Jack Bean Seeds Research Articles

Early feeding experience in chicks and piglets: effect of social factors

Young animals learn feeding through trial and error processes and from social models, especially the parental one (i.e. Suboski and Bartshumas, 1984). Under practical conditions the mother-young bond has been reduced by early weaning in piglets or deleted in artificially hatched chicks. In both cases feeding experiences develop in groups of congeners of the same age. Negative reinforcements of feeding experiences have been demonstrated with diets containing concanavalin A from jackbean seeds (Canavalia ensiformis) (Leon et al., 1991). Preliminary trials suggested that piglets react similarly to jackbean containing diets. The aim of the present experiment was to investigate the capacity of piglets and chicks to influence the feeding behaviour of conspecifics according to their previous feeding experience (negative with concanavalin A, or neutral) and their social reactivity.

Isolation and Analysis of cDNA Encoding a Precursor of Canavalia ensiformis Asparaginyl Endopeptidase (Legumain)

Recently, asparaginyl endopeptidase was purified to homogeneity from jack bean (Canavalia ensiformis) seeds, and its NH2-terminal amino acid sequence was determined for 25 residues [Abe, Y. et al. (1993) J. Biol. Chem. 268, 3525-3529]. On the basis of this sequence information, we searched for seed cDNAs encoding this enzyme. Seven clones were obtained and sequenced. By combining four of them, we obtained a cDNA for a precursor of the enzyme containing the reported NH2-terminal sequence. The other three clones seemed to be for precursors of its isozymes. When the deduced amino acid sequences of these enzyme precursors were compared with those in the GeneBank, EMBL, and NBRF databases, only one protein was found with a homologous sequence. It was a precursor of hemoglobinase from a blood fluke (Schistosoma mansoni). Significant homology was observed only in the range of sequence for the mature form. Although hemoglobinase and asparaginyl endopeptidase behave as cysteine proteinases, their protein natures are distinct from either papain-type proteinases or clostripain. Various plant seeds have been reported to contain asparaginyl endopeptidases. This may be the first report, however, that deals with the primary structure of such a proteinase.

Polyphenols, Phytate, Cyanogenic Glycosides, and Trypsin Inhibitor Activity of Several Philippine Indigenous Food Legumes

The levels of antinutritional and toxic factors were determined in several Philippine indigenous legumes, namely, hyacinth bean (Dolichos lablab L.) lima bean (Phaseolus lunatus L.), sabawel or velvet bean (Mucuna curanii), sam-samping (Clitoria ternatea), rice bean (Vigna umbellata), jackbean (Canavalia ensiformis), and swordbean (Canavalia gladiata). Low levels of condensed tannins (0–2.48 mg catechin /g) and protein-precipitable polyphenols (0.16–0.77 mg tannic acid/gram) were observed in raw mature seeds of the six legumes analyzed in this preliminary study. Seeds of hyacinth bean, jackbean, and lima bean had relatively high levels of phytate phosphorus ranging from 6.6 to 11.6 mg of phytate P/g of sample. Lower values ( <5) were obtained for sabawel and swordbean as well as some varieties of mungbean used as controls. The indigenous legumes analyzed had low levels of less than 50 μg/g of cyanide in the seeds, immature and mature leaves, and immature pods using two methods of analysis. Among the legumes investigated, hyacinth bean had the highest trypsin inhibitor activity ranging from 14 to 27 units/mg sample for four accessions. Accession 6-1 of sabawel had 20 units/mg. Sword bean and jack bean had low levels of 1 to 5 units/mg except for one jack bean accession (A-1) which had 9 units/mg. Rice bean also had low levels of trypsin inhibitor activity, 5–7 units/mg.

