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PubMed1994

A single gene (Eu4) encodes the tissue-ubiquitous urease of soybean.

We sought to determine the genetic basis of expression of the ubiquitous (metabolic) urease of soybean. This isozyme is termed the metabolic urease because its loss, in eu4/eu4 mutants, leads to accumulation of urea, whereas loss of the embryo-specific urease isozyme does not. The eu4 lesion eliminated the expression of the ubiquitous urease in vegetative and embryonic tissues. RFLP analysis placed urease clone LC4 near, or within, the Eu4 locus. Sequence comparison of urease proteins (ubiquitous and embryo-specific) and clones (LC4 and LS1) indicated that LC4 and LS1 encode ubiquitous and embryo-specific ureases, respectively. That LC4 is transcribed into poly(A)+ RNA in all tissues was indicated by the amplification of its transcript by an LC4-specific PCR primer. (The LS1-specific primer, on the other hand, amplified poly(A)+ RNA only from developing embryos expressing the embryo-specific urease.) These observations are consistent with Eu4 being the ubiquitous urease structural gene contained in the LC4 clone. In agreement with this notion, the mutant phenotype of eu4/eu4 callus was partially corrected by the LC4 urease gene introduced by particle bombardment.

Amino Acid Sequence↗
PubMed2004

Urease-glutamic dehydrogenase biosensor for screening heavy metals in water and soil samples.

A screen-printed three-electrode amperometric biosensor based on urease and the nicotinamide adenine dinucleotide hydrogen (NADH)-glutamic dehydrogenase system was developed and applied to the screening of heavy metals in environmental samples. The development of an amperometric sensor for the monitoring of urease activity was feasible by coupling the urea breakdown reaction catalysed by urease to the reductive ammination of ketoglutarate catalysed by glutamic dehydrogenase (GLDH). The ammonia provided by the urea conversion is required for the conversion of ketoglutarate to glutamate with the concomitant oxidation of the NADH cofactor. NADH oxidation is monitored amperometrically at 0.3 V (vs. Ag/AgCl) after urease immobilization onto the screen-printed three-electrode configuration. Immobilization of urease on the surface of screen-printed electrodes was performed by entrapment in alginate gel and adsorption on the electrode in a nafion film. Low sensitivity to inactivation by metals was recorded after urease entrapment in alginate gel with detection limits of 2.9 and 29.8 mg L(-1) for Hg(II) and Cu(II), respectively. The use of the negatively charged nafion film created a more concentrated environment of cations in proximity to the enzyme, thus enhancing the urease inhibition when compared to gel entrapment. The calculated detection limits were 63.6 and 55.3 microg L(-1) for Hg(II) and Cu(II), respectively, and 4.3 mg L(-1) for Cd(II). A significant urease inactivation was recorded in the presence of trace amounts of metals (microg L(-1)) when the enzyme was used free in solution. Analysis of water and soil samples with the developed nafion-based sensor produced inhibition on urease activity according to their metal contents. The obtained results were in agreement with the standard methods employed for sample analysis. Nevertheless, the use of the amperometric assay (with free urease) proved more feasible for the screening of trace amounts of metals in polluted samples.

Biosensing Techniques↗
PubMed1993

Urease activity in Streptococcus salivarius at low pH.

Arginine metabolism to alkali by the arginine deiminase system in oral bacteria increases their acid tolerance. The potential of urease activity in Streptococcus salivarius to fulfil a similar role was examined. In cell extracts between pH 5.0 and 8.0, urease activity was over 80% the maximal rate. The urease rate was zero at pH 4.3, and at pH 3.6 the enzyme was rapidly inactivated (t 1/2 of 0.6 min). The pH range of intact cells was broader. In Strep. salivarius cells acidified to pH 2.6 for 5 min, urease was completely retained and the ureolytic pH rise was rapid. There was no urease activity after acidification to pH 2. In cells acidified to maintain the pH between 3.3 and 4, viability was maintained for a short period (extrapolation indicated 20 min) and then decreased. This acidification induced alkali generation or acid removal that decreased in parallel to loss of viability. A small fraction (10%) of the urease was rapidly inactivated, after which both the remaining urease and pH response decreased at a similar rate to cell viability (t 1/2 of 15-20 min), but for at least 1 h following acidification, a rapid ureolysis induced rise in pH to above 7. In cells held at pH 3.6 and treated to compromise their membranes by freeze-thawing or transient acidification to pH 2.3, 70-80% of the urease was lost rapidly and the remainder inactivated at a rate similar to that in intact cells. Therefore, although at pH below 4, S. salivarius urease is outside its pH activity range and the free enzyme is rapidly inactivated, intact cells the urease is protected and ureolytic generation of ammonia is capable of substantially raising the pH for at least 1 h while the cell population is being progressively killed by acid.

