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The behavior of an enzyme/membrane system containing urease is studied when an external electric field is applied. The device using a potential difference across the enzyme/membrane system is first described. Optimal operating conditions with respect to substrate concentration, ionic strength and pH are studied. Possible mechanisms of the change in membrane activity by electric field are discussed.
Urease (urea aminohydrolase, EC 3.5.1.5) has been immobilized on o-alkylated nylon tubes in an enzymatically active form. The measured activity of the enzyme coil has been shown to be dependent on the flow rate of substrate through it. The apparent Km decreased and V increased as flow rates were increased. It is suggested that diffusional control is responsible for these observations. Their implications in flow-through immobilized enzyme analyses are discussed.
A purification procedure is described for Aspergillus urease, the most important step being affinity chromatography on hydroxyurea Sepharose. The enzyme exists as a single active species of Mr 240,000. The pure enzyme has an activity of 670 mumol urea hydrolysed/min, has a Km of 10(-3) M, an optimum pH of 8.5 and a sub-unit Mr of 40,000.
Biopsy and serum specimens were obtained from 95 patients undergoing endoscopy at the University of Zimbabwe Medical School. Common presenting features were epigastric pain, bleeding and dyspepsia. Ulcers were detected in 16 patients (17%), and were more common in men (24%) than in women (7%). Histological examination of biopsies showed that all 95 patients had spiral-shaped organisms that were indistinguishable microscopically from Helicobacter pylori, though the numbers of organisms varied considerably. There was evidence that the degree of inflammation in the mucosa was related to the numbers of H. pylori-like organisms (HPLO) present. Fifty-one biopsy specimens (55%) gave a positive rapid urease test (RUT), with colour change occurring within 4 h. In all but one case, the gastric mucosa from these patients contained moderate to numerous HPLO. We defined the 'gold standard' of H. pylori-associated gastritis as the presence of both moderate to numerous HPLO and moderate to severe inflammation in the gastric mucosa. Using these criteria, RUT had a sensitivity of 67% and a specificity of 68%. Sera from 92 patients were tested for immunoglobulin G antibodies reactive with a glycine-extract antigen of H. pylori, using an enzyme-linked immunosorbent assay (ELISA). Sera giving an indeterminate reaction in the ELISA were also tested by Western blotting. In all, 36 sera (39%) gave a positive ELISA or Western blot reaction. There was poor correlation between serology and RUT results, with only 57% of biopsy specimens from seropositive patients giving a positive RUT, compared with 45% from seronegative patients. Positive serology was found in only 35 patients (61%) with histological evidence of H. pylori-associated gastritis, and the specificity of the test was only 54%. When used in combination with the RUT result, however, 79% of patients with a positive RUT and positive serology had histological evidence of H. pylori-associated gastritis. There was a general trend for increased seroprevalence in patients with mild to moderate atypia. These findings indicate that serology, using an antigen derived from the type strain of H. pylori, is unreliable in detecting H. pylori infection in Zimbabwe. Current studies are aimed at characterizing antigens from organisms isolated from Zimbabwean patients.
Ammonia, released in the gastric mucosa by the action of Helicobacter pylori urease on transuded plasma urea, curtails the biosynthesis of mucus and/or causes the mucus to be disassembled at the mucosal surface. These changes facilitate colonisation by H pylori and may promote gastric ulcer formation.
Urease enzyme was immobilized in photographic gelatin by chemical cross-linking using formaldehyde, glutaraldehyde and chromium (III) acetate. The effects of enzyme and cross-linker concentrations, temperature, incubation time and pH on urea hydrolysis were investigated. Effect of reuse on the activity of immobilized enzyme was also studied. Glutaraldehyde (0.004 M) was the most suitable cross-linker; relative activities within 2.5 months after 24 reuses were stable (about 78%).
