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PubMed2002

Detection of Helicobacter pylori using nested polymerase chain reaction and rapid urease test in gastric biopsy samples.

BACKGROUND/AIMS: The purpose of this study was to compare nested polymerase chain reaction and rapid urease test findings in the diagnosis of Helicobacter pylori infection. METHODS: Two gastric biopsy specimens were obtained from each of 64 patients and polymerase chain reaction and rapid urease test were performed. DNA extraction was followed by amplification with two primer pairs from the urease A gene of Helicobacter pylori genome. RESULTS: Fourty two patients (65.6%) had a positive rapid urease test result while 22 (34.4%) had a negative result for Helicobacter pylori. Sixty of 64 patients (93.8 %) had a positive result with the nested polymerase chain reaction method. Four patients with negative nested polymerase chain reaction results also had negative rapid urease test results. All of the eighteen patients with rapid urease test-negative results were positive with nested polymerase chain reaction. The number of nested polymerase chain reaction-positive patients (93.8%) was significantly higher than rapid urease test-positive (65.6%) and first round polymerase chain reaction-positive (53.1%) patients (P<0.001). CONCLUSIONS: Our results indicate that the nested polymerase chain reaction is more specific and sensitive than the rapid urease test for detecting Helicobacter pylori in gastric biopsy samples.

Journal Article↗
PubMed2004

Recombinant antigen from Helicobacter pylori urease as vaccine against H. pylori-associated disease.

It is well documented that the enzymatic active site of Helicobacter pylori urease is present in the beta-subunit. An important sequence of 135 amino acids of the beta-subunit was determined from the structure of H. pylori urease and by a homology-based study of the urease of other bacteria and plants. The sequence (UreB) was expressed in Escherichia coli as a recombinant fusion protein with glutathione-S-transferase (GST). Seventeen monoclonal antibodies, UA-1-17, were produced using the UreB-GST as the immunogen. The obtained monoclonal antibodies showed a high specificity to UreB, and some of the MAbs cross-reacted with Jack bean urease. About 70% of the established MAbs displayed an inhibitory effect on the enzymatic activity of the urease. Among them, UA-15 MAb could reduce the activity by 53% and it immunologically binds to the bacterium infecting the human stomach mucosa. The antiserum induced by immunization with a recombinant UreB-GST into rabbits displayed a specific binding to mucosal surfaces of the human stomach infected with the pathogen H. pylori. Moreover, the antiserum suppressed the enzymatic activity of H. pylori urease, while the purified H. pylori urease could not induce such an antiserum.

Amino Acid Sequence↗
PubMed1992

A survey of urease-positive Vibrio parahaemolyticus strains isolated from traveller's diarrhea, sea water and imported frozen sea foods.

The frequency of urease-positive Vibrio parahaemolyticus among isolates from patients, imported frozen sea foods and the environment (sea water) was studied. The highest isolation frequency of urease-positive V. parahaemolyticus was found in clinical isolates (11.2% out of 204 strains examined). Urease-positive V. parahaemolyticus was found in 5.7% of 88 frozen sea food-isolates examined, but no strains isolated from sea water were urease-positive. The isolates were further examined for the production of thermostable direct hemolysin (Vp-TDH) and its related hemolysin (Vp-TRH). Both are possible pathogenic toxins produced by mostly clinical isolates of V. parahaemolyticus. Urease-positive strains have a tendency to associate with clinical isolates producing both or neither Vp-TDH and Vp-TRH. Rabbit ligated ileal loops test was performed with several strains of urease-positive and -negative clinical isolates, and we found that some strains producing urease, even those which do not produce Vp-TDH or Vp-TRH, caused intestinal fluid accumulation.

Diarrhea↗
PubMed1995

Irritant and protective action of urea-urease ammonia in rat gastric mucosa. Different effects of ammonia and ammonium ion.