Dental Plaque↗
PubMed1996

Evaluation of a new reagent strip rapid urease test for detection of Helicobacter pylori infection.

BACKGROUND: Rapid urease tests are commonly used as a convenient method to detect Helicobacter pylori infection. Our previous experiments demonstrated enhanced efficacy of agar gel rapid urease test compared with reagent strip rapid urease tests. We evaluated the efficacy of PyloriTek, a new reagent strip rapid test for detecting H. pylori infection. METHODS: Gastric antral mucosal biopsy specimens were obtained for comparison between agar gel rapid urease tests and PyloriTek (200 specimens). The rapid urease test to be used first was selected randomly. H. pylori status was determined using the Genta stain. Culture was performed to confirm H. pylori status when false rapid urease tests were suspected. RESULTS: One hundred patients were studied; 68 had H. pylori infection. There were two false-negative and one false-positive PyloriTek when scored at 1 hour, compared with only one false-positive and no false-negative tests at 2 hours. With the agar gel rapid urease tests, there were no false-positive tests and 5 false-negative tests when scored at 1 hour, 2 false-negative tests at 12 hours and 1 at 24 hours; there were no false-positive tests. At 1 hour, 3% (95% CI = 1% to 9%) of PyloriTek tests had an erroneous categorization of H. pylori status compared with 5% for the agar gel rapid urease tests (95% CI = 1.6% to 11%) (p > 0.7). CONCLUSION: The new reagent strip rapid urease test, PyloriTek, is rapid and comparable in accuracy to agar gel rapid urease tests for detecting H. pylori Infection.

Adult↗
PubMed1999

Effect of immersion of biopsy forceps in formalin on tissue urease activity.

BACKGROUND AND AIMS: It is routine practice to wash biopsy forceps that have been immersed in formalin solution before taking gastric biopsies to test for urease activity as formalin is thought to inactivate the urease enzyme. The aim of this study was to assess the effect of pre-immersion of biopsy forceps in formalin solution on the ability to detect Helicobacter pylori urease activity in biopsies obtained with the same forceps. METHODS: Two hundred consecutive patients undergoing gastroscopy who had macroscopic evidence of possible H. pylori infection had an initial antral biopsy taken using sterile forceps for determining biopsy urease activity. The same forceps were then used to obtain an antral biopsy for histological examination. The forceps were then used, without washing off any adherent formalin solution, to obtain a further antral biopsy for urease testing. RESULTS: The concordance rate for urease tests, with or without formalin exposure, was 100% (95% confidence interval (CI) 98.2-100%). Fifty-six of 200 patients (28%) were found to have urease-positive biopsies. Of these, 52/56 (92.9%) had identifiable H. pylori on histopathology. One hundred and forty-four of 200 patients (72%) were found to have urease-negative biopsies. Of these, seven (4.9%) had identifiable H. pylori on histopathology. Six of seven (85.7%) had only a small number of organisms identified. The sensitivity and specificity for the urease test compared with the histopathology as a reference standard was 88.1% (95% CI 79.9-96.4%) and 97.2% (95% CI 94.4-99.9%), respectively. CONCLUSION: Immersion of biopsy forceps in formalin did not reduce the ability to detect urease activity in gastric biopsies taken subsequently.

Biopsy↗
PubMed1996

Helicobacter pylori urease is a potent stimulus of mononuclear phagocyte activation and inflammatory cytokine production.