The indirect interactions between the carbonic anhydrase (CA) and urease (UR) are investigated in the present work using rate determinations detected by combined potentiometric measurements. It is shown that, in accord with the mass-action law for the two enzyme catalyzed reactions, the two enzymes assume a synergic pattern: the increase in the rate of removal of CO2 from the solution facilitated by CA increases the rate of production of NH3 consequent from urea dissociation. The experimental system which has been set up to monitor these interactions consists of a potentiometric apparatus to follow the gaseous exchanges of CO2 and NH3 which take place from a buffered solution containing both CA and UR. The results of the present work are consistent with, and add a further support to the finding of Dodgson and Forster, who first demonstrated in vivo the existence of an indirect linkage between urea production and CA catalytic activity.
Urea-sensitive sensors, based on immobilized urease and the differential antimony electrode, have been constructed. The calibration curve of the sensor is linear up to 1.6-2.0 mmol litre-1 in the stationary or kinetic mode of operation. The sensors retain 50% of their sensitivity for 16-17 days. The analyser for urea, constructed on the basis of the sensor, ensures quick and precise urea determination in pure blood.
Sodium saccharin, at concentrations similar to those in the urine of rats fed 1-5% sodium saccharin in their diet, markedly inhibited urease, and 3 proteases in vitro and sodium ion did not appear to play a role in enzyme inhibition. These observations suggest that enzyme inhibition of any of a large number of enzymes may play a role in the tumorigenesis of the urinary bladder by saccharin.
A monoclonal antibody, CP11, has been produced which is directed against the ureas of Campylobacter pylori. This antibody has been used to look for antigenic cross-reactivity, in other ureolytic and non-ureolytic campylobacters, by immunohistological techniques. It has also been used to investigate the helical-shaped organisms found in the stomach of the human, monkey and cat (CS1) and the ileum of the rat (ST1). Interestingly the antibody cross-reacted with the gastric helical organisms from the human, monkey and cat but not with the rat helical organism. No cross-reactivity was observed with C. mustelae or the other ureolytic campylobacters, C. nitrofigilis and the urease positive thermophilic campylobacters. These results are discussed in relation to the phylotaxonomy of these organisms.
We report the sequence of ureG, an accessory gene that is a part of the ure gene cluster of uropathogenic Proteus mirabilis and required for full enzymatic activity of urease. The 615-bp open reading frame predicts a M(r) 22,374 polypeptide, which contains a consensus amino acid (aa) sequence for ATP-binding. The polypeptide shares sequence homology with UreG of Escherichia coli (93% of identical aa), Klebsiella aerogenes (59%) and Helicobacter pylori (59%).
A cosmid gene library of chromosomal DNA from Yersinia enterocolitica A2635 (serogroup O:8) was constructed in Escherichia coli. Subcloning of a urease-positive (Ure+) clone revealed a region of 6.6 kb that was sufficient for expression of Ure activity in E. coli. Sequencing of this fragment disclosed seven ORFs transcribed in the same direction. On the basis of homology to known Ure, these were designated ureA, ureB, ureC, ureE, ureF, ureG and ureD, which are predicted to encode polypeptides of 11.1, 17.9, 61.0, 29.5, 25.0, 24.1 and 36.4 kDa, respectively. The polypeptides encoded by the ure gene complex of Y. enterocolitica are significantly divergent from those encoded by the ure operons of other Enterobacteriaceae, which appear to be closely related to each other. This suggests that the ure genes were acquired by Y. enterocolitica from an unrelated organism or alternatively, that they diverged from those of other Enterobacteriaceae some considerable time ago.
Eighty-five clinical urine samples and nineteen urine samples previously found by other laboratories to suggest genetic metabolic defects were prepared for trimethylsilylation by treatment with urease, followed by azeotropic dehydration. The "Target Analyte Search" program provided with the VG Trio 2 gas chromatograph-mass spectrometer required 6 min to quantify 103 compounds relative to endogenous urinary creatinine. This technique has been used to confirm diagnoses including cystinuria, lysinuria, medium-chain acyldehydrogenase deficiency, ornithine transcarbamylase deficiency, aspartylglucosaminuria, methylmalonic, propionic and glutaric acidurias.
