C. R. Carlini, Department of Biophysics, IB, and Graduate Program in Cellular and Molecular Biology, Center of Biotechnology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil, CEP 91.501-970. Fax: + 55 51 3316 7003, Tel.: + 55 51 3316 7606, E-mail: email@example.com
In this work we compared two plant ureases, jackbean urease (JBU) and embryo-specific soybean urease (SBU) and a bacterial (Bacillus pasteurii) urease, for kinetic parameters and other biological properties described recently for ureases that are independent of the ureolytic activity. The insecticidal effect of ureases was investigated in feeding trials with the cotton sucker bug, Dysdercus peruvianus (Hemiptera) as an insect model. Contrasting with B. pasteurii urease (PBU), both plant ureases presented potent insecticidal activity, with LD50 values of 0.017% (w/w) and 0.052% (w/w) for JBU and SBU, respectively. The insecticidal property of JBU or SBU was not affected by treatment with p-hydroxymercuribenzoate, an irreversible inhibitor of ureolytic activity of both proteins. Also, contrasting with canatoxin – a urease isoform from jackbean seeds that displays a toxic effect in mice (LD50 = 2 mg·kg−1) – no lethality was seen in mice injected intraperitoneally with JBU or SBU (20 mg·kg−1). Similarly to canatoxin, the three enzymes promoted aggregation of blood platelets (EC50 = 400.0 µg·mL−1, 22.2 µg·mL−1, 15.8 µg·mL−1 for BPU, SBU and JBU, respectively). This platelet activating property was also independent of urease activity. Comparison of the kinetic properties indicated that SBU is fivefold less susceptible than JBU to inhibition by acetohydroxamic acid, a chelator of Ni+2 and Zn+2 ions. The ureases also showed different susceptibility to agents that modify cysteine residues, such as p-hydroxymercuribenzoate and p-benzoquinone. Altogether, these data emphasize that biological properties that are independent of ureolytic activity are not restricted to jackbean ureases and that these proteins may have a role in plant defense against insect predators.
Ureases (urea amidohydrolase; EC 188.8.131.52) are nickel dependent enzymes  that catalyze the hydrolysis of urea to form ammonia and carbon dioxide. Ureases have been isolated from a wide variety of organisms including plants, fungi and bacteria . While fungal and plant (e.g. jackbean and soybean) ureases are homo-oligomeric proteins of ≈ 90 kDa subunits, bacterial ureases are multimers of two or three subunit complexes [3,4]. The UreA, UreB and UreC subunits of Bacillus pasteurii and most other bacterial ureases are colinear with the single subunit of fungal and plant ureases, the major difference being two gaps, between UreA and UreB and between UreB and UreC. Helicobacter pylori urease has two subunits, one being a fusion of UreA and UreB [2,3]. So far only bacterial ureases have had their 3D crystallographic structure successfully resolved, e.g. Klebsiella aerogenes (1FWJ), Bacillus pasteurii (4UBP) and Helicobacter pylori (1E9Z). However, the high sequence similarity of all ureases indicates they are variants of the same ancestral protein and are likely to possess similar tertiary structures and catalytic mechanisms .
Urease activity enables bacteria to use urea as a sole nitrogen source. Some bacterial ureases play an important role in the pathogenesis of human and animal diseases such as those from Proteus mirabilis and Helicobacter pylori.
Despite the abundance of urease in some plant tissues, e.g. seeds of members of the families Fabaceae (Leguminosae) and Curcubitaceae, and its ubiquity in virtually all plants [3,4], little has been revealed about its physiological roles. Soybean contains two distinct urease isoenzymes: an ubiquitous urease that is synthesized in all tissues examined and an embryo-specific urease that is confined to the developing embryo and is retained in the mature seed where its activity is roughly 1000-fold greater than that of the ubiquitous urease in many tissues [5,6]. One role of the ubiquitous urease, in recycling metabolically derived urea, has been demonstrated in a number of experimental conditions [4,7–9]. In spite of the high concentration of the protein in the seeds, it has been suggested that the embryo-specific urease plays no role in nitrogen assimilation from urea [4,7,10]. To our knowledge, no recent work has addressed the question of the physiological relevance of this highly active enzyme.