BACKGROUND & AIMS: Helicobacter pylori surface proteins induce the production of proinflammatory mediators by mononuclear phagocytes, but the protein responsible for this stimulation has not been identified. This study determined whether urease, the major component of the soluble proteins extracted from H. pylori grown in culture, activates mononuclear phagocytes and stimulates them to produce proinflammatory cytokines. METHODS: Primary human blood monocytes were incubated with column-purified H. pylori urease and assayed by flow cytometry, Immunoassay, and reverse-transcription polymerase chain reaction for phenotypic, functional, and molecular evidence of activation. RESULTS: H. pylori urease induced monocyte expression of surface interleukin 2 receptors and increased expression of HLA-DR, phenotypic changes consistent with activation. Urease also stimulated dose-dependent production of interleukin 1 beta, interleukin 6, interleukin 8, and tumor necrosis factor alpha peptides and messenger RNA. These urease-induced phenotypic and functional changes were inhibited by preincubation of the urease with antisera to H. pylori whole bacteria, purified urease, or the 31-kilodalton subunit of urease. CONCLUSIONS: Among the soluble proteins released by H. pylori, urease is capable of activating monocytes for proinflammatory cytokines production. The local production of cytokines by urease-stimulated mononuclear phagocytes may play a central role in the development of H. pylori gastroduodenal inflammation.

Base Sequence↗
PubMed2004

Analysis of two alleles of the urease gene from potato: polymorphisms, expression, and extensive alternative splicing of the corresponding mRNA.

Globally, urea is the most widely used nitrogen fertilizer and is made accessible to plants via the urease reaction. However, sequence information for the plant enzyme is scarce. A cDNA encoding urease from soybean (Glycine max) has been cloned and sequence information has been obtained for two alleles (11 and 19 kbp, respectively) of the potato (Solanum tuberosum, cv. Desiree) urease gene and the corresponding cDNAs. It was found that urease is encoded by a single copy gene in several solanaceous species, and maps to chromosome V in potato. By contrast, the presence of two urease genes was reported for soybean. Comparative analysis of 11 kbp overlapping allelic DNA allowed the quantification of single nucleotide polymorphisms and revealed the presence of a truncated Ty1-copia retrotransposon in one of the alleles. Both alleles contained a copy of a terminal-repeat retrotransposon in miniature (TRIM). 25-50% of urease pre-mRNAs from both alleles were alternatively spliced in a variety of different ways. The retrotransposons had no effect on splicing. While urease is expressed in all tissues tested, its mRNA and protein are of low abundance. The TATA-less urease promoter appears to drive low-level housekeeping transcription. An in silico analysis showed that eukaryotic urease protein sequences are very similar to sequences from prokaryotic enzymes, conserving all residues of known functional importance. It is therefore likely that all known ureases are structurally similar, employing the same catalytic mechanism.

Alleles↗
PubMed1999

The marine cyanobacterium Synechococcus sp. WH7805 requires urease (urea amidohydrolase, EC 3.5.1.5) to utilize urea as a nitrogen source: molecular-genetic and biochemical analysis of the enzyme.

Cyanobacteria assigned to the genus Synechococcus are an important component of oligotrophic marine ecosystems, where their growth may be constrained by low availability of fixed nitrogen. Urea appears to be a major nitrogen resource in the sea, but little molecular information exists about its utilization by marine organisms, including Synechococcus. Oligonucleotide primers were used to amplify a conserved fragment of the urease (urea amidohydrolase, EC 3.5.1.5) coding region from cyanobacteria. A 5.7 kbp region of the genome of the unicellular marine cyanobacterium Synechococcus sp. strain WH7805 was then cloned, and genes encoding three urease structural subunits and four urease accessory proteins were sequenced and identified by homology. The WH7805 urease had a predicted subunit composition typical of bacterial ureases, but the organization of the WH7805 urease genes was unique. Biochemical characteristics of the WH7805 urease enzyme were consistent with the predictions of the sequence data. Physiological data and sequence analysis both suggested that the urease operon may be nitrogen-regulated by the ntcA system in WH7805. Inactivation of the large subunit of urease, ureC, prevented WH7805 and Synechococcus WH8102 from growing on urea, demonstrating that the urease genes cloned are essential to the ability of these cyanobacteria to utilize urea as a nitrogen source.

