Histochemical and biochemical urease localization in the periplasm and outer membrane of two Proteus mirabilis strains

Proteus mirabilis, a gram-negative bacillus, is often implicated in the formation of infectious kidney stones. As ureolytic activity of this organism is thought to play a major role in its pathogenesis, we adapted our recently described urease localization technique to visualize urease activity in vivo. Urease activity was ultrastructurally localized in two clinically isolated P. mirabilis strains by precipitating the enzymatic reaction product (ammonia) with sodium tetraphenylboron. Subsequent silver staining of the cells revealed urease activity to be predominantly associated with the periplasm and outer membranes of each strain. Biochemical measurements of urease activity in P. mirabilis cell fractions correlated well with histochemical observations in that the majority of urease activity was associated with the periplasm. Membrane-bound urease activity of these strains was associated mainly with the peptidoglycan in the detergent-insoluble (outer membrane) fraction.     

The effects of tetraphenylboron on energy-linked reactions in spinach chloroplasts

1. The inhibitory action of tetraphenylboron, a lipid-soluble anion, on the proton uptake, the photophosphorylation and the light-induced quenching of the fluorescence of 9-aminoacridine by spinach chloroplasts was studied.2. The inhibition of the three processes by tetraphenylboron was transient; the proton uptake was affected to a much smaller extent than either the photophosphorylation or the fluorescence quenching.3. The inhibitory effects of tetraphenylboron on the proton uptake and the fluorescence quenching of 9-aminoacridine were qualitatively the same in CF₁-depleted chloroplasts, that were recoupled with N,N′-dicyclohexylcarbodiimide (DCCD).4. The reversal of the fluorescence quenching of 9-aminoacridine upon addition of tetraphenylboron in the light was found to be very fast, being completed within the response time of the apparatus.5. The presence of tetraalkylammonium salts in the incubation medium prevented the inhibitory effect of tetraphenylboron.6. Tetraphenylboron disappeared from the chloroplast suspension in a light-dependent irreversible way; in the dark no ‘ptake’ of tetraphenylboron could be detected.7. The effects of tetraphenylboron may be explained by the presence of groups with a high affinity for tetraphenylboron in the membrane; these groups become protonated upon illumination of the chloroplasts.     

Isolation of choline esters from aqueous solutions by extraction with sodium tetraphenylboron in organic solvents

1. The method is based on the observation that choline esters and sodium tetraphenylboron (Kalignost) form complexes that are insoluble in water but soluble in organic solvents such as nitriles, higher ketones and benzyl alcohol. 2. The extraction procedure is an example of liquid cation exchange where tetraphenylboron is the cation-exchange group. 3. The proportion of choline esters extracted depends on the type and total amount of cation in the aqueous phase and the amount of sodium tetraphenylboron in the organic solvent. 4. The proportion of choline esters extracted is independent of the choline ester concentration, the pH (between 8 and 3) and the relative volumes of the two phases. 5. The affinity of sodium tetraphenylboron for choline esters increases with an increase in the size of the acyl group. 6. The choline ester extracted can be released into an aqueous solution by treatment with strong acids, silver salts and anion-exchange resins.     

Selective silver staining of urease activity in polyacrylamide gels

A selective method for staining urease activity bands in nondenaturing polyacrylamide gels is described. It is based on the deposition of silver at the urease bands after incubation of gels in the presence of urea and photographic developers. Its highly sensitivity (up to 0.015 enzyme units, corresponding to 5 ng of purified urease) is based on both the silver deposition enhancement methodology and the developers used. The selectivity of the procedure is based on the local pH increase catalytically produced by the enzyme in the presence of urea. The densitometric scan of the enzyme bands gives a linear response at least in the range 0.015-0.300 urease units. This selective staining method is about 2.5 times more sensitive than the standard silver staining of proteins, in terms of detectable urease amount.     

The self-association of jack bean urease and its modification by silver ions.