The effects of urea-urease-ammonia on the rat gastric mucosa were examined and compared with those of NH4OH and NH4Cl. The mucosal application of urea with urease produced a reduction in potential difference (PD) in a dose-related manner for urea, and a significant drop was observed by > 0.1% urea in the presence of 100 units urease. Such PD reduction was also observed when the mucosa was exposed to either NH4OH (> 0.03%) or NH4Cl (> 1%); delta PD (20 mV) caused by 0.3% NH4OH and 3% NH4Cl was equivalent to that induced by 0.5% urea+urease (100 units). The combined oral administration of urea (approximately 6%) and urease (100 units) did not induce any macroscopic damage in the gastric mucosa. NH4Cl given orally had no or little effect on the mucosa at any dose levels even at 10%, while NH4OH given orally caused hemorrhagic lesions in the mucosa at the dose of > 0.3%. In contrast, both urea+urease and NH4Cl given prior to HCl/ethanol protected the gastric mucosa against damage in a dose-related manner, and a significant effect was obtained by urea at > 0.5% and by NH4Cl at > 1%. NH4OH was also effective in reducing the severity of HCl/ethanol-induced gastric lesions at lower dose (0.3%). The protective effect of urea+urease was attenuated significantly by prior administration of indomethacin or coadministration of hydroxyurea, while that of NH4Cl or NH4OH was mitigated by indomethacin.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonia↗
PubMed2002

Characterization of the urease gene cluster from Rhizobium leguminosarum bv. viciae.

Moderate levels of urease activity (ca. 300 mU mg(-1)) were detected in Rhizobium leguminosarum bv. viciae UPM791 vegetative cells. This activity did not require urea for induction and was partially repressed by the addition of ammonium into the medium. Lower levels of urease activity (ca. 100 mU mg(-1)) were detected also in pea bacteroids. A DNA region of ca. 9 kb containing the urease structural genes ( ureA, ureB and ureC), accessory genes ( ureD, ureE, ureF, and ureG), and five additional ORFs ( orf83, orf135, orf207, orf223, and orf287) encoding proteins of unknown function was sequenced. Three of these ORFs ( orf83, orf135 and orf207) have a homologous counterpart in a gene cluster from Sinorhizobium meliloti, reported to be involved in urease and hydrogenase activities. R. leguminosarum mutant strains carrying Tn 5 insertions within this region exhibited a urease-negative phenotype, but induced wild-type levels of hydrogenase and nitrogenase activities in bacteroids. orf287 encodes a potential transmembrane protein with a C-terminal GGDEF domain. A mutant affected in orf287 exhibited normal levels of urease activity in culture cells. Experiments aimed at cross-complementing Ni-binding proteins required for urease and hydrogenase synthesis (UreE and HypB, respectively) indicated that these two proteins are not functionally interchangeable in R. leguminosarum.

Bacterial Proteins↗
PubMedDate not supplied

Comparison of immunohistochemistry and silver stain for the diagnosis of pediatric Helicobacter pylori infection in urease-negative gastric biopsies.

We compared immunohistochemical and silver stains of pediatric gastric biopsy sections for the identification of Helicobacter pylori infection with chronic inflammation and a negative urease screening test. Thirty-seven patients (age range 10 months to 21 years) whose gastric antral biopsies were negative for the rapid urease test (CLO(R)) but positive for lymphocytic infiltration were selected for a retrospective study. Specimens had been subjected to a rapid urease test (CLO(R)) and hematoxylin and eosin staining, and Dieterle silver staining and immunohistochemical staining specific for H. pylori were also performed. Twelve additional patients with urease-positive biopsies were used as controls. With Dieterle staining, 8/37 (22%) urease-negative biopsies contained organisms morphologically compatible with H. pylori, 21/37 (56%) contained organisms not compatible with H. pylori, and 8/37 (22%) were negative for organisms. Immunostaining confirmed 6/8 (75%) Dieterle-positive cases as being H. pylori, was negative in 2/8 (25%) Dieterle-positive cases, and was positive in 2/8 (25%) Dieterle-negative cases. Biopsies from 8/12 (67%) urease-positive specimens contained organisms seen with both Dieterle and immunohistochemical stains, and 4/12 (33%) were negative with both stains. Although both stains yielded comparable results with H. pylori-positive biopsies, Dieterle staining was potentially confusing because of nonspecific staining of other organisms. A significant proportion of (CLO(R))-negative biopsies was positive for H. pylori with special stains. We therefore recommend the use of immunohistochemical staining rather than silver staining in the evaluation of urease-negative gastric biopsies demonstrating chronic inflammation in children.

Adolescent↗
PubMed2004

Comparative study of controlled pore glass, silica gel and poraver for the immobilization of urease to determine urea in a flow injection conductimetric biosensor system.

