Ureases display biological effects independent of enzymatic activity: Is there a connection to diseases caused by urease-producing bacteria?

Ureases are enzymes from plants, fungi and bacteria that catalyze the hydrolysis of urea to form ammonia and carbon dioxide. While fungal and plant ureases are homo-oligomers of 90-kDa subunits, bacterial ureases are multimers of two or three subunit complexes. We showed that some isoforms of jack bean urease, canatoxin and the classical urease, bind to glycoconjugates and induce platelet aggregation. Canatoxin also promotes release of histamine from mast cells, insulin from pancreatic cells and neurotransmitters from brain synaptosomes. In vivo it induces rat paw edema and neutrophil chemotaxis. These effects are independent of ureolytic activity and require activation of eicosanoid metabolism and calcium channels. Helicobacter pylori, a Gram-negative bacterium that colonizes the human stomach mucosa, causes gastric ulcers and cancer by a mechanism that is not understood. H. pylori produces factors that damage gastric epithelial cells, such as the vacuolating cytotoxin VacA, the cytotoxin-associated protein CagA, and a urease (up to 10 percent of bacterial protein) that neutralizes the acidic medium permitting its survival in the stomach. H. pylori whole cells or extracts of its water-soluble proteins promote inflammation, activate neutrophils and induce the release of cytokines. In this paper we review data from the literature suggesting that H. pylori urease displays many of the biological activities observed for jack bean ureases and show that bacterial ureases have a secretagogue effect modulated by eicosanoid metabolites through lipoxygenase pathways. These findings could be relevant to the elucidation of the role of urease in the pathogenesis of the gastrointestinal disease caused by H. pylori.

Partial purification and some physicochemical properties of phospholipases A2 from the venom of the bushmaster snake (Lachesis muta)

Screening of the biochemical-pharmacological properties of the crude venom from the snake Lachesis muta indicated the presence of phospholipase A2 (PLA2; 5260 U/mg protein), procoagulant (2630 U/mg protein), platelet aggregating (43 U/mg protein) and caseinolytic activities (6670 U/mg protein). These activities were separated by filtration of the crude venom on Sephacryl S-200. The material containing PLA2 activity was further fractioned by DEAE-cellulose ion exchange chromatography into four active fractions (F-I to F-IV, containing 1.7, 1.2, 0.3, and 0.05 per cent of the crude venom protein, respectively) by stepwise elution with buffers of increasing ionic strength. All fractions presented a molecular weight of approximately 15,000 and isoelectric points in the range pH 4.6-6.0. In addition to their indirect hemolytic activity, the partially purified fractions inhibited platelet aggregation induced either by collagen or thrombin. p-Bromophenacyl bromide-treated fractions lost both phospholipase A2 activity and their inhibitory effect on collagen-induced platelet aggregation

Purification, physicochemical characterization and N-terminal-amino acid sequence of a phospholipase A2 from Bothrops jararaca venom

Snake venoms usually contain multiple molecular forms of phospholipase A2 enzymes (phosphatide acyl hydrolase, E.C. 3.1.1.4; PLA2). Phospholipases A2 induce a wide range of pharmacological effects which may depend or not on the hydrolysis of phospholipids. In this study, a PLA2 from Bothrops jararaca venom was purified to homogeneity by gel filtration on a Sephacryl S-200 column, followed by FPLC reverse-phase chromatography on a Pep-RPC HR 5/5 column (yield 1.63 per cent of venom protein). The PLA2 activity of the fractions was determined by indirect hemolysis using hen's egg yolk lecithin as substrate. The enzyme is an acidic protein with PI 4.5 and an apparent molecular weight of 14,200, as estimated by gel filtration on a Superose 12 FPLC column. Similar properties have been described for PLA2 from other snake venoms. The N-terminal-sequence of the purified protein was NLMQFETMIMXXAGQ. These partial sequence data show a high degree of homology between the B. jararaca PLA2 and the enzymes from other snake venoms as well as bovine pancreatic PLA2

Canatoxin induces activation on mice peritoneal macrophages

Canatoxin (CNTX), the toxic protein from Canavalia ensiformis seeds, injected into the peritoneal cavities of mice (10 micrograms/cavity) induced a significant neutrophil migration (10.5 +/- 0.5 x 10(6) cells/cavity) after 4 h. A later migratory effect (48 h) on mononuclear cells, predominantly macrophages, was also observed (controls: 7 +/- 0.9; CNTX: 17 +/- 2.0 x 10(6) cells/cavity). These CNTX-elicited macrophages, when compared to resident cells (R) or cells elicited by thioglycollate (TG), had an increased content of the lysosomal enzyme N-acetyl-beta-D-glucosaminidase (R: 4.5 +/- 0.5; TG: 7.2 +/- 1.0; CNTX: 20.2 +/- 3.0 mU/10(6) cells) and a greater (> or = 100%) phagocytic activity. The data suggest that CNTX-stimulated macrophages presented some characteristics of activated cells