42] Legumain: Asparaginyl endopeptidase

Publisher Summary This chapter describes asparaginyl endopeptidase from jack bean ( C. ensiformis ). During the characterization and purification of legumain from jack bean seeds, DNP-Pro-GIu-Ala-Asn-Val-Ile-Arg-NH 2 (DNP, dinitrophenyl) and DNP-Pro-GIu-Ala-Asn-NH 2 , which contain a sequence similar to that around one of the processing sites in proconcanavalin A, are used as substrates. The presence of the DNP group with absorption near 370 nm allows easy monitoring of separation between an enzymatic reaction product, DNP-Pro-Glu-Ala-Asn, and either of the substrates by high-performance liquid chromatography (HPLC), even when the enzyme in a crude extract of seeds is assayed. The addition of 2-mercaptoethanol to the incubation mixture is essential to yield the full activity. The final preparation of jack bean legumain obtained after the second gel-permeation chromatography on G3000SW gives a single peak in HPLC on a reversed-phase column and a single protein band with mobility corresponding to a molecular mass of about 37,000 Da on sodium dodecyl sulfate (SDS)-polyacrylamide gel after electrophoresis under reducing conditions. The inhibition profile of jack bean legumain suggests that the enzyme is a cysteine-type endopeptidase.

Asparaginyl endopeptidase of jack bean seeds. Purification, characterization, and high utility in protein sequence analysis.

Asparaginyl endopeptidase was highly purified from mature seeds of the jack bean (Canavalia ensiformis). The final enzyme preparation showed a single peak in high-performance liquid chromatography on a reversed-phase column, and the material in the peak gave the following NH2-terminal amino acid sequence on Edman degradation for 25 cycles: H-Glu-Val-Gly-Thr-Arg-Trp-Ala-Val-Leu-Val-Ala-Gly-Ser-Asn-Gly-Tyr-Gly-Asn-Tyr- Arg-His-Gln-Ala-Asp-Val-. Behavior of the enzyme toward various protease inhibitors suggested that it belongs to a family of cysteine proteases. Strict substrate specificity of this enzyme was verified by the use of 14 polypeptide substrates including those derived from proteins. Almost all the peptide bonds on the carboxyl side of Asn residues were susceptible to the enzyme. The exceptions were cases where the residue was at the NH2 terminus or the second position from the NH2 terminus of substrates and where it was N-glycosylated Asn. Peptide bonds on the carboxyl side of any other amino acid residues were not cleaved. These properties promise the high utility of this novel endopeptidase in protein sequence analysis. Identity of jack bean asparaginyl endopeptidase with a processing enzyme responsible for maturation of concanavalin A from its precursor is also discussed.

Preliminary crystallographic studies of urease from jack bean and from Klebsiella aerogenes

Ureases from both jack bean ( Canavalia ensiformis) seeds and Klebsiella aerogenes have been crystallized by the hanging drop method. The plant-derived urease crystals are regular octahedra analogous to those obtained by Sumner. Preliminary X-ray diffraction studies show that the crystals belong to the cubic space group F4 132, with a = 364 A ̊ , and appear to contain one or two subunits in the asymmetric unit. Using a synchrotron source, the crystals diffract to near 3·5 Å resolution. Crystals of urease from K. aerogenes belong to the cubic space group I23 or I2 13, with a = 170·8 A ̊ and appear to contain a single catalytic unit per asymmetric unit. The crystals diffract to better than 2·0 Å resolution and are well suited for structural analysis.

A novel protease from jack bean seeds: Asparaginyl endopeptidase

A novel protease from jack bean seeds: Asparaginyl endopeptidase

Pharmacological characterization of rat paw edema induced by canatoxin, the toxic protein from Canavalia ensiformis (jack bean) seeds

Intraplantar injection of canatoxin (CNTX) induced a dose-dependent rat hind-paw edema which was distinguished by two phases. In the first phase, observed between 0 and 2 hr after CNTX injection, there was an increase in paw volume, with no apparent involvement of inflammatory phagocytic cells. The second phase, beginning at about the 3rd hr, was characterized by an intense cellular infiltration at the site of administration which was coincident with a further increase in paw swelling. The edema was maximum at 6 hr after injection and disappeared within 48 hr at doses of 50–100 μg, while at doses of 200–300 μg edema was present in excess of 48 hr. The pharmacological studies suggested that CNTX-induced edema is a multi-mediated phenomenon. Histamine, serotonin, PAF and prostaglandins are likely involved in the first phase. Lipoxygenase metabolites, probably leukotrienes, seem to play a major role in the second phase, and may account for the development of cellular infiltration in the inflammatory site.