At low ionic strength urease has been found to dissociate at protein concentrations below 1 × 10⁸m. The inhibition of enzyme activity by Ag⁺ has been used to demonstrate this. The inhibition by Ag⁺ has been shown to be independent of dissociation but, at dilutions where dissociation occurs, silver ion modifies the process. Urease is aggregated by Ag⁺ at high Ag⁺:protein ratios. Such inactive aggregates can be solubilized and reactivated by dithiothreitol. Further evidence has been obtained indicating the similarity of the (8n) and (16n) forms of urease. The phenomena of inhibition and aggregation in the presence of the heavy metal ion have been shown to be separate processes.     

Genetic tests of the roles of the embryonic ureases of soybean

We assayed the in vivo activity of the ureases of soybean (Glycine max) embryos by genetically eliminating the abundant embryo-specific urease, the ubiquitous urease, or a background urease. Mutant embryos accumulated urea (250-fold over progenitor) only when lacking all three ureases and only when developed on plants lacking the ubiquitous urease. Thus, embryo urea is generated in maternal tissue where its accumulation is not mitigated by the background urease. However, the background urease can hydrolyze virtually all urea delivered to the developing embryo. Radicles of 2-day-old germinants accumulated urea in the presence or absence of the embryo-specific urease (2 micromoles per gram dry weight radicle). However, mutants lacking the ubiquitous urease exhibited increased accumulation of urea (to 4-5 micromoles urea per gram dry weight radicle). Thus, the ubiquitous and not the embryo-specific urease hydrolyzes urea generated during germination. In the absence of both of these ureases, the background urease activity (4% of ubiquitous urease) may hydrolyze most of the urea generated. A pleiotropic mutant lacking all urease accumulated 34 micromoles urea per gram dry weight radicle (increasing 2.5-fold at 3 days after germination). Urea (20 millimolar) was toxic to in vitro-cultured cotyledons which contained active embryo-specific urease. Cotyledons lacking the embryo-specific urease accumulated more protein when grown with urea than with no nitrogen source. Among cotyledons lacking the embryo-specific urease, fresh weight increases were virtually unchanged whether grown on urea or on no nitrogen and whether in the presence or absence of the ubiquitous urease. However, elimination of the ubiquitous urease reduced protein deposition on urea-N, and elimination of both the ubiquitous and background ureases further reduced urea-derived protein. The evidence is consistent with the lack of a role in urea hydrolysis for the embryo-specific urease in developing embryos or germinating seeds. Because the embryo-specific urease is deleterious to cotyledons cultured in vitro on urea-N, its role may be to hydrolyze urea in wounded or infected embryos, creating a hostile environment for pest or pathogen. While the ubiquitous urease is operative in leaves and in seedlings, all or most of its function can be assumed by the background urease in embryos and in seedlings.     

Acridine-based (thio)semicarbazones and hydrazones: Synthesis,in vitro urease inhibition,molecular docking and in-silico ADME evaluation

Urease is a bacterial enzyme that is responsible for virulence of various pathogenic bacteria such as Staphylococcus aureus, Proteus mirabilis, Klebsiella pneumoniae, Ureaplasma urealyticum, Helicobacter pylori and Mycobacterium tuberculosis. Increased urease activity aids in survival and colonization of pathogenic bacteria causing several disorders especially gastric ulceration. Hence, urease inhibitors are used for treatment of such diseases. In search of new molecules with better urease inhibitory activity, herein we report a series of acridine derived (thio)semicarbazones (4a-4e, 6a-6l) that were found to be active against urease enzyme. Molecular docking studies were carried out to better comprehend the preferential mode of binding of these compounds against urease enzyme. Docking against urease from pathogenic bacterium S. pasteurii was also carried out with favorable results. In silico ADME evaluation was done to determine drug likeness of synthesized compounds.     

Soybean Roots Retain the Seed Urease Isozyme Synthesized during Embryo Development

