Soybean (Glycine max (L) Merril) seeds are known to contain different proteins displaying antinutritional and/or toxic effects, such as soybean agglutinin (an N-acetylgalactosamine-specific lectin), proteinase inhibitors (Kunitz-and Bowman-Birk-type trypsin and chymotrypsin inhibitors) and urease (seed and tissue isoforms). Two other toxic proteins were previously isolated from soybeans, soyatoxin (21 kDa) and soybean toxin (18.4 kDa), which are immunologically related to canatoxin, a toxic protein from Canavalia ensiformis (jackbean) seeds. In this work we have screened crude extracts from seeds of six different soybean cultivars, which together represent most of the crop harvested in the southern region of Brazil, for the presence of urease, trypsin inhibitory and haemagglutination activities, intraperitoneal toxicity in mice and immunoreactivity against anti-canatoxin antibodies. Significant differences were found in the contents of proteinase inhibitors, lectin, urease activity and lethality in mice. The relevance of these findings to the agronomic qualities and to the choice of soybean cultivars to be used as food or feed is discussed.
Ureases, nickel-dependent enzymes that catalyze the hydrolysis of urea into ammonia and bicarbonate, are widespread in plants, bacteria, and fungi. Previously, we cloned a cDNA encoding a Canavalia ensiformis urease isoform named JBURE-II, corresponding to a putative smaller urease protein (78kDa) when compared to other plant ureases. Aiming to produce the recombinant protein, we obtained jbure-IIb, with different 3' and 5' ends, encoding a 90kDa urease. Three peptides unique to the JBURE-II/-IIb protein were detected by mass spectrometry in seed extracts, indicating that jbure-II/-IIb is a functional gene. Comparative modeling indicates that JBURE-IIb urease has an overall shape almost identical to C. ensiformis major urease JBURE-I with all residues critical for urease activity. The cDNA was cloned into the pET101 vector and the recombinant protein was produced in Escherichia coli. The JBURE-IIb protein, although enzymatically inactive presumably due to the absence of Ni atoms in its active site, impaired the growth of a phytopathogenic fungus and showed entomotoxic properties, inhibiting diuresis of Rhodnius prolixus isolated Malpighian tubules, in concentrations similar to those reported for JBURE-I and canatoxin. The antifungal and entomotoxic properties of the recombinant JBURE-IIb apourease are consistent with a protective role of ureases in plants.
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