Urease conjugated enzyme linked immunosorbent assays (ELISA) were developed for the detection of human IgM and IgG antibodies against Mycoplasma pneumoniae. Results obtained by ELISA were compared with complement fixation test (CFT); which showed that of the 214 serum specimens tested, 80 were found to have antibody against M. pneumoniae. ELISA revealed that 70 of these specimens were IgG antibody, and 27 of them also contain IgM antibody. CFT failed to detect the presence of antibody against M. pneumoniae in five serum specimens tested. However, by using ELISA, three of them were found to have IgG and IgM antibodies. and the other two sera have IgG antibody only. Four out of the five specimens tested were the first serum specimens collected from patients with clinical and serological evidence of M. pneumoniae infection. In addition, 28 serum specimens, including 10 sera containing IgM rheumatoid factors and sera known to contain IgM antibody to other infectious organisms, were also tested for IgM antibody against M. pneumoniae by ELISA. None of these specimens showed a nonspecific reaction. ELISA had a sensitivity of 87.5% and a specificity of 96.3% when compared with CFT. Thus, ELISA developed in our laboratory is a specific test, and the results indicated that IgM ELISA might be used as a rapid diagnosis for M. pneumoniae infection.
The tryptophan uptake into forebrain and brainstem was measured in urease-injected rats and in controls after prior application of sodium benzoate or hippurate. Benzoate led to an increased brain uptake index of tryptophan. This effect was additive to the increase in tryptophan uptake effected by the provoked hyperammonemia. In contrast, hippurate did not alter tryptophan uptake across the blood-brain barrier. We conclude that benzoate should be monitored especially when applied intravenously to patients with hyperammonemic coma.
Aggregation of jack bean urease (JBU) is associated with alterations of its biological properties, notably the ureolytic and entomotoxic activities. We investigated the influence of metals on protein oligomerization and biological properties. Besides protein aggregation, Cu(2+) induces inhibition of both ureolytic and insecticidal activities of JBU. Chemical modification of histidine residues in JBU with diethylpyrocarbonate (DEPC) decreases its affinity for Cu(2+) and inhibits oligomerization induced by this metal. Furthermore, this modification protects the insecticidal properties of JBU from being inactivated by Cu(2+). Although DEPC-treated JBU displayed lower ureolytic activity, the modified protein is less susceptible to inhibition by Cu(2+) when compared to native enzyme. Our findings show that Cu(2+) promotes JBU aggregation and differently of other heavy metals studied here, it apparently inhibits the ureolytic activity by inducing protein polymerization along with blockage of sulfhydryl groups.
An amperometric biosensor has been developed for the quantitative determination of urea in aqueous solution. The principle is based on the use of pH-sensitive redox active dissolved hematein molecule. The enzyme, urease (Urs), was covalently immobilized on a conducting copolymer poly (N-3-aminopropyl pyrrole-co-pyrrole) film, electrochemically prepared onto an indium-tin-oxide (ITO)-coated glass plate. The covalent linkage of enzyme and porous morphology of the polymer film lead to high enzyme loading and an increased lifetime stability of the enzyme electrode. Amperometric response was measured as a function of concentration of urea, at fixed bias voltage of 0.0 V vs. Ag/AgCl in a phosphate buffer (pH 7.0). The electrode gives a linear response range of 0.16-5.02 mM for urea in aqueous medium. The response time is 40 s reaching to a 95% steady-state current value, and 80% of the enzyme activity is retained for about 2 months.
Aggregation of jack bean urease (JBU) is involved in many alterations of its biological properties, notably the ureolytic and entomotoxic activities. In order to investigate this phenomenon, protein aggregates were characterized by dynamic (DLS) and static light scattering (SLS) spectroscopies through determination of apparent hydrodynamic radii, the average molecular masses, radii of gyration and second virial coefficients. No effect of disulfide reducing agents on protein association was observed contrasting with previous reports implicating their function in the prevention of JBU aggregation. The influence of freeze-thawing cycles on protein aggregation was also investigated. Our results showed that after freeze-thawing cycles the native form of JBU with apparent hydrodynamic radius of 7 nm and radius of gyration of 12 nm is replaced by high-order oligomers and this aggregation is not reverted neither by dithiothreitol (DTT) treatment nor by high concentration of salts. Altogether the data help to understand the complex behavior of JBU in solution and may correlate with the diversity of biological properties of this enzyme.