Effect of concanavalin A from jackbean seeds on short-term food intake regulation in chicks and laying hens

A balanced diet containing 30% jackbean seed meal induces a 50% reduction in food intake in young chicks (7 or 14 days old) after a delay of 30–45 min from the onset of normal consumption. This reduction in food intake, which is the major cause of growth depression in chicks fed on jackbean (JB), is consistent (14 experiments) and can be used as a routine procedure for testing various fractions of JB seeds. Concanavalin A (Con A) was isolated and identified as the major cause of this early reduction in food intake. Dose-response curves show a linear decrease of food intake up to 0.85% of Con A in the diet. From 0.85 to 2.55% of Con A in the diet, the response is less marked. This response is much faster (a few minutes) in adult hens which have been previously exposed to JB than in naive birds (1 h). After repeated distribution, the diet containing JB is probably recognised by cephalic organs (smell or taste), although the initial effect of Con A on food intake is probably independent of taste or smell control. A single oral administration of purified Con A significantly affects the intake of a regular diet for > 6 h. Addition of D (+) mannose partly counteracts the effect of Con A on food intake, both in young chicks and adult hens. The effect of Con A on the initial stage of food intake in chicks is probably not initially associated with taste or smell detection; it is associated with the haemagglutinating capacity of the diet and is dependent on the carbohydrate-binding capacity of this lectin, perhaps involving the glycosylated proteins and lipids of the gut cells.

A jack bean protein similar to pea seed ferritin and unrelated to concanavalin A is the only iron storage protein in jack bean seeds, although concanavalin A can form ferritin-like iron cores in vitro

Iron storage in living organisms is performed by ferritins. These proteins are built up from 24 subunits organized in a spherical shell forming a coat to a core of bioavailable iron. Recently, it was observed that concanavalin A from jack bean can form in vitro iron cores similar to those of animal ferritins (Yariv, J., Kalb, A. J., Helliwell, J. R., Papiz, M. Z., Bauminger, E. R., and Nowik, I. (1988) J. Biol. Chem. 263, 13508-13510). From this observation and from the comparison of the three-dimensional structures of horse spleen ferritin and of the form of concanavalin A which forms soluble polynuclear iron complexes, these authors suggested that concanavalin A is the apoferritin of jack bean seeds. On the basis of immunological and biochemical results, we report here that a protein purified from jack bean seeds, unrelated to concanavalin A and similar to plant seed ferritins, is responsible for iron storage in jack bean seeds. Furthermore, concanavalin A does not contain iron in vivo. Therefore we conclude that a unique protein, ferritin, stores iron in vivo in jack bean seeds and that concanavalin A provides an unusual model for studying the formation of iron cores inside a protein shell.

Subtilisin inhibitors in Canavalia and Vicia faba seeds. A comparative study

AbstractSubtilisin isoinhibitors (SI) were isolated from jack bean (Canavalia ensiformis L) and broad bean (Vicia faba L) seeds. Jack beans contain three isoinhibitors (pI 6.6, 6.3 and 6.0) that constitute 0.021 g per 100 g of dry seeds, while the two active proteins from broad beans (pI 5.7 and 5.1) represent 0.028%. The molecular weight, determined by gel filtration, is around 8000 D in both legumes. Large variations in specific activity of SI against subtilisin Carlsberg were detected in six species of the Canavalia genus, whereas only slight changes were found among six cultivars of C ensiformis. Antibodies raised against SI isolated from jack beans are specific for SI from different varieties of this and other species of the Canavalia genus. However, they do not recognise SI from broad beans and other legume seeds tested. In broad beans the variability between cultivars is significant. Cv Canaria has two active bands and four are detected in the ‚dark’︁ variety, which is 2.7 times less active than the former. SI are specific towards microbial serine proteases, showing high affinity for proteinase K. No appreciable activity against animal proteases or plant thiol enzymes can be detected. SI are also inactive against 4 alpha‐amylases of different origins. Reversible limited proteolysis of the reactive site bond suggests that SI interact with subtilisin by the ‚standard mechanism’︁ usually accepted for the inhibition of trypsin by its proteinaceous inhibitors.