Amino Acid Sequence↗
PubMed2006

Urease-positive bacteria other than Helicobacter pylori in human gastric juice and mucosa.

BACKGROUND AND AIM: Many bacteria carry the urease enzyme in different human ecosystems, but Helicobacter pylori is the only known bacterium showing urease activity in gastric ecosystems. For this reason, the rapid urease test (RUT) on gastric biopsies and urea breath test (C-UBT) are used to detect H. pylori infection. The aim of this study was to evaluate the presence of urease-positive bacteria other than H. pylori in gastric juice and mucosa in hypochlorhydric subjects. METHODS: Twenty-five hypochlorhydric and 10 normochlorhydric patients were analyzed for the presence of H. pylori and bacterial overgrowth both in gastric juice and on the mucosa. During upper gastrointestinal endoscopy at 8.00 a.m. gastric juice samples and biopsy specimens were taken from the antrum and corpus. All samples were analyzed using standard microbiological procedures like aerobic/anaerobic growth, gram-staining, gas chromatography, API test, 96-clone method, and selective medium to search for specific bacteria. In addition, all strains isolated were screened for urease activity using the CP-test. Urease positive strains were tested for the capacity to survive in an acid environment with or without urea (10 mM/L), at pH 7, 4, 3, and 2, respectively, at different times (0, 20, 30, and 60 min). RESULTS: Six hypochlorhydric patients had 10 strains of urease-positive non-H. pylori bacteria among which Staphylococcus capitis urealiticum showed the strongest urease activity. CONCLUSIONS: Hypochlorhydric patients present many urease-positive bacteria other than H. pylori. The strong urease activity may be responsible for false positive results at RUT or UBT test in patients with suspected H. pylori infection.

Adult↗
PubMed1987

Genetic and biochemical diversity of ureases of Proteus, Providencia, and Morganella species isolated from urinary tract infection.

Bacterial urease, particularly from Proteus mirabilis, has been implicated as a contributing factor in the formation of urinary and kidney stones, obstruction of urinary catheters, and pyelonephritis. Weekly urine specimens (n = 1,135) from 32 patients, residing at two chronic-care facilities, with urinary catheters in place for greater than or equal to 30 days yielded 5,088 phenotypically and serotypically diverse bacterial isolates at greater than or equal to 10(5) CFU/ml. A total of 86% of specimens contained at least one urease-positive species, and 46% of 3,939 gram-negative bacilli were urease positive. For investigation of genetic relatedness of urease determinants, whole-cell DNA from 50 urease-positive isolates each of Providencia stuartii, Providencia rettgeri, P. mirabilis, Proteus vulgaris, and Morganella morganii were hybridized with a urease gene probe derived from within the urease operon of Providencia stuartii BE2467. The percentage of strains hybridizing with the gene probe was 98 for Providencia stuartii, 100 for Providencia rettgeri, 70 for P. mirabilis, 2 for M. morganii, and 0 for P. vulgaris. Electrophoretic mobilities of ureases from representative isolates revealed nine different patterns among the five species. The urease gene probe hybridized with fragments of HindIII-digested chromosomal DNA from all isolates except M. morganii. Fragment sizes differed between species. Molecular sizes of the enzymes, determined by Sephacryl S-300 chromatography, were found to be 280 kilodaltons (kDa) (P. mirabilis), 323 to 337 kDa (Providencia stuartii, Providencia rettgeri, P. mirabilis, P. vulgaris), 620 kDa (providencia rettgeri), and greater than 700 kDa (M. morganii, Providencia rettgeri). Kms ranged from 0.7 mM urea for M. morganii to 60 mM urea for a P. mirabilis isolate. In general, P. mirabilis ureases demonstrated lower affinities for substrate but hydrolyzed urea at rates 6- to 25-fold faster than did enzymes from other species, which may explain the frequent association of this species with stone formation.

Aged↗
PubMed1992

Characteristics of Helicobacter pylori variants selected for urease deficiency.