Roots of young soybean (Glycine max [L.] Merr.) plants (up to 25 days old) contain two distinct urease isozymes, which are separable by hydroxyapatite chromatography. These two urease species (URE1 and URE2) differ in: (a) electrophoretic mobility in native gels, (b) pH dependence, and (c) recognition by a monoclonal antibody specific for the seed (“embryo-specific”) urease. By these parameters root URE1 urease is similar to the abundant embryo-specific urease isozyme, while root URE2 resembles the “ubiquitous” urease which has previously been found in all soybean tissues examined (leaf, embryo, seed coat, and cultured cells). The embryo-specific and ubiquitous urease isozymes are products of the Eu1 and Eu4 structural genes, respectively. Roots of the eu1-sun/eu1-sun genotype, which lacks the embryo-specific urease (i.e. `seed urease-null'), contain no URE1 urease activity. Roots of eu4/eu4, which lacks ubiquitous urease, lack the URE2 (leaflike) urease activity. From these genetic and biochemical criteria, then, we conclude that URE1 and URE2 are the embryo-specific and ubiquitous ureases, respectively. Adventitious roots generated from cuttings of any urease genotype lack URE1 activity. In seedling roots the seedlike (URE1) activity declines during development. Roots of 3-week-old plants contain 5% of the total URE1 activity of the radicle of 4-day-old seedlings, which, in turn, has approximately the same urease activity level as the dormant embryonic axis. The embryo-specific urease incorporates label from [³⁵S]methionine during embryo development but not during germination, indicating that there is no de novo synthesis of the embryo-specific (URE1) urease in the germinating root. We conclude that the seedlike urease (URE1) found in roots of young soybean plants is a remnant of the Eu1-encoded, abundant, embryo-specific urease which accumulates in the embryonic root axis during seed development.     

Expression of urease does not affect the ability of Bordetella bronchiseptica to colonise and persist in the murine respiratory tract

To investigate the role played by urease during the Bordetella bronchiseptica infection process, the ability to colonise and persist in the mouse respiratory tract of a urease-negative B. bronchiseptica BB7865 and a BB7865 derivative constitutively expressing urease was compared with that of the wild-type strain. The results obtained showed that neither constitutive expression nor abolishment of urease activity had any significant effect on the course of B. bronchiseptica infection. Therefore, under our experimental conditions, urease is not essential for B. bronchiseptica to colonise and persist within the murine host.     

Soybean Leaf Urease: A Seed Enzyme?

The soybean (Glycine max L. [Merrill]) var Itachi has 0.2 to 0.3% the urease activity found in developing embryos of a normal line, Prize. The hydroxyurea sensitivity and pH preference of this basal seed urease indicate that it represents a unique enzyme rather than an unusually low level of the normal seed urease. Itachi's seed urease is less sensitive to hydroxyurea inhibition (65-80% inhibition) than Prize seed urease (85-95% inhibition) and is more active at pH 6.1 and 8.8 than at 7.4, whereas the normal seed urease is least active at pH 8.8. Both properties of the basal seed urease are in agreement with the behavior of the leaf urease in extracts of Prize and Itachi leaves.

Neither the leaf urease nor the Itachi seed urease is immuneprecipitated by affinity-purified seed urease antibodies. However, when antibody is in excess, Staphylococcus aureus (Cowan) cell walls containing protein A can precipitate soluble antibody-urease complexes (47-68% of total enzyme) from both leaf (Itachi and Prize) and Itachi seed extracts. Under identical conditions, greater than 90% of Prize seed urease is precipitated. At a 100-fold dilution of antibody, 60% of Prize seed urease is still antibody-complexed while the antibody recognition of the leaf or Itachi seed urease is reduced to 2 to 24%.

The cell culture urease also resembles leaf urease by the criteria of pH preference, hydroxyurea sensitivity, and recognition by seed urease antibodies. In the presence of cycloheximide, nickel stimulates cell culture urease levels (14- or 35-fold depending on assay pH) indicating that cell cultures make a preponderance of apourease under nickel-limiting conditions.

Inasmuch as the ureases of leaf, cell culture, and Itachi seeds are more closely related to each other than they are to the abundant (Prize) seed urease, suggests that the three tissues either contain an identical urease or related tissue-specific isozymes. This second form of urease may have an assimilatory role since it is found in both leaf and seed sink tissues and is required for urea assimilation in cell culture (Polacco 1977 Plant Physiol 59: 827-830).

     

A rapid head-space method for ethyl alcohol determination in saliva samples

At neutral pH, tetraphenylboron dissolved in 3-heptanone extracts acetylcholine but not acetylcarnitine from aqucous media. At acid pH, both acetylcholine and acetylcarnitine are extracted. A pH curve of the efficiency of extraction of acetylcholine and acetylearnitine is given and the method of chemical synthesis of [1-acetyl-¹⁴C]carnitine is deseribed.     

