A study of maturation events in jackbeans (Canavalia ensiformis).

Changes in cell volume and 42K+ efflux associated with concentrative alanine uptake were studied in isolated rat hepatocytes suspended in Krebs-Ringer bicarbonate buffer. After addition of 10 mM-alanine, cellular water volume increased by 15% and the rate constant of 42K+ efflux by 250%. Alanine-induced 42K+ efflux was abolished by quinine and was strongly decreased when the cell-volume increase was counteracted by sucrose. The results suggest that K+ efflux during alanine uptake is implicated in a volume-regulatory response.     

Inducible Formation of Urease in Canavalia ensiformis

Increase in urease activity in leaves of Canavdlia ensifomis has been demonstrated. The activity of excised leaves increased by about 100 percent when 1.5 × 10^?1M urea was added externally as inducer. Glycine-1-¹⁴C was used to investigate whether the increase in activity was dependent on de novo protein synthesis. The Incorporation of labeled amino acid into urease was twofold higher in induced samples than in non-induced ones. This indicates that the increase in activity is connected with de nova protein synthesis. The once increased activity was always followed by a rapid decrease. The urease activity was lost constantly with time after the external addition of ammonia in vivo. The inhibitory action of ammonia on urease fa vitro was eliminated by dialysis. Accordingly it may be concluded that the loss of activity was dependent on the product repression by ammonia.     

Binding of the Kunitz-type trypsin inhibitor DE-3 from Erythrina caffra seeds to serine proteinases: a comparative study.

The effect of pH and temperature on kinetic and thermodynamic parameters (i.e., k(on),k(off),Ka,delta G0, delta H0 and delta S0 values) for the binding of the Kunitz-type trypsin inhibitor DE-3 from Erythrina caffra seeds (ETI) to bovine beta-trypsin, bovine alpha-chymotrypsin, the human tissue plasminogen activator, human alpha-, beta- and gamma-thrombin, as well as the M(r) 33,000 and M(r) 54,000 species of the human urinary plasminogen activator (also named urokinase) has been investigated. At pH 8.0 and 21.0 degrees C: (i) values of the second-order rate constant (K(on)) for the proteinase:ETI complex formation vary between 8.7 x 10(5) and 1.4 x 10(7)/M/s; (ii) values of the dissociation rate constant (k(off)) for the proteinase: ETI complex destabilization range from 3.7 x 10(-5) to 1.4 x 10(-1)/s; and (iii) values of the association equilibrium constant (Ka) for the proteinase:ETI complexation change from < 1.0 x 10(4) to 3.8 x 10(11)/M. Thus, differences in k(off) values account mostly for the large changes in Ka values for ETI binding. The affinity of ETI for the serine proteinases considered can be arranged as follows: bovine beta-trypsin > human tissue plasminogen activator > bovine alpha-chymotrypsin > human alpha-, beta- and gamma-thrombin approximately M(r) 33,000 and M(r) 54,000 species of the human urinary plasminogen activator. Moreover, the serine proteinase:ETI complex formation is an endothermic, entropy-driven, process.(ABSTRACT TRUNCATED AT 250 WORDS)     

The alpha-mannosidase from Canavalia ensiformis seeds: chemical and kinetic properties and action on animal lymphocytes

The alpha-mannosidase from Canavalia ensiformis was characterized with respect to molecular mass, glycoprotein nature, amino-acid composition, enzymic properties and action on animal cells. The enzyme is composed of two pairs of subunits (molecular mass 44 and 66 kDa) which form a tetramer (220 kDa). The larger subunit is glycosylated, the smaller one is not. Both subunits have similar amino-acid compositions. The larger subunit contains a surplus of alanine, aspartic acid/asparagine, histidine, phenylalanine and tyrosine, the smaller one a surplus of glutamic acid/glutamine, serine and threonine. The enzyme is subject to product inhibition by mannose. It stimulates the proliferation of B-lymphocytes from nude mice.     

洋刀豆植物资源的综合开发利用

本文通过对洋刀豆的生物学特征以及其中的营养成分和药用成分的系统介绍 ,为其植物资源在食品及医药工业和农业生态环境治理中的综合开发利用提供参考     

Urease in jack-bean (Canavalia ensiformis (L.) DC) seeds is a cytosolic protein

Urease (EC 3.5.1.5) is abundantly present in the seeds of many species of Leguminosae. There is at present conflicting information in the literature about its subcellular location and status as a glycoprotein. We have made a study of the subcellular location of urease in jack-bean cotyledons using an immunocytochemical approach; in addition, we studied the biosynthesis and glycoprotein nature of the enzyme using several biochemical approaches. All the results are in agreement with the interpretation that the seed urease is not a glycoprotein, is synthesized on free polysomes, and is present in the cytosol of the storage parenchyma cells.Abbreviations ConA Concanavalin A - ER endoplasmic reticulum - IgG immunoglobulin G - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis On leave from Laboratoire de Photobiologie (CNRS-UA 203), Faculté des Sciences de Rouen, F-76130 Mont Saint Aignan, France     

Anti-inflammatory and anti-necrotic effects of lectins from Canavalia ensiformis and Canavalia brasiliensis in experimental acute pancreatitis

Glycoconjugate Journal - Lectins isolated from Canavalia ensiformis (ConA) and Canavalia brasiliensis (ConBr) are promising molecules to prevent cell death. Acute pancreatitis, characterized by...     