Recently, our group has shown that canatoxin, an isoform of jackbean urease consisting of a dimer of 95 kDa subunits, displays several biological properties independent of its ureolytic activity, such as activation of blood platelets and interaction with glycoconjugates [11–15]. Moreover, canatoxin is lethal to rats and mice when injected intraperitoneally (LD50 2.0 mg per kg body weight) and presents insecticidal activity when fed to some groups of insects, suggesting that ureases may be involved in plant defense [16–18]. The kissing bug Rhodnius prolixus, and three economically important crop pests, the cowpea weevil Callosobruchus maculatus, the Southern green soybean stinkbug Nezara viridula and the cotton stainer bug Dysdercus peruvianus are highly susceptible to the entomotoxic effect of canatoxin .
In order to investigate if ureases from other sources share, with jackbean ureases, the property of inducing biological effects not related to their ureolytic activity, we have tested soybean embryo-specific urease (SBU) and Bacillus pasteurii urease (BPU)  for their lethality in mice and for their insecticidal and platelet aggregating activities. Kinetic parameters and susceptibility of SBU and BPU to different inhibitors were also compared with those of the jackbean urease (JBU).
Material and methods
The protein content of samples was determined by their absorbance at 280 nm or, alternatively, by the method of Spector .
Bacillus pasteurii urease
A commercially available preparation of BPU (U-7127, Sigma Chemical Co.) was used in all experiments without further purification. The freeze-dried protein was resuspended in 20 mm sodium phosphate, pH 7.5, 1 mm EDTA, 2 mm 2-mercaptoethanol to give 0.5 mg protein per mL solutions.
Purification of jackbean urease
The jackbean enzyme was purified from jackbean meal based on the method of Blakeley et al.  with modifications. Dry seeds (Casa Agrodora, São Paulo, Brazil) were powdered and 50 g of defatted meal were extracted with buffer A (20 mm sodium phosphate, pH 7.5, 1 mm EDTA, 2 mm 2-mercaptoethanol) for 1 h at 4 °C. The meal was removed by centrifugation (30 000 g, 20 min, 4 °C), and 28% (v/v) ice-cold acetone (final concentration) was added to the supernatant. The suspension was kept at 4 °C overnight and the precipitated proteins were removed by centrifugation (30 000 g, 20 min, 4 °C). The concentration of acetone in the supernatant was then increased to 31.6% (v/v) and, after stirring at room temperature for 10 min, the precipitate was removed by centrifugation (30 000 g, 20 min, 4 °C). The supernant was dialysed against buffer B (20 mm sodium phosphate, pH 7.5, 1 mm EDTA, 5 mm 2-mercaptoethanol) and then mixed with 25 mL of Q-Sepharose resin (Amersham-Biotech Pharmacia) equilibrated in buffer B. After stirring in a beaker for 30 min in an ice bath, the mixture was filtered and the resin was washed with 100 mm NaCl in buffer B to remove the nonretained proteins. Elution of an urease-enriched fraction was achieved by adding 300 mm NaCl to buffer B. The active fraction was concentrated using a CentriPrep cartridge (Millipore). The urease-enriched material was then applied into a Superose 6 HR 10/30 gel filtration column (Amersham-Biotech Pharmacia) equilibrated in 20 mm sodium phosphate, pH 7.5, 1 mm EDTA, mounted in a FPLC system. The peak fraction containing urease activity was dialysed against 20 mm sodium phosphate, pH 7.0, 500 mm NaCl (buffer C) and then submitted to affinity chromatography on 10 mL of a Co+2 loaded iminodiacetic acid-Sepharose resin equilibrated in buffer C. Highly purified urease was recovered in the nonretained fraction (Fig. 1).
Purification of soybean urease
A new method for purifying soybean embryo-specific urease was developed based on the procedure of Blakeley et al. . Briefly, dry seeds of soybean (cultivar EM66, Crisciumal, RS, Brazil) were powdered and 25 g of defatted meal were extracted with buffer A for one hour at 4 °C. The meal was removed by centrifugation (30 000 g, 20 min, 4 °C), and 20% (v/v) ice-cold acetone (final concentration) was added to the supernatant. The suspension was kept at 4 °C for 2 h and the precipitated proteins were removed by centrifugation (30 000 g, 20 min, 4 °C). The supernatant was dialysed against buffer B and then mixed with 15 mL of Q-Sepharose resin (Amersham-Biotech Pharmacia) equilibrated in buffer B. After stirring in a beaker for 30 min, the mixture was filtered and the resin was washed with 150 mm NaCl in buffer B to remove the nonretained proteins. Elution of an urease-enriched fraction was achieved by adding 300 mm NaCl to buffer B. The gel filtration column and the affinity chromatography in immobilized Co+2 were performed as described for JBU. As for JBU, SBU did not bind to immobilized Co+2 in the affinity chromatography step. Purified SBU showed a major band in SDS/PAGE analysis (Fig. 1).
