding urease in fission yeast.

Cloning and sequencing of the ure1+ gene of Schizosaccharomyces pombe indicated that it encodes the urease which had been biochemically identified. The fission yeast urease has a one-subunit structure like those from plants but different from bacterial ureases which are composed of two or three distinct subunits. Genetic analyses showed that the ure1+ gene product is actually involved in urea metabolism.

Amino Acid Sequence↗
PubMed2002

Molecular characterization of Bacillus pasteurii UreE, a metal-binding chaperone for the assembly of the urease active site.

The present study describes the cloning, isolation, and thorough biochemical characterization of UreE from Bacillus pasteurii, a novel protein putatively involved in the transport of Ni in the urease assembly process. A DNA fragment of the B. pasteurii urease operon, containing all four accessory genes (ureE, ureF, ureG, and ureD) required for the incorporation of Ni ions into the active site of urease, was cloned, sequenced, and analyzed. B. pasteurii ureE was cloned, and the UreE protein (BpUreE) was over-expressed and purified to homogeneity. The identity of the recombinant protein was determined by N- and C-terminal sequencing and by mass spectrometry. BpUreE has a chain length of 147 amino acids, and features a p I value of 4.7. As isolated, BpUreE contains one Zn(II) ion per dimer, while no Ni(II) is present, as shown by mass spectrometry and atomic absorption spectroscopy. BpUreE behaves as a dimer independently of the presence of Zn(II), as shown by gel filtration and mass spectrometry. Paramagnetic NMR spectroscopy on concentrated (2 mM) UreE solutions reveals a one Ni atom per tetramer stoichiometry, with the Ni(II) ion bound to histidines in an octahedral coordination environment. BpUreE has a high sequence similarity with UreE proteins isolated from different biological sources, while no sequence homology is observed with proteins belonging to different classes. In particular, BpUreE is most similar to UreE from Bacillus halodurans (55% identity). A multiple sequence alignment reveals the presence of four strictly conserved residues (Leu55, Gly97, Asn98, His100; BpUreE numbering), in addition to position 115, conservatively occupied by an Asp or a Glu residue. Several secondary structure elements, including a betaalphabetabetaalphabeta "ferredoxin-like" motif, are highly conserved throughout the UreE sequences.

Amino Acid Sequence↗
PubMed1990

Flow injection analysis of serum urea using urease covalently immobilized on 2-fluoro-1-methylpyridinium salt-activated fractogel and fluorescence detection.

Serum samples were analyzed for their urea content using fluorescence flow injection analysis incorporating an immobilized urease bioreactor and a gas permeable separator. The urease was immobilized under mild and facile conditions to a hydrophilic 2-fluoro-1-methylpyridinium-activated support. The ammonia released as a result of urease-catalyzed urea hydrolysis diffused through a gas permeable membrane into a constant stream of o-phthaldehyde solution to form a highly fluorescent product with lambda ex at 340 nm and lambda em at 455 nm. Up to 25 serum samples can be analyzed per hour. The within-day coefficient of variation (CV) was 1.12% and the day-to-day CV was 1.25% for serum containing 10.50 mg urea nitrogen dl-1. The bioreactor shows excellent storage (at 4 degrees C) and operational stabilities (at 37 degrees C).

Ammonia↗
PubMed1982

An investigation of the use of urease-antibody conjugates in enzyme immunoassays.

The development of urease (E.C.3.5.1.5) as a label for enzyme immunoassay (EIA) procedures is described and the use of such conjugates illustrated with examples. Urease catalyzes the hydrolysis of urea to carbon dioxide and ammonia. The production of ammonia may be detected readily by a pH shift which we have found best indicated by the vivid colour change (yellow to purple) of bromocresol purple incorporated in the substrate solution. This enzyme-substrate system offers a number of important advantages. The substrate in aqueous solution is stable, titration end points are sharp and readily visible and the enzyme is not inhibited by sodium azide. Thus, test reagents may be prepared with this preservative and stored ready to use. Urease of high specific activity is commercially available and because it does not occur in mammalian tissues, it is suitable for use in EIA tests to detect cell-associated antigens and their antibodies. Finally, the enzyme reaction may be stopped by the addition of organomercurial preservatives, thus allowing storage of developed tests for later examination.

Alkaline Phosphatase↗
PubMed1991

Description of a urease-based microELISA for the analysis of limiting dilution microcultures.