The urease of Helicobacter pylori is suspected to play a role in the pathogenesis of gastritis. Although all clinical isolates of H. pylori are urease positive (U+), we have selected and characterized several spontaneously arising urease-negative (U-) variants from wild-type strain 60190. Urease-negative variants were identified by growth in medium containing 60 mM urea and arose at a frequency of 10(-5) to 10(-6). The urease activity of the wild-type strain inhibited growth of this strain in the presence of 60 mM urea. U- variants retained the U- phenotype for more than 100 passages on medium with or without urea. The urease activities of the original U+ and derived U- cells were 9.55 to 16.7 and 0.01 to 0.17 U/mg of protein, respectively. Colonial growth and other biochemical characteristics were identical for the strains. U- variants showed three classes of whole-cell sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles: (i) identical to U+; (ii) change in the migration of the 61-kDa urease subunit; and (iii) lack of 61- and 30-kDa subunits. These differences were confirmed by immunoblotting and by protein separation using fast protein liquid chromatography. The U+ strain but not U- variants tolerated exposure to pH 4.0 for 60 min in the presence of urea. Supernatants of the U+ strain and U- variants contained vacuolating cytotoxin activity for HeLa cells in similar titers. By enzyme-linked immunosorbent assay, human serum samples recognized water extract from the U+ strain significantly better than extract from a U- variant lacking urease subunits. In conclusion, this study demonstrates that U- H. pylori variants may arise spontaneously, that urease activity enhances survival at acid pH, and that urease and cytotoxin activities are disparate phenotypes.

Bacterial Proteins↗
PubMed1994

Immunological and molecular characterization of Helicobacter felis urease.

Urease activity has recently been shown to be an important virulence determinant for Helicobacter pylori, allowing it to survive the low pH of the stomach during colonization. Experimental murine infection with Helicobacter felis is now being used as a model for H. pylori infection to study the effects of vaccines, antibiotics, and urease inhibitors on colonization. However, little information comparing the ureases of H. felis and H. pylori is available. Urease was partially purified from the cell surface of H. felis ATCC 49179 by A-5M agarose chromatography, resulting in an eightfold increase in specific activity over that of crude urease. The apparent Km for urea for the partially purified urease was 0.4 mM, and the enzyme was inhibited in a competitive manner by flurofamide (50% inhibitory concentration = 0.12 microM). Antiserum to whole cells of H. pylori recognized both H. pylori and H. felis urease B subunits. Antiserum raised against H. felis whole cells recognized the large and small autologous urease subunits and the cpn60 heat shock molecule in both H. felis and H. pylori. However, this antiserum showed only a weak reaction with the B subunit of H. pylori urease. Two oligomeric DNA sequences were used as probes to evaluate the relatedness of H. felis and H. pylori urease gene sequences. One 30-mer from the ureA sequence, which had been shown previously to be specific for H. pylori, failed to hybridize to H. felis genomic DNA. A probe to the putative coding sequence for the active site of the H. pylori ureB subunit hybridized at low intensity to a 2.8-kb fragment of BamHI-HindIII-digested H. felis DNA, suggesting that the sequences were homologous but not identical, a result confirmed from the recently published sequences of ureA and ureB from H. felis.

Base Sequence↗
PubMed2003

Dependence of Helicobacter pylori urease activity on the nickel-sequestering ability of the UreE accessory protein.

The Helicobacter pylori ureE gene product was previously shown to be required for urease expression, but its characteristics and role have not been determined. The UreE protein has now been overexpressed in Escherichia coli, purified, and characterized, and three altered versions were expressed to address a nickel-sequestering role of UreE. Purified UreE formed a dimer in solution and was capable of binding one nickel ion per dimer. Introduction of an extra copy of ureE into the chromosome of mutants carrying mutations in the Ni maturation proteins HypA and HypB resulted in partial restoration of urease activity (up to 24% of the wild-type levels). Fusion proteins of UreE with increased ability to bind nickel were constructed by adding histidine-rich sequences (His-6 or His-10 to the C terminus and His-10 as a sandwich fusion) to the UreE protein. Each fusion protein was overexpressed in E. coli and purified, and its nickel-binding capacity and affinity were determined. Each construct was also expressed in wild-type H. pylori and in hypA and hypB mutant strains for determining in vivo urease activities. The urease activity was increased by introduction of all the engineered versions, with the greatest Ni-sequestering version (the His-6 version) also conferring the greatest urease activity on both the hypA and hypB mutants. The differences in urease activities were not due to differences in the amounts of urease peptides. Addition of His-6 to another expressed protein (triose phosphate isomerase) did not result in stimulation of urease, so urease activation is not related to the level of nonspecific protein-bound nickel. The results indicate a correlation between H. pylori urease activity and the nickel-sequestering ability of the UreE accessory protein.