Stage-specific gut proteinases of the cotton stainer bug Dysdercus peruvianus: role in the release of entomotoxic peptides from Canavalia ensiformis urease

Canavalia ensiformis ureases are toxic to insects of different orders. The entomotoxicity of urease is due to a 10 kDa internal peptide released by proteinases in the insect digestive tract. We previously observed that, given orally, urease is toxic to nymphs of Dysdercus peruvianus, but does not affect adults. Here we characterized the major proteolytic activities of D. peruvianus midgut homogenates and investigated their in vitro-catalyzed release of the 10 kDa entomotoxic peptide from urease. Cysteine, aspartic and metalloproteinases are present in both homogenates. Variations in optimal pH and susceptibility to inhibitors indicated differences in the enzyme profiles in the two developmental stages. Only nymph homogenates released approximately 10 kDa fragment(s) from urease, recognized by antibodies against the entomotoxic peptide. Fluorogenic substrates containing urease partial sequences flanking the N-terminal or the C-terminal portion of the entomotoxic peptide were efficiently cleaved by homogenates from nymphs, but much more slowly by the adult homogenate. Different classes of enzymes in the homogenates cleaved both substrates suggesting that in vivo the release of the entomotoxic peptide results from the concerted action of at least two different proteinases. Our findings support the view that a differential processing of ingested urease by the insects explains at least in part the lack of toxicity in adults.     

Purification and characterization of a basic amino acid-specific peptidase from seeds of jack bean (Canavalia ensiformis)

A peptidase was purified from seeds of Canavalia ensiformis by extraction with water, ammonium sulfate precipitation, and successive chromatographies on DEAE-Toyopearl 650M, butyl-Toyopearl 650M, and G-3000 SW columns. The enzyme has an apparent molecular weight of 41,000. Activity is maximal at pH 9 and 60 degrees C. The enzyme hydrolyzed synthetic substrates at Arg-X and Lys-X bonds more rapidly than bovine trypsin did, and did not cleave protein or ester substrates. The enzyme was inhibited by alkylamines and several serine protease inhibitors such as diisopropylfluorophosphate, chymostatin, leupeptin, and benzamidine. Cysteine protease-, metalloprotease-, and proteinous trypsin inhibitors were ineffective. Inhibition by alkylamines was dependent on length of the alkyl chains. From the substrate specificity and susceptibility to chemicals, the enzyme is a unique peptidase with trypsin-like specificity.     

Renal alterations promoted by the lectins from Canavalia ensiformis (ConA) and Dioclea guianensis (DguiL) seeds

The lectin from the seeds of Canavalia ensiformis (ConA) and Dioclea guianensis (DguiL) was tested upon its renal effects using the isolated perfusion rat kidney method. Both lectins (10 microg/ml) affected perfusion pressure and renal vascular resistance, but DguiL showed a much greater action than ConA. However, ConA, but not DguiL, affected potassium tubular transport.     

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.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzylaminopurine - CNTX canatoxin - Con A concanavalin A - CRM cross-reactive material - DEAE-cellulose diethylaminoethyl-cellulose - IgG immunoglobulin G - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - Tris tris (hydroxymethyl) aminomethane     

Ammonia Assimilation in Canavalia ensiformis Plants under Water and Salt Stress

Nitrogen fixation and ammonia assimilation in nodules have beenthoroughly studied under stress conditions, but the behaviorof enzymes involved in ammonia assimilation to organic compoundsin plants of the Leguminosae family subjected to stress stillremains to be conclusively established. We found that understress conditions, C. ensiformis plants can switch from theirusual pathway of assimilation to an alternative one dependingon the nature of the stress and the tissue in which the processtakes place. In roots, it switches from the glutamate dehydrogenase(GDH) pathway to the glutamine synthetase (GS)/glutamate synthase(GOGAT) cycle under water stress but not under salt stress.However, in leaves under salt stress, GDH activity is maintainedbut GS activity markedly decreases (Received March 24, 1987; Accepted March 4, 1988)     

Interaction of the alpha-mannosidase from Canavalia ensiformis with the lectin from the same plant, concanavalin A

The specific interaction between the lectin concanavalin A and the alpha-mannosidase from the Leguminosa Canavalia ensiformis was studied by means of laser nephelometry and affinity chromatography. Both proteins react optimally within a certain stoichiometrical range. Interaction is restricted to a narrow pH interval (around pH 5) and to low ionic strengths (less than 10mM NaCl). Neither the sugar-binding site of the lectin nor the catalytic and the