Limiting dilution analysis has been a valuable approach for both determining the frequency of cell subpopulations elicited during immune responses, as well as for the analysis of immunoregulatory circuits. We describe a simple, visually scored spot test for evaluating the response of Mishell-Dutton microcultures used in limiting dilution analysis. This spot test is based on a microELISA using immunoreagents conjugated to the enzyme, urease, as an alternative to the hemolytic spot test. The assay as performed in Terasaki trays requires minute quantities (less than 10 microliters) of culture supernatant, yet the ELISA yields a distinct color difference between tray wells containing culture supernatants derived from responding (purple) and nonresponding (yellow) microcultures. Although designed to be scored rapidly by visual inspection, the assay can be quantified by manual alignment of the Terasaki tray wells on commercially available ELISA plate readers with an accuracy and reproducibility comparable to assays performed in 96-well ELISA plates. Determination of anti-sheep RBC responses in limiting dilution Mishell-Dutton microcultures with both the hemolytic spot test and the urease-microELISA spot test showed a very close correlation between the results of the two assays. However, the urease-microELISA should be amenable for use with antigens not readily conjugated to an indicator RBC, and should be useful in those situations where determination of the antibody subclass(es) produced by responding microcultures is desired.

Animals↗
PubMed1992

Preliminary crystallographic studies of urease from jack bean and from Klebsiella aerogenes.

Ureases from both jack bean (Canavalia ensiformis) seeds and Klebsiella aerogenes have been crystallized by the hanging drop method. The plant-derived urease crystals are regular octahedra analogous to those obtained by Sumner. Preliminary X-ray diffraction studies show that the crystals belong to the cubic space group F4(1)32, with a = 364 A, and appear to contain one or two subunits in the asymmetric unit. Using a synchrotron source, the crystals diffract to near 3.5 A resolution. Crystals of urease from K. aerogenes belong to the cubic space group I23 or I2(1)3, with a = 170.8 A and appear to contain a single catalytic unit per asymmetric unit. The crystals diffract to better than 2.0 A resolution and are well suited for structural analysis.

Bacterial Proteins↗
PubMed1996

Optimization studies on the features of an activated charcoal-supported urease system.

The adsorption of urease onto a well-defined solid support, petroleum-based activated charcoal, has been achieved to provide the enzymatic hydrolysis of urea. In order to produce a biocompatible surface, the enzyme support system has been coated with hexamethyldisiloxane through plasma polymerization. The quality of the resulting coat was tested by electronic spectroscopy for chemical analysis and scanning electron microscopy techniques. Studies on the adsorption of urease, and activity and stability of the enzyme on the support have been in the direction to optimize the features of the charcoal-supported urease and improve its availability for further use in clinical applications.

Adsorption↗
PubMed1992

Measurement of pH to quantify urease activity.

The rate of change of pH caused by the hydrolysis of urea was measured for urease solutions of 18 different concentrations. Concentrations were converted into an activity, A (measured in IU/cm3), by using a titrimetric method to assay the urease sample. For activities in the range 0.6-38 IU/cm3, A was related to the initial rate of change of pH, (dpH/dt)0, (measured in s-1) by the empirical relationship: A = 549(dpH/dt)0-1423(dpH/dt)2(0). Values of (dpH/dt)0 were sensitive to changes in the urease activity of about 0.6 IU/cm3.

Hydrogen-Ion Concentration↗
PubMed1988

One-step affinity purification of urease from jack beans.

Jack bean (Canivalia ensiformis) urease (EC3.5.1.5) was purified in one-step by ligand affinity chromatography using epoxy-activated Sepharose 6B-urea. The yield of the purified enzyme was about 80% with a specific activity of about 500 U/mg of protein. The enzyme was apparently homogeneous when analyzed by SDS-PAGE and native PAGE. The protein band on native PAGE coincided with the stained band of urease activity. The affinity column could be regenerated and reused several times without any loss of binding capacity and resolution. Affinity gels containing either acetamide or semicarbazide as affinity ligands were also found to be useful for the isolation of urease.

Chromatography, Affinity↗
PubMed1990

Cloning of the genes encoding urease from Proteus vulgaris and sequencing of the structural genes.

A fragment of chromosomal DNA from proteus vulgaris encoding urease was cloned and expressed in Escherichia coli. A 3 kbp region was sequenced and revealed three open reading frames with homology to jack bean (Canavalia ensiformis) urease. The smallest protein (11 kDa) was homologous to the N-terminus of the plant enzyme and the largest polypeptide (61 kDa) corresponded to the C-terminus. The large protein contained conserved regions and a cysteine residue which is known to be catalytically important in the plant enzyme. A protein of 12 kDa showed homology to residues 132 to 237 of jack bean urease.

Amino Acid Sequence↗