Canatoxin, a toxic protein from jack beans (Canavalia ensiformis), is a variant form of urease (EC 3.5.1.5): biological effects of urease independent of its ureolytic activity.

Canatoxin is a toxic protein from Canavalia ensiformis seeds, lethal to mice (LD(50)=2 mg/kg) and insects. Further characterization of canatoxin showed that its main native form (184 kDa) is a non-covalently linked dimer of a 95 kDa polypeptide containing zinc and nickel. Partial sequencing of internal peptides indicated homology with urease (EC 3.5.1.5) from the same seed. Canatoxin has approx. 30% of urease's activity for urea, and K(m) of 2-7 mM. The proteins differ in their affinities for metal ions and were separated by affinity chromatography on a Zn(2+) matrix. Similar to canatoxin, urease activates blood platelets and interacts with glycoconjugates. In contrast with canatoxin, no lethality was seen in mice injected with urease (10 mg/kg). Pretreatment with p-hydroxymercuribenzoate irreversibly abolished the ureolytic activity of both proteins. On the other hand, p-hydroxymercuribenzoate-treated canatoxin was still lethal to mice, and both treated proteins were fully active in promoting platelet aggregation and binding to glycoconjugates. Taken together, our data indicate that canatoxin is a variant form of urease. Moreover, we show for the first time that these proteins display several biological effects that are unrelated to their enzymic activity for urea.

Canatoxin-, concanavalin A- and canavalin-cross-reactive materials during maturation of Canavalia brasiliensis (Mart.) seeds.

The distribution of three cross-reactive materials (CRMs), a toxic protein analogous to canatoxin, CNTX-CRM, a lectin analogous to concanavalin A, Con A-CRM, and a major storage protein, canavalin-CRM, was investigated during successive stages of maturation of Canavalia brasiliensis Mart. seeds. The data obtained by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunological analyses indicated that these proteins share extensive homology with the analogous proteins found in Canavalia ensiformis seeds. The changes in CNTX-CRM and Con A-CRM levels throughout the maturation process were assayed by rocket immunoelectrophoresis. Synthesis of Con A-CRM was detectable at 30 days post-anthesis (DPA) while its hemagglutinating activity appeared only at 35 DPA. The CNTX-CRM was detected as a biologically active protein from 30 DPA onwards. The behavior of CNTX-CRM during maturation of C. brasiliensis seeds was quite distinct from that of Con A-CRM, pointing to different biological roles of these proteins in the seed.

The presence of concanavalin A and canatoxin in Canavalia ensiformis DC tissue culture.

Isolated embryos, cotyledons and embryos plusa fragment of cotyledon from seeds of Canavalia ensiformis (jack bean) were cultured in vitro. Concanavalin A and canatoxin cross-reactive material were detected by double immunodiffusion tests. Canatoxin was detectable until 30 days in cultures of embryos, embryos plus cotyledons and hypocotyls. Concanavalin A was also present in all cultures being detected until 90 days in cultures treated with 6-benzylaminopurine. No concanavalin A was detected in root cultures. Concanavalin A was present in cell suspensions until 45 days of culture; the culture medium contained neither concanavalin A nor canatoxin. Tissue cultures thus can produce Con A and CNTX and will be an important research tool for studying the biosynthesis of such substances.