Electrophoresis in 10% polyacrylamide minigels containing 0.1% sodium dodecyl sulfate  were run at 20 mA for 2–3 h. The gels were stained with Coomassie Blue R-250.
Assay of biological activities of ureases
Toxic activity was expressed as LD50 and defined as lethality of mice within 24 h after intraperitoneal injection of single doses (20 mg·kg−1, equivalent to 10 LD50 of canatoxin) of the samples . Institutional (IB-UFRGS) protocols designed to minimise suffering and limit the number of animals killed, were followed throughout the experiments.
Platelet-rich plasma was prepared from rabbit blood collected from the ear central artery in the presence of sodium citrate to a final concentration of 0.313% (v/v). Blood samples were then centrifuged at 200 g for 20 min at room temperature, to give a platelet-rich plasma suspension . Platelet aggregation and shape change were monitored turbidimetrically , using a Lumi-Aggregometer apparatus (Chrono-Log Co., Havertown, PA, USA) and light transmission across the rabbit platelet-rich plasma suspension was registered on a chart recorder for 3 min. Platelet aggregation assays were also performed on a SpectraMax microplate reader (Molecular Devices, Sunnyvale, CA, USA) as described previously . The use of microplate assays has been shown to give results similar to those obtained with Born's aggregometry. Briefly, urease samples (previously dialysed against phosphate buffered saline) in 96-well flat-bottomed plates were prepared to a final volume of 50 µL with saline. Aggregation was triggered by the addition of 100 µL of platelet suspension. Controls were run by adding platelet-poor plasma. The plate was incubated for 2 min at 37 °C before commencing agitation, and readings were taken at 650 nm every 11 s for 20 min. Change in turbidity was measured in absorbance units and results are expressed as the area under the aggregation curves.
The insecticidal activity of ureases was evaluated in feeding trials with the cotton stainer bug Dysdercus peruvianus (Hemiptera), which is an economically important crop pest. Groups of 15 second instar insects (from a colony housed in this laboratory) were fed on cotton seed meal mixed with freeze-dried urease in a final protein concentration of 0.02–0.1% (w/w). For this, solutions of ureases were added to cotton seed meal, the mixtures were homogenized, freeze-dried, put inside gelatin capsules and then offered to the insects. Control insects fed on cotton seed meal containing equivalent volumes of freeze dried buffer A alone or containing 20 µmp-hydroxymercuribenzoate. For proteins treated with 50 µmp-HMB, excess reagent was removed by dialysis against buffer A prior to the bioassays. The insects were kept at 26 °C, 70–80% air humidity, 12-h dark : 12-h light cycle and examined every 2 days during 20 days for lethality, body weight and developmental stage (the insect goes through five instar stages before becoming adult). The results are mean and SEM of triplicates and expressed as survival rate and percentage of body weight of the control insects. LD50 values were calculated by linear regression of survival rates after 20 days plotted against five doses of the ureases tested in the feeding trials.
The ammonia released was measured colorimetrically . One unit of urease releases one µmol ammonia per minute, at 37 °C, pH 7.5. Kinetic parameters (Km and specific activity) were calculated as by Cleland . For inhibitory studies, the proteins were incubated with p-hydroxymercuribenzoate (p-HMB), acetohydroxamic acid (AHA) and p-benzoquinone (p-BQ), or the corresponding diluents, for 18–24 h at 4 °C.