Alterations in rat carbohydrate metabolism induced by canatoxin as a probable consequence of primary hypoxia

Canatoxin, a protein displaying lipoxygenase-activating properties isolated from Canavalia ensiformis seeds, induces hypoxia and hyperglycemia in male rats. Liver glycogen, blood glucose and lactate levesls were measured in male and female rats after canatoxin (50 mU, iv) injection. Increased levels of serum glutamic oxaloacetic transaminase activity were used as an indicator of hepatic injury. There was no sex-related difference observable during canatoxin-induced hypoxia but male and female rats did whow different patterns of metabolic change and hepatic injury after toxin observed in male rats while female rats showed only hypoglycemia and glycogenolysis. Pretreatment of male rats with either glucose, diazepam or hexamethonium abolished both the hypoxia and hepatic injury and the metabolic alterations produced by toxin injection. The results suggest that the metabolic alterations and hepatic injury detected after canatoxin injection may be a consequence of primary hypoxia

Convulsions induced by canatoxin in rats are probably a consequence of hypoxia

Canatoxin, a convulsant neurotoxin from the seeds of Canavalia ensiformis, induces lipoxygenase-dependent hypoxia and sex-related alterations of carbohydrate metabolism in rats which are blocked by glucose, diazepam and hexamethonium. The present study analyzes the possible causal relationship between the convulsant action of canatoxin and its effects on carbohydrate metabolism. The incidence of canatoxin-induced convulsions was greater in male than in female rats. Pretreatment of male rats with drugs that block hypoxia, such as glucose (2.5 g/kg,iv,15 min), diazepam (5 mg/kg,ip, at 48 h, 24 animals against convulsions, respiratory distress and death. These results suggest that canatoxin/induced convulsions are probably the consequence of hypoxia and both effects are mediated by lipoxygenase activation

Effect of canatoxin on the circulating levels of gonadotropins and prolactin in rats

1. The effects of canatoxin, the toxic principle from Canavalia ensiformis seeds which has lipoxygenase-activating properties, were evaluated in rats using radioimmunoassay techiniques to measure plasma levels of prolactin (PRL), progesterone, follicle stimulating (LH) and luteinizing (LH) hormones. 2. The chronic administration of canatoxin (50, 100 or 200 microng/Kg daily for 12 days, ip) to female rats induced a sharp rise in plasma LH and FSH concentrations whit no changes in progesterone level. A fall in circulating PRL was also observed. The frequency of proestrus and weight gain increased in rats treated with the higest dose of toxin used, but there was no alteration in weight of uterus or ovaries. 3. The increases in gonadotropin levels with canatoxin are consistent with the lipoxygenase-activating properties of the toxin, but do not explain why plasma PRL concentrations decreased in canatoxin-treated rats. 4. Since the animals in the control group had high PRL and low LH levels and since canatoxin increased LH and decreased PRL in the circulation, a possible stress-prevention effect is discussed for the toxin. 5. This study supports previous suggestions of central actions for canatoxin, and indicates the hypophysis and/or hypothalamus as one of the target sites for the toxin in the central nervous system

The secretory effect of canatoxina ou rat brain synaptosomes involves a lipoxygenase-mediated pathway

Canatoxin (CNTX), a neurotoxic protein, is known to activate platelet secretion and aggregation in vitro through a lipoxygenase-dependent pathway. This study shows that CNTX also induces time and dose-dependent serotonin secretion from rat brain synaptosomes. The secretory effect induced by 6 micronM CNTX was similar to that elicited by 150 mM KCl. Nordihyderoguaiaretic acid (500 micronM) completely abolished CNTX-induced serotonin release while 150 micronM indomethacin had no effect. These data suggest the involvement of the lipoxygenase pathway in neurotransmitter release elicited by CNTX as occurs in the platelet

Biochemical and pharmacological screening of snake (Bothrops) venoms: characterization of components acting on blood coagulation and platelet aggregation

Six venoms from snakes of the genus Bothrops were tested for coagulation, platelet aggregation and phospholipase A2 (PLA2) activity. Almost all showed pro-coagulant and PLA2 activities while pro-aggregating properties were found only for some venoms. Bothrops jararaca venom showed different protein peaks associated with these activities. The pro-aggregating activity was inhibited by EDTA, leupeptin and mepacrine while the PLA2 activity was blocked by p-bromophenacyl bromide and 2-mercaptoethanol. Venom screening tests for clotting and platelet aggregation may represent a valuable tool for snake taxonomy and for monitoring the quality of antisera