Effects of heat treatments and germination on trypsin inhibitor activity and polyphenols in jack bean (Canavalia ensiformis L. DC).

The application of dry heat to seeds and meal was not effective in inactivating the TI and reducing the polyphenol content. Soaking for 24 h followed by cooking for 20 min, was equally effective in destroying the TI activity. Germination of jack bean seeds for 40 h decreased the levels of TI and polyphenols by 31% and 35%, respectively.

Blood glucose alterations induced in rats by canatoxin, a protein isolated from jack bean ( Canavalia ensiformis) seeds

Canatoxin, a lethal convulsant protein isolated from the seeds of Canatoxin ensiformis (jack bean), induced a biphasic alteration in blood glucose levels when injected i.v. into rats and mice. After administration of subconvulsant doses of canatoxin the rats showed initially hyperglycemia during the first 30 min, followed by a long-lasting hypoglycemia. Return to basal glucose levels was not seen up to 15 days. The hyperglycemic effect was dose-dependent and occurred in starved and well-fed animals. A parallelism between convulsant and hyperglycemic activity was observed throughout all canatoxin purification steps. Mice were about 20-fold less sensitive (on a weight basis) than rats to canatoxin-induced hyperglycemia. The hyperglycemic phase was not affected by pretreatment of the rats with alphaor beta-adrenergic blockers or chlorpromazine, but was potentiated by reserpine and haloperidol. Diazepam and hexamethonium were able to block the hyperglymic phase of canatoxin's effect. The results suggest that the hyperglycemic alterations induced by canatoxin in rats and mice are probably mediated via the central nervous system.

A concanavalin A-like lectin in the developing seed of Canavalia ensiformis

During the early stages of seed development (Stage A) in Canavalia ensiformis (jackbean), the presence of an α-d-mannoside specific lectin(s) is shown by hemagglutination assays, although the usual subunit of concanavalin A (Mr 26 000), is not detectable by SDS-polyacrylamide gel electrophoresis. By employing affinity chromatography on Sephadex G-50 we have identified a Con A-like lectin with specificity to α-d-mannoside and α-d-glucoside in the seeds at early stages of development. Its native Mr has been estimated by gel filtration to be ca 122 000 with subunits of ca 28 500 as determined by SDS-polyacrylamide gel electrophoresis. It accounts for the hemagglutinating activity in the immature seeds of jackbean. In the later stage of seed development (Stage B), the high hemagglutination titre values are consistent with the presence of significant amounts of both this Con A-like lectin and Con A, whereas the equally high titre values in the dry mature seeds (Stage C) may be attributable essentially to Con A since the Mr, 28 500 polypeptide is present only as a very minor component. Immunological and peptide mapping studies reveal a very close structural relationship between this lectin and Con A. These similarities and the inverse relationship in the relative abundance of these two proteins during different stages of seed development indicate an apparent precursor-product relationship between these two lectins.

Foliage spray tests with L-canavanine for control ofSpodoptera litura

Ricinus communis (castor) leaves were sprayed with the non-protein amino acid L-canavanine fromCanavalia ensiformis (jack bean) seeds at various concentrations ranging from 50 to 1000 ppm and fed to larvae ofSpodoptera litura F. Larval growth was observed until pupation, or for a maximum of 30 days. Growth of 1st instar larvae was retarded at the ⩳200 ppm level. Feeding treated food to older larvae (early 3rd instar, 50–60 mg body weight) at the 1000 ppm level reduced growth rate significantly, but not at 500 ppm.

Effect of different sources of urease on the treatment time and digestibility of urea-ammonia treated rice straw

Chopped (2–3 cm) rice straw (var. H 4) was treated with urea solution (4 g/100 g dry matter in 100 ml water) alone or with a source containing urease enzyme. Five sources containing urease were used: soya bean seeds, watermelon seeds, pumpkin seeds, jack bean seeds and winged bean seeds. Each of the sources was included at 3 levels: 2, 4 or 8 g/100 g treated-straw dry matter. Untreated straw and those treated with urea solution alone for periods up to 21 days were used as controls. The urease sources, their levels, treatment time and their interactions produced significant ( P < 0·01) effects. Inclusion of a urease reduced the treatment time to less than 5 days and at the same time produced IVO MD values similar to 14 to 21 days treatment without urease. Untreated straw had an IVO MD of 40%.