Kinetic parameters and inhibitors of urea hydrolysis
Table 1 shows the kinetic parameters for the three ureases, JBU, SBU and BPU. Purified JBU and SBU displayed ureolytic specific activities at pH 7.5 of 22.2 ± 0.7 and 14.2 ± 0.6 U·mg−1, respectively. Susceptibility of the ureolytic activity to different inhibitors was also evaluated (Fig. 2, Table 1). The inhibitors tested were p-HMB and p-BQ, two cysteine-binding inhibitors, and AHA, a chelator of Ni+2 and Zn+2 ions. JBU was fivefold more susceptible to AHA than SBU. Although p-HMB and p-BQ have the same mechanism of action, different inhibition patterns were seen for JBU and SBU, two highly similar enzymes.
Table 1. Comparative data on physicochemical and biological properties of soybean embryo-specific urease (SBU), jackbean urease (JBU) and B. pasteurii urease (BPU). ND, not determined.
a Values of IC50 were taken from Fig. 2 and are expressed as mol of inhibitor per mol of enzyme.
Molecular mass, SDS/PAGE
11,13 and 61 kDa [2,3] (chains A, B and C, respectively)
As described for canatoxin [16,17], JBU and SBU were also highly toxic to the cotton stainer bug Dysdercus peruvianus in feeding trials, with calculated LD50 values of 0.017% and 0.052% (w/w) of protein added to the cotton meal, respectively (not shown). The time dependency of the entomotoxic effect was similar for both proteins, with a lag-phase of 3–4 days for death of the first insects, and reaching maximal lethality in about two weeks (Fig. 3). Contrasting to the plant ureases, Bacillus pasteurii urease was not toxic to the insects in the feeding trials at 0.1% (w/w) concentration (not shown). After treating JBU and SBU with p-HMB, an irreversible urease inhibitor, their insecticidal property was re-evaluated. The results showed that p-HMB-treated JBU or SBU maintained full toxic activity in the insect (Figs 3B,D and 4), while the enzymatic activity of the proteins was abolished (Fig. 4). Both plant ureases were detrimental for the development of the surviving insects, which showed decreased body weight and delayed progress through the instar stages (Fig. 5).
Similarly to both jackbean ureases, canatoxin  and JBU , SBU and BPU also induced aggregation of rabbit platelets (Fig. 6). EC50 for the platelet aggregation was estimated to be 400.0, 22.2 and 15.8 µg·mL−1, for BPU, SBU and JBU, respectively. As described for canatoxin and JBU , SBU was also still fully able to activate platelets after treatment with 50 µmp-HMB (Table 1).
Lethality in mice
Canatoxin is lethal to rats and mice (LD50 2 mg·kg−1 for mice), while JBU is not . Similarly to JBU, no signs of toxicity were seen after 7 days in animals injected intraperitoneally with 20 mg SBU per kg of body weight. BPU was not tested for intraperitoneal toxicity in mice.
Table 1 summarizes the data on kinetic parameters and biological activities of the three ureases analyzed in this work.
Despite their highly conserved structures and similar mechanisms of catalytic action, little is known about the physiological role of ureases in the source organisms.
The wide distribution of ureases in leguminous seeds as well as the accumulation pattern of the protein during seed maturation is suggestive of an important physiological role. As soybean mutants lacking the embryo-specific urease do not exhibit any of the abnormalities associated with loss of the ubiquitous urease, this enzyme probably has no essential physiological function . Studies with developing cotyledons of pea  and soybean seedlings [28,29] indicated that urease(s) play little or no role in embryo nutrition. The obvious question from this observation is why the developing soybean embryo would invest in a very active ureolytic protein when it never ‘sees’ urea.
Canatoxin, first isolated as a highly toxic protein  and identified recently as an isoform of jackbean urease , displays insecticidal activity against insects of Coleoptera (beetles) and Hemiptera (bugs) orders, such as the cowpea weevil, Callosobruchus maculatus, the kissing bug, Rhodnius prolixus, the cotton stainer bug, Dysdercus peruvianus and the green soybean stinkbug, Nezara viridula. The entomotoxic property of canatoxin is independent of its enzymatic activity and requires the proteolytic activation of the protein by insect cathepsin-like digestive enzymes in order to produce entomotoxic peptide(s) . The more abundant isoform of urease, here designated JBU, was previously shown to be as lethal as canatoxin in feeding trials with the kissing bug Rhodnius prolixus.