Amino Acid Sequence↗
PubMed2000

Helicobacter pylori urease binds to class II MHC on gastric epithelial cells and induces their apoptosis.

Infection by Helicobacter pylori leads to injury of the gastric epithelium and a cellular infiltrate that includes CD4+ T cells. H. pylori binds to class II MHC molecules on gastric epithelial cells and induces their apoptosis. Because urease is an abundant protein expressed by H. pylori, we examined whether it had the ability to bind class II MHC and induce apoptosis in class II MHC-bearing cells. Flow cytometry revealed the binding of PE-conjugated urease to class II MHC+ gastric epithelial cell lines. The binding of urease to human gastric epithelial cells was reduced by anti-class II MHC Abs and by staphylococcal enterotoxin B. The binding of urease to class II MHC was confirmed when urease bound to HLA-DR1-transfected COS-1 (1D12) cells but not to untransfected COS-1 cells. Urease also bound to a panel of B cell lines expressing various class II MHC alleles. Recombinant urease induced apoptosis in gastric epithelial cells that express class II MHC molecules, but not in class II MHC- cells. Also, Fab from anti-class II MHC and not from isotype control Abs blocked the induction of apoptosis by urease in a concentration-dependent manner. The adhesin properties of urease might point to a novel and important role of H. pylori urease in the pathogenesis of H. pylori infection.

Alleles↗
PubMed1998

Usefulness of brushing urease test for diagnosis of Helicobacter pylori infection.

BACKGROUND AND AIMS: Gastric brushing cytology is an accurate technique for rapidly detecting Helicobacter pylori infection, but it is not routinely employed since the presence of personnel experienced in this field, is necessary in the endoscopy suite. To evaluate the diagnostic usefulness of rapid urease test carried out on cytological brushing. PATIENTS: A total of 143 consecutive patients with suspected Helicobacter pylori infection, referred for elective gastroscopy. METHODS: For each patient, 2 brushings were carried out and 4 biopsies were taken from antral mucosa during gastroscopy. The former brushing was smeared on a slide, and stained by a rapid staining set for blood smears. The latter was used for rapid urease test, by shaking the brush into the urea broth. Two biopsies were used for rapid urease test and two for histologic examination. Histology was considered as the gold standard. RESULTS: Of 143 patients, 73 were diagnosed as Helicobacter pylori infected using histology. Six brushing slides were inadequate due to insufficient cytology material. Biopsy-rapid urease test and brushing-rapid urease test had similar sensitivity (87.3% vs 83.5%), specificity (98.4% vs 96.8%) and overall accuracy (92.3% vs 89.5%). In 62 Helicobacter pylori infected patients, both rapid urease test techniques were positive. Brushing-rapid urease test became positive in a significantly shorter time than biopsy-rapid urease test (22 +/- 54 minutes vs 39 +/- 63 minutes; p < 0.01). CONCLUSIONS: Brushing-rapid urease test is as accurate as biopsy-rapid urease test in detecting Helicobacter pylori infection, but it is significantly faster. Special care should be taken to carry out brushing adequately, to minimize the occurrence of false negatives.

Adult↗
PubMed1994

Threonine is present instead of cysteine at the active site of urease from Staphylococcus xylosus.