A soybean seed urease-null produces urease in cell culture

Itachi, a soybean (Glycine max [L.] Merr.) variety with 0.2% normal seed urease activity, was recovered from a screen of 6,000 entries in the United States Department of Agriculture soybean germplasm collection. No urease antigen in Itachi seed extracts was detected by double diffusion or by rocket immunoelectrophoresis. Native gels stained for protein or ureolytic activity revealed no detectable urease holoenzyme. An anti-urease antibody affinity column was used to remove all detectable urease activity and antigen from `wild type' (cv. Prize) seed extracts. Affinity column effluent and nonchromatographed Itachi extracts both lack a species which comigrates with purified urease subunits in sodium dodecylsulfate polyacrylamide gels. Inability to detect urease antigen or urease protein suggests that during development of Itachi seeds there is no synthesis of urease protein or that, at most, its synthesis is 0.2% of wild type (Prize).     

Targeting Helicobacter pylori urease activity and maturation: In-cell high-throughput approach for drug discovery

Background

Helicobacter pylori is a bacterium strongly associated with gastric cancer. It thrives in the acidic environment of the gastric niche of large portions of the human population using a unique adaptive mechanism that involves the catalytic activity of the nickel-dependent enzyme urease. Targeting urease represents a key strategy for drug design and H. pylori eradication.

Immunological features and efficacy of a multi-epitope vaccine CTB-UE against H. pylori in BALB/c mice model

Epitope vaccine is a promising option for prophylactic and therapeutic vaccination against Helicobacter pylori infection. Urease is an essential virulence factor and colonization factor for H. pylori. In this study, we constructed a multi-epitope vaccine named CTB-UE with mucosal adjuvant cholera toxin B subunit (CTB) and tandem copies of Th and B cell epitopes from H. pylori urease A and B subunits. The immunogenicity, specificity, ability to induce neutralizing antibodies against H. pylori urease, and prophylactic and therapeutic efficacy of the CTB-UE vaccine were evaluated in BALB/c mice model after purification. The experimental results indicated that CTB-UE could induce comparatively high levels of specific antibodies against native H. pylori urease, UreA, UreB, or the selected B cell epitopes UreA_183–203 and UreB_327–334 involved with the active site of urease and showed an effectively inhibitory effect on the enzymatic activity of urease. Besides, oral prophylactic or therapeutic immunization with CTB-UE significantly decreased H. pylori colonization compared with oral immunization with rUreB or PBS, and the protection was correlated with antigen-specific CD4⁺ T cells and IgG, IgA, and mucosal sIgA antibody responses. This CTB-UE vaccine may be a promising vaccine candidate for the control of H. pylori infection.     

The genetics and biochemistry of urease in Ustilago violacea

Two complementing loci in different linkage groups of the basidiomycete Ustilago violacea are involved in urease activity: a structural one (ure-1) and a second inferred to involve a permease (ure-2) locus. Two types of complementing mutations occur in the structural locus: null activity (ure-1a) and obviously reduced activity (ure-1b). The ure-2 mutants lacked urease activity in vivo on the phenol red-urea test medium, but gave extracts with wild-type activity. Extracts from wild-type strains gave one site of urease activity after polyacrylamide gel electrophoresis. A number of ure-1b mutants and active revertants from ure-1a mutants yielded electrophoretically variant urease sites. The results are discussed in terms of enzyme polymorphism in haploid eukaryotes by one (missense) or two (null, then missense) mutations.     