This study compared the responses of three enzyme reactors containing urease immobilized on three types of solid support, controlled pore glass (CPG), silica gel and Poraver. The evaluation of each enzyme reactor column was done in a flow injection conductimetric system. When urea in the sample solution passed though the enzyme reactor, urease catalysed the hydrolysis of urea into charged products. A lab-built conductivity meter was used to measure the increase in conductivity of the solution. The responses of the enzyme reactor column with urease immobilized on CPG and silica gel were similar and were much higher than that of Poraver. Both CPG and silica gel reactor columns gave the same limit of detection, 0.5 mM, and the response was still linear up to 150mM. The analysis time was 4-5 min per sample. The enzyme reactor column with urease immobilized on CPG gave a slightly better sensitivity, 4% higher than the reactor with silica gel. The life time of the immobilized urease on CPG and silica gel were more than 310h operation time (used intermittently over 7 months). Good agreement was obtained when urea concentrations of human serum samples determined by the flow injection conductimetric biosensor system was compared to the conventional methods (Fearon and Berthelot reactions). These were statistically shown using the regression line and Wilcoxon signed rank tests. The results showed that the reactor with urease immobilized on silica gel had the same efficiency as the reactor with urease immobilized on CPG.

Biosensing Techniques↗
PubMed2004

Multi-step analysis of Hg2+ ion inhibition of jack bean urease.

We performed a multi-step analysis of the inhibition of jack bean urease by Hg(2+) ions that included residual activity measurements after incubation of the enzyme with the metal ion, reactivation of Hg(2+)-inhibited urease, protection of urease with thiol reagents prior to incubation with Hg(2+), progress curve analysis, and spectroscopic assay of thiol groups in urease-Hg(2+) complexes with a cysteine selective agent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Hg(2+) ions were found to form stable complexes with urease that could rapidly be reversed only by the treatment with dithiotreitol, and not by dilution or dialysis. The residual activity data interpreted in terms of the Hill equation revealed the multisite Hg(2+) inhibition of urease, and along with the DTNB thiol-assay they demonstrated the involvement in the reaction with Hg(2+) of six cysteine residues per enzyme subunit, including the active-site flap cysteine. The molar ratios of the inhibitor and enzyme imply that the inhibition consists of the formation of RSHgX complexes, X being a water molecule or an anion. The time-dependent Hg(2+) inhibitory action on urease determined in the system without enzyme preincubation was best described by slow-binding mechanism with the steady-state inhibition constant K(i) = 1.9 nM (+/-10%).

Cysteine↗
PubMed1999

Oral immunization with urease and Escherichia coli heat-labile enterotoxin is safe and immunogenic in Helicobacter pylori-infected adults.

BACKGROUND & AIMS: Oral immunization with Helicobacter pylori urease can cure Helicobacter infection in animals. As a step toward therapeutic immunization in humans, the safety and immunogenicity of oral immunization with recombinant H. pylori urease were tested in H. pylori-infected adults. METHODS: Twenty-six H. pylori-infected volunteers were randomized in a double-blind study to four weekly oral doses of 180, 60, or 20 mg of urease with 5 microg heat-labile enterotoxin of Escherichia coli (LT), LT alone, or placebo. Side effects and immune responses were evaluated weekly after immunization, and gastric biopsy specimens were obtained after 1 month and 6 months for histology and quantitative cultures. RESULTS: Diarrhea was noted in 16 of 24 (66%) of the volunteers who completed the study. Antiurease serum immunoglobulin A titers increased 1. 58-fold +/- 0.37-fold and 3.66-fold +/- 1.5-fold (mean +/- SEM) after immunization with 60 and 180 mg urease, respectively, whereas no change occurred in the placebo +/- LT groups (P = 0.005). Circulating antiurease immunoglobulin A-producing cells increased in volunteers exposed to urease compared with placebo (38.9 +/- 13. 6/10(6) vs. 5.4 +/- 3.1; P = 0.018). Eradication of H. pylori infection was not observed, but urease immunization induced a significant decrease in gastric H. pylori density. CONCLUSIONS: H. pylori urease with LT is well tolerated and immunogenic in H. pylori-infected individuals. An improved vaccine formulation may induce curative immunity.

Administration, Oral↗
PubMed2001

Urea permeation and hydrolysis through hollow fiber dialyzer immobilized with urease.