Here we have analyzed the insecticidal properties of three ureases, JBU, SBU and BPU, using the cotton stainer bug Dysdercus peruvianus as the insect model. Only the plant ureases were toxic in the feeding trials. JBU, with a LD50 of 0.017% (w/w) was as toxic as canatoxin , whereas both jackbean ureases are three-fold more potent than SBU, with a LD50 of 0.052% (w/w). Besides lethality, both ureases induced severe detrimental effects in surviving insects, reducing gain in body weight and delaying the developmental stages of nymphs into adults. The insecticidal effect of JBU and SBU was not altered after treating the proteins with p-HMB, clearly indicating that this feature is independent of their ureolytic activity (Figs 3 and 4). The lack of insecticidal activity of Bacillus pasteurii urease may be explained by its three-chain structure. Part of the region comprising the sequence of the entomotoxic peptide released from canatoxin (, patent pending) by insect cathepsins is absent in microbial ureases, corresponding in plant ureases to a fragment located between the UreB and UreC chains of Bacillus pasteurii urease. Altogether, our findings suggest that insecticidal activity is a characteristic of plant ureases and provide compelling evidence for a possible defense role of these proteins. Additional studies are under way in our laboratory to characterize and to study the mode of action of entomotoxic ureases in order to establish their biotechnological potential against phytophagous insects.
Contrasting with canatoxin, which is highly toxic in rats and mice , both JBU and SBU were not lethal to mice when given intraperitoneally (maximal dose tested 20 mg·kg−1). Thus, there is no correlation between the insecticidal activity of ureases and the intraperitoneal toxicity in mice, until now a property displayed only by canatoxin. It is plausible to think that this unique feature of canatoxin may be related to its dimeric form, as compared to the hexameric JBU and the embryo-specific SBU, making it more difficult for the larger proteins to be absorbed from the site of injection into the blood stream.
All three ureases studied here shared with canatoxin the ability of inducing activation of rabbit blood platelets [12–15]. JBU and SBU showed similar potency as inducers of platelet aggregation (Fig. 6), with EC50 = 22.2 and 15.8 µg·mL−1 for SBU and JBU, respectively. BPU, on the other hand, showed a 20-fold lower potency, with EC50 of 400 µg·mL−1. The time pattern of platelet response to the ureases was very similar, showing a collagen-type shape change reaction. As already described for canatoxin and JBU , this activity was retained in p-HMB treated SBU confirming it is independent of the enzymatic activity.
These newly described properties of plant and microbial ureases may shed new light on the physiological roles of these proteins in the source organisms. The involvement of plant ureases in the bioavailability of nitrogen is still controversial. Brodzik et al.  reported no significant alteration in the growth pattern of tobacco plants expressing Helicobacter pylori urease, which caused a two-fold increase in the ureolytic activity and an eight-fold increase in ammonia levels of the transgenic plants as compared to controls. However, these authors did not test the transgenic plants for their resistance to insects or phytopathogens. Polacco and Holland  have proposed that plant ureases may have a role in plant defense, assuming the released ammonia would have a deleterious effect upon predators. Altogether, our data reinforce the possibility of plant ureases having a protective role through an entirely different mechanism, unrelated to the release of ammonia.
We also compared the kinetic properties of these enzymes on the hydrolysis of urea and susceptibility to different urease inhibitors. Even the highly homologous JBU and SBU (86% identity and 92% similarity in a blast analysis) have different susceptibility to inhibition by p-HMB, AHA or p-BQ. Our data showed that JBU is fivefold more sensitive than SBU to inhibition by AHA, a Ni+2 and Zn+2 ions chelator , suggesting a different environment for the nickel atoms within the catalytic site. JBU and SBU also showed different susceptibility to two cysteine-reactive urease inhibitors, p-HMB [15,32] and p-BQ .
Taken together, our data show that ureases from plant and microbial sources belong to a group of multifunctional proteins with at least two distinctive domains: a thiol-dependent domain containing the ureolytic active site and a thiol-independent domain involved in toxic effects in insects (and mice, only for canatoxin) and the activation of blood platelets. Further elucidation of the 3D structures of plant enzymes should provide new insights for understanding the structural basis of the multiple biological effects displayed by ureases.
This work was supported by Conselho Nacional Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Ensino Superior (Procad-CAPES-MEC), Fundação de Amparo a Pesquisa do Estado do Rio Grande do Sul (FAPERGS) and Programa de Apoio a Núcleos de Excelência (MCT-FINEP-CNPq).