DNA sequence analysis of the structural urease genes from Staphylococcus xylosus revealed that three enzyme subunits are encoded in the order of 11,000, 15,400 and 61,000 (mol. mass), which correspond to the single polypeptide chain of jack bean urease (90,800). Comparing the deduced amino acid sequence of S. xylosus urease with the amino acid sequence of jack bean urease an overall portion of 56% identical residues was found. For S. xylosus urease a subunit structure of (alpha beta gamma)4 was proposed, based on the comparison of the deduced amino acid content of the enzyme subunits with the total amino acid content of the purified enzyme. The staphylococcal enzyme contained no cysteine, as deduced from DNA sequence and confirmed by the determination of the total amino acid content in the purified enzyme. Instead of cysteine, known to be catalytically essential in the plant enzyme, and conserved among all bacterial ureases analyzed so far, threonine was found in S. xylosus. This amino acid-exchange was located within a highly conserved domain of 17 amino acids, supposed to be part of the active site. Sequence analysis of the respective region of Staphylococcus saprophyticus urease showed that it also contains threonine instead of cysteine. In contrast to jack bean urease S. xylosus urease was not affected by the SH-group inhibitor dipyridyl disulfide but was completely inhibited by the serine protease inhibitor phenylmethanesulfonyl fluoride. The presented results indicate that in these staphylococcal strains urea hydrolysis might function in a manner similar to the peptide bond cleavage by chymotrypsin.

Amino Acid Sequence↗
PubMed1987

Recovery of a soybean urease genomic clone by sequential library screening with two synthetic oligodeoxynucleotides.

We report the first isolation of a low-copy-number gene from a complex higher plant (soybean) genome by direct screening with synthetic oligodeoxynucleotide (oligo) probes. A synthetic, mixed, 21-nucleotide (nt) oligo (21-1) based on a seven amino acid (aa) sequence from soybean seed urease, was used to screen genomic libraries of soybean (Glycine max [L.] Merr.) in the lambda Charon 4 vector. Twenty homologous clones were recovered from a screen of 500,000 plaques. These were counterscreened with embryo-specific cDNA (15-2 cDNA) made by priming with a second, mixed 15-nt oligo (15-2), based on a Jack bean (Canavalia ensiformis) urease peptide [Takishima et al., J. Natl. Def. Med. Coll. 5 (1980) 19-23]. Five out of 20 clones were homologous to 15-2 cDNA and proved to be identical. Nucleotide sequence analysis of representative clone E15 confirmed that it contained urease sequences. Subclones of E15 homologous to the oligo probes contain a deduced amino acid sequence which matches 108 of 130 aa residues of an amino acid run in a recently published [Mamiya et al., Proc. Jap. Acad. 61B (1985) 359-398] complete protein sequence for Jack-bean seed urease. Using clone E15 as a probe of soybean embryonic mRNA revealed a homologous 3.8-kb species that is the size of the urease messenger. This species is absent from mRNA of embryos of a soybean seed urease-null mutant. However, both urease-positive and urease-null genomes contain the 11-kb DNA fragment bearing urease sequences.

Amino Acid Sequence↗
PubMed2002

Purification and characterization of urease from dehusked pigeonpea (Cajanus cajan L) seeds.

Urease has been purified from the dehusked seeds of pigeonpea (Cajanus cajan L.) to apparent electrophoretic homogeneity with approximately 200 fold purification, with a specific activity of 6.24 x10(3) U mg(-1) protein. The enzyme was purified by the sequence of steps, namely, first acetone fractionation, acid step, a second acetone fractionation followed by gel filtration and anion-exchange chromatographies. Single band was observed in both native- and SDS-PAGE. The molecular mass estimated for the native enzyme was 540 kDa whereas subunit values of 90 kDa were determined. Hence, urease is a hexamer of identical subunits. Nickel was observed in the purified enzyme from atomic absorption spectroscopy with approximately 2 nickel ions per enzyme subunit. Both jack bean and soybean ureases are serologically related to pigeonpea urease. The amino acid composition of pigeonpea urease shows high acidic amino acid content. The N-terminal sequence of pigeonpea urease, determined up to the 20th residue, was homologous to that of jack bean and soybean seed ureases. The optimum pH was 7.3 in the pH range 5.0-8.5. Pigeonpea urease shows K(m) for urea of 3.0+/-0.2 mM in 0.05 M Tris-acetate buffer, pH 7.3, at 37 degrees C. The turnover number, k(cat), was observed to be 6.2 x 10(4) s(-1) and k(cat)/K(m) was 2.1 x 10(7) M(-1) s(-1). Pigeonpea urease shows high specificity for its primary substrate urea.

Amino Acid Sequence↗