The surface of commercial polyacrylonitrile hollow fibers was hydrolyzed and covalently bonded with urease by using glutaraldehyde. Mini-modules assembled with these modified hollow fibers were then used to study the effect of concentration of glutaraldehyde, pH, and temperature on the catalysis of urea. The immobilized amount increased with the concentration of glutaraldehyde. However, urease immobilized with 5% glutaraldehyde had higher activity (0.38 micromol/min/mg-urease) than with other concentrations. The activity of the immobilized urease remained at above 0.32 micromol/min/mg-urease over wider applicable ranges of temperature (4-70 degreesC) and pH (5-8) compared to free urease. For comparison, the activity of immobilized urease was 0.35 micromol/min/mg-urease, while that for non-immobilized urease was 0.33 micromol/min/mg-urease at pH 7 and 20 degreesC. The removal of urea using urease-immobilized dialyzer was demonstrated with in-vitro dialysis and showed faster removing rate of urea than a regular dialyzer by 2.7 times.

Catalysis↗
PubMed2005

Influence of the primary structure of enzymes on the formation of CaCO2 polymorphs: a comparison of plant (Canavalia ensiformis) and bacterial (Bacillus pasteurii) ureases.

The influence of the primary structures of plant (Canavalia ensiformis) and bacterial (Bacillus pasteurii) ureases on the precipitation of calcium carbonate polymorphs in solutions of calcium salts and urea at room temperature was investigated. Despite a similar catalytic function in the decomposition of urea, these ureases exerted different influences on the crystal phase formation and on the development of unusual morphologies of calcium carbonate polymorphs. Spherical and uniform vaterite particles were precipitated rather than calcite in the presence of Bacillus urease, while the presence of Canavalia urease resulted in the precipitation of calcite only. Vaterite particles were shown to be built up of nanosized crystallites, proving the importance of nanoscale aggregation processes on the formation of colloidal carbonates. Reduction of the concentration of Bacillus urease in the reacting solution results in the formation of calcite crystals with a more complex surface morphology than the ones obtained by Canavalia urease. These differences may be explained by dissimilarities in the amino acid sequences of the two examined ureases and their different roles in nucleation and physicochemical interactions with the surface of the growing crystals, during the precipitation processes. This study exemplifies the diversity of proteins produced by different organisms for the same function, and the drastic effects of subtle differences in their primary structures on crystal phase formation and growth morphology of calcium carbonate precipitates, which occur as inorganic components in a large number of biogenic structures.

Amino Acid Sequence↗
PubMed1998

Effect of potent urease inhibitor, fluorofamide, on Helicobacter sp. in vivo and in vitro.

The therapeutic potential of urease inhibition of Helicobacter pylori has been studied by examining the effect of the potent urease inhibitor, fluorofamide (N-(diaminophosphinyl)-4-fluorobenzenamide), on urease activity and bacterial survival in vivo and in vitro. In culture, acid protection in H. pylori was shown to be due to changes in the pH of the medium brought about by the release of ammonia. Both the acid protection and the ammonia release were completely blocked by fluorofamide at low doses (ED50 = approximately 100 nM). However, fluorofamide was unstable under acidic conditions (T1/2 = 5.7 min at pH 2). Despite this, fluorofamide was the best available compound to test in vivo. In ferrets naturally infected with H. mustelae, a single dose (50 mg/kg, per os) of fluorofamide completely inhibited bacterial urease. In repeat dosing studies, fluorofamide (50 mg/kg per os, three times a day) was compared with the Helicobacter triple therapy regime (amoxycillin, metronidazole, and bismuth subcitrate). Fluorofamide failed to eradicate the H. mustelae infection, compared to 80% eradication with triple therapy. However, histological samples showed a profound reduction in bacterial numbers following fluorofamide treatment. A combination of fluorofamide and amoxycillin was dosed to ferrets (seven days of treatment with 50 mg/kg fluorofamide plus 10 mg/kg amoxycillin per os twice a day); however, this failed to eradicate the infection, despite there being a reduction in bacterial numbers in 3/5 ferrets after 21 days after dosing stopped. It was concluded that urease inhibitors (either alone or in combination with antibiotics) are unlikely to have therapeutic potential for Helicobacter pylori infections. This is probably because, in vivo, some bacteria (perhaps dormant forms) are not entirely dependent upon urease for survival. However, given the acid instability of fluorofamide, the possibility that more stable urease inhibitors might have therapeutic potential, cannot be excluded.

Amoxicillin↗
PubMed2004

Immobilization of pigeonpea (Cajanus cajan) urease on DEAE-cellulose paper strips for urea estimation.