Soybean (Glycine max) urease: Significance of sulfhydryl groups in urea catalysis

The soybean urease (urea amidohydrolase; EC 3.5.1.5) was investigated to elucidate the presence of sulfhydryl (–SH) groups and their significance in urea catalysis with the help of various –SH group specific reagents. The native urease incubated with 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) showed exponential increase in the absorbance, thereby revealing the presence of –SH groups. A total of 34 –SH groups per hexamer enzyme molecule were estimated from the absorption studies which represents nearly six –SH groups per subunit. The time-dependent inactivation of urease with DTNB, p-chloromercuribenzoate (p-CMB), N-ethylmaleimide (NEM) and iodoacetamide (IAM) showed biphasic kinetics, where half of the enzyme activity was lost more rapidly than the other half. This study reveals the presence of two categories of “accessible” –SH groups, one category being more reactive than the other. The inactivation of urease by p-CMB was largely reversed on treatment with cysteine, which might be due to unblocking of –SH group by mercaptide exchange reaction. Finally, when NEM inactivated urease was incubated with sodium fluoride, a time-dependent regain of activity was observed with higher concentrations of fluoride ion.     

Production of large quantities of 32P-labeled RNA tumor virus nucleic acid.

A rapid thin-layer chromatographic method on Gelman I.T.L.C. type SA plates for the determination of choline acetylase activity has been developed. The mobile phase consists of sodium tetraphenylboron dissolved in acetonitrile. The R_f values for acetyl coenzyme A and acetate are about 0.1, while that for acetylcholine is about 0.9. The method compares favorably with other radiometric methods as far as recovery, separation, and simplicity are concerned.     

Edwardsiella ictaluri Encodes an Acid-Activated Urease That Is Required for Intracellular Replication in Channel Catfish (Ictalurus punctatus) Macrophages

Genomic analysis indicated that Edwardsiella ictaluri encodes a putative urease pathogenicity island containing the products of nine open reading frames, including urea and ammonium transporters. In vitro studies with wild-type E. ictaluri and a ureG::kan urease mutant strain indicated that E. ictaluri is significantly tolerant of acid conditions (pH 3.0) but that urease activity is not required for acid tolerance. Growth studies demonstrated that E. ictaluri is unable to grow at pH 5 in the absence of urea but is able to elevate the environmental pH from pH 5 to pH 7 and grow when exogenous urea is available. Substantial production of ammonia was observed for wild-type E. ictaluri in vitro in the presence of urea at low pH, and optimal activity occurred at pH 2 to 3. No ammonia production was detected for the urease mutant. Proteomic analysis with two-dimensional gel electrophoresis indicated that urease proteins are expressed at both pH 5 and pH 7, although urease activity is detectable only at pH 5. Urease was not required for initial invasion of catfish but was required for subsequent proliferation and virulence. Urease was not required for initial uptake or survival in head kidney-derived macrophages but was required for intracellular replication. Intracellular replication of wild-type E. ictaluri was significantly enhanced when urea was present, indicating that urease plays an important role in intracellular survival and replication, possibly through neutralization of the acidic environment of the phagosome.Identification of virulence factors is vitally important to an understanding of the pathogenesis of Edwardsiella ictaluri and to the development of methods for controlling the spread of disease. Although the pathogenesis of E. ictaluri was reviewed in 1993 (28, 31), recent reports demonstrated that E. ictaluri is a facultative intracellular pathogen (3) and that a type III secretion system is required for intracellular survival and replication within channel catfish head kidney-derived macrophages (HKDM) (30). Using signature-tagged mutagenesis (STM) in an immersion challenge model for E. ictaluri, Thune et al. (30) identified 50 transconjugants carrying transposon insertions in genes required for survival and replication in the channel catfish host. Two of those mutants had insertions in genes encoding homologs of UreG and UreF, proteins that are essential for the production of an active urease enzyme in other bacteria (6, 10, 14, 26). UreG is a GTP-binding accessory protein that functions in energy-dependent assembly of the urease holoenzyme (19), while UreF is a urease accessory protein that functions in the generation or delivery of carbon dioxide to the urease metallocenter assembly site (19). Both the ureG and ureF mutant strains were further characterized in a competitive challenge with the wild-type (WT) parental strain and were confirmed to be significantly attenuated (30). The identification of two mutants with insertions in urease-associated genes suggests an important role for urease activity in E. ictaluri pathogenesis, despite the fact that E. ictaluri is urease negative in standard biochemical tests. Consequently, the objectives of this study are to characterize the E. ictaluri urease pathogenicity island (PAI), to evaluate conditions for E. ictaluri urease activity, and to establish a possible role for urease in E. ictaluri pathogenesis.