Polypeptide ligation occurs during post-translational modification of concanavalin A.

Lectins are proteins with multivalent carbohydrate-binding sites, which confer the ability to agglutinate. The seeds of legumes are particularly rich in lectins, for example, concanavalin A (Con A) comprises up to 15% of the protein in the cotyledons of jack bean (Canavalia ensiformis) seeds. The amino acid sequences of Con A and several other legume lectins have been partially or fully determined, and comparison of these sequences from different species reveals a circular homology (Fig. 1A); rearrangements within the genome have been suggested to explain this. We report here that the circular homology displayed by Con A is due to a post-translational transposition and ligation within the initial polypeptide. This type of modification has not been reported previously for eukaryotes, although it has been suggested to occur in bacteriophage lambda.

Alteration of quaternary structure and biological activity of concanavalin A

A major molecular species of concanavalin A (Con A), a mitogenic lectin from jack bean seeds, has a quaternary structure composed of four homologous subunits and a tetravalent sugar-binding ability. We show that the tetrameric Con A can be converted into a monovalent monomeric form by either photochemical alkylation or hydrogen peroxide/dioxane oxidation of about two tryptophan residues. A divalent dimeric derivative of Con A is also prepared by sulfomethylamidation of about four carboxyl groups. Chemical properties and mitogenic and hemagglutinating activities of these new Con A derivates are compared with those of the tetravalent Con A, as well as of the Con A derivatives that have appeared in the literature on cell biological studies. The significance of the lectin valences in lymphocyte activation and hemagglutination is also discussed.

Urease from leaves of Glycine max and Zea mays

Extracts of Glycine max and Zea mays leaves catalysed the release of 14CO 2 from [ 14C] urea with multiple pH maxima (5.5 and 9.0 for G. max; 5.5, 7.5 and 8.8 for Z. mays). Evidence was obtained that the principal activities, at pH 5.5 and 8.8–9.0 catalysed the same reaction stoichiometry as did urease purified from jackbean seeds (EC 3.5.1.5). The urease activities with these pH optima were not resolved by ammonium sulphate fractionation, DEAE-cellulose chromatography, or gel filtration chromatography. Many structural analogues of urea inhibited leaf urease, the most effective being amino acid hydroxamates, hydroxyurea and selenourea. Allantoic acid and ureidoglycolate are probably not alternative substrates because they showed at most only weak competitive inhibition with respect to radioactive urea.

Interaction of Agrobacterium with potato lectin and concanavalin A and its effect on tumor induction in potato

Fluorescent-labeled concanavalin A, a d-glucose- and d-mannose-specific lectin from jackbean seeds, adhered to cells of Agrobacterium tumefaciens strains B6M and NT-1 (B6) (oncogenic on potato) and NT- I (nononcogenic on potato), but not to cells of 19 other Agrobacterium strains. The mean number of radioiodinated concanavalin A molecules bound per bacterial cell was 27 600 for B6M, 30 000 for NT-1 (B6) and 50 600 for NT-1. Concanavalin A precipitated lipopolysaccharides from strains B6M, NT-1, 2R and B695 (intact cells of the latter 2 strains did not bind the lectin). Lipopolysaccharides from strains B6 and C-58 and extracellular polysaccharides from all 6 strains were not precipitated by the lectin. When supplied at 3 × 10 8 molecules per bacterial cell, concanavalin A specifically reduced the number of tumors produced on potato by strain B6M by about 25%, but had no significant effect on the tumorigenicity of strain B6. Potato lectin, which is specific for oligomers of N-acetyl- d-glucosamine and was purified from potato tubers, did not adhere to cells of any of the 22 Agrobacterium strains. The lectin did not precipitate lipopolysaccharides or extracellular polysaccharides from 6 strains, and it did not influence the ability of strain B6 to produce tumors on potato tuber discs.