Pigeonpea ( Cajanus cajan ) urease was immobilized on 1 cmx1 cm DEAE-cellulose paper strips. The optimum immobilization (51% activity) was observed at 4 degrees C, with a protein concentration of 1.0 mg/strip. The apparent optimum pH shifted from 7.3 to 6.8. Immobilized urease showed an optimal stability temperature of 67 degrees C, compared with 47 degrees C for the soluble urease. Time-dependent kinetics of the thermal inactivation of the immobilized urease were examined and found to be monophasic as compared with the soluble enzyme, which was biphasic. The Michaelis constant ( K (m)) for the DEAE-cellulose-immobilized urease was found to be 4.75 mM, 1.5 times higher than the soluble enzyme. Immobilized strips stored at 4 degrees C showed an increased half-life ( t (1/2)=150 days). There was practically no leaching of the enzyme from the immobilized strips over a period of 2 weeks. These strips were used for estimating the urea content of blood samples; the results obtained matched well with those obtained in a clinical laboratory through an Autoanalyzer(R) (Zydus Co., Rome, Italy). The easy availability of pigeonpea urease, the ease of its immobilization on DEAE-cellulose strips and the significantly lower cost of urease described in the present study makes it a suitable product for future applications in diagnostics.

Cajanus↗
PubMed1996

The role of Helicobacter pylori urease in the pathogenesis of gastritis and peptic ulceration.

Helicobacter pylori produces a 550 kDa, multimeric, nickel-containing urease that catalyses the hydrolysis of urea to yield ammonia and carbonic acid. The ure gene cluster, comprised of seven genes, encodes the two structural subunits UreA (26.5 kDa) and UreB (60.3 kDa), and five accessory proteins: UreI, UreE, UreF, UreG and UreH. Accessory proteins are required for nickel ion insertion into the apoenzyme. The native protein consists of six copies each of UreA and UreB; two nickel ions are coordinated into each UreB active site. Urease is found in the cytosol, but may also localize on the surface (although this may be an artefact) and elicits a strong serum immunoglobulin response. Urease aids in colonization of the host by neutralizing gastric acid and providing ammonia for bacterial protein synthesis. Host defences are avoided by urease by continuing to neutralize acid locally and by shedding urease, which may be bound by immunoglobulin, from the surface of the bacterium. Host tissues can be damaged directly by the urease-mediated generation of ammonia and indirectly by urease-induced stimulation of the inflammatory response, including recruitment of leukocytes and triggering of the oxidative burst in neutrophils.

Gastritis↗
PubMed2004

Characterization of urease genes cluster of Streptococcus thermophilus.

AIMS: The milk acidification rate of Streptococcus thermophilus strains can be affected by several factors, one of which is the hydrolysis of urea by the urease complex. To evaluate the technological suitability of S. thermophilus strains deprived of urease activity in milk fermentation, the genetic cluster related to urease enzymatic activity has been characterized in the type strain DSM 20167T. METHODS AND RESULTS: Amplification of the urease genes of S. thermophilus DSM 20167T was developed on the basis of the urease gene cluster of the phylogenetically related S. salivarius. Nucleotide sequencing revealed the presence of eight open reading frames, which were most homologous to ureABC (structural genes) and ureI, ureEFGD (accessory genes) of S. salivarius and other ureolytic bacteria. Reverse transcriptase PCR experiments were in agreement with an operon organization for the eight genes (ureIABCEFGD). A food grade mutant A16 (DeltaureC3) with a 693 bp in-frame deletion in ureC gene exhibited a urease negative (Ure-) phenotype. Unlike the wild-type strain, the acidification rate of the mutant in reconstituted skimmed milk was not affected by the presence of urea or nickel ions. A small-scale yoghurt fermentation trials were carried out using the wild-type or the Ure- mutant A16 (DeltaureC3) in co-culture with Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 in presence of urea. The result obtained underlines that when the Ure- mutant was used as a co-starter the acidification rate was higher than that obtained using the wild-type strain. CONCLUSIONS: The study provides the first genetic characterization and the technological implication of S. thermophilus DSM 20617T urease activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The detrimental effect of ureolytic activity on the rate of milk acidification was evaluated and superseded using a food-grade Ure- recombinant strain. Small-scale yoghurt production trials highlighted the positive role of a Ure-S. thermophilus mutant as a co-starter in milk fermentations. Moreover, the vector pMI108 developed for the construction of the Ure- strain, should be considered as a potential tool for the generation of Ure- dairy S. thermophilus strains selected for other relevant technological properties but characterized by the undesirable ureolytic phenotype.

Animals↗
PubMed1994

Inhibitory effect of penicillin on caecal urease activity in chickens fed on a low protein diet plus urea.

1. The effects of dietary penicillin on the urease activities of small intestine, large intestine, caecum and their contents, liver and kidney in chickens fed a diet containing 5 g/kg protein plus urea were examined. 2. About 0.88 of the total urease activity determined was observed in intestinal contents, of which 0.95 of the activity was accounted for by caecal contents, 0.05 by colo-rectal contents and none by small intestinal contents. Intestinal tissues (caecum included), liver and kidney accounted for 0.03, 0.06 and 0.02, respectively, of the total urease activity. 3. Dietary penicillin decreased urease activity to 0.17 in caecal contents and to 0.05 in colo-rectal contents of the corresponding control values (P < 0.01). The urease activity of caecal tissue was lowered by penicillin to half that of control activity (P < 0.05) but none of the activities of other tissues were affected. 4. It is concluded that, even when the urease activity is stimulated by dietary urea, 20 mg/kg dietary penicillin can strikingly lower it in the caecum, where most of the urease activity in the chicken body is to be found.

Animals↗
PubMed2006

Inhibition of jack bean urease by tetrachloro-o-benzoquinone and tetrachloro-p-benzoquinone.

Tetrachloro-o-benzoquinone (TCoBQ) and tetrachloro-p-benzoquinone (TCpBQ) were studied as inhibitors of jack bean urease in 20 mM phosphate buffer, pH 7.0, 1 mM EDTA, 25 degrees C. The mechanisms of inhibition were evaluated by analysis of the progress curves obtained with two procedures: the reaction initiated by addition of the enzyme and the reaction initiated by addition of the substrate after preincubation of the enzyme with the inhibitor. The obtained results were characteristic of slow-binding inhibition. The effects of different inhibitor concentrations on the initial and steady-state velocities obeyed the relationships of two-step enzyme-inhibitor interaction, qualified as mechanism B. It was found that TCoBQ and TCpBQ are strong urease inhibitors. TCpBQ is more effective than TCoBQ with the overall inhibition constant of K(i)* = 4.5 x 10(-7) mM. The respective inhibition constant of TCoBQ was equal to: K(i)* = 2.4 x 10(-6) mM. The protective experiment proved that the urease active site is involved in the tetrachlorobenzoquinone inhibition process. High effectiveness of thiol protectors against inhibition by TCoBQ and TCpBQ indicates the strategic role of the active site sulfhydryl group in the blocking process. The stability of the complexes: urease-TCoBQ and urease-TCpBQ was tested in two ways: by dilution or addition of dithiothreitol. No recovery of urease activity bound in the urease-inhibitor complexes proves that the complexes are stable and strong.

Canavalia↗
PubMed2002

Purification and characterization of urease isolated from the pathogenic fungus Coccidioides immitis.

Coccidioides immitis, the causative agent of San Joaquin Valley fever (coccidioidomycosis), produces a urease which has been suggested to contribute to the virulence of this fungal pathogen. Urease catalyzes the hydrolysis of urea and has been proposed to at least partly account for alkalinity of the microenvironment in which C. immitis grows due to the release of ammonia and ammonium ions. The C. immitis urease was purified to homogeneity (1048-fold) from the mycelial cytosol by chromatographic fractionation. The sequence of 12 N-terminal amino-acid residues of the purified, native polypeptide was identical to that predicted by the translated urease gene sequence which has been reported. The isolated enzyme exhibited a specific activity in the presence of urea of 1750 micromol min(-1) mg(-1) protein, has a native molecular mass of 450 kDa, revealed a Km for urea of 4.1 mM, had a pH optimum of 8.0 and is heat stable. Hydroxyurea, acetohydroxamic acid (AHA) and boric acid each inhibited activity of the purified enzyme. Urease activity was enhanced by the presence of 5-10 mM concentrations of Mg2+ or Mn2+, but inhibited by Li+, Ni2+, Cu2+ or Zn2+. The reversible urease inhibitor, AHA, blocked enzyme activity in the crude mycelial cytosolic fraction when added at a concentration of 10 mM. On the other hand, 10 mM AHA added to 4-day-old mycelial cultures only partially decreased the amount of ammonium detected in the culture medium. It is evident, therefore, that C. immitis urease activity does not account for the total amount of ammonia secreted during in vitro growth of the pathogen. Other metabolic sources of ammonia, which may also contribute to the virulence of C. immitis, are under investigation.

Amino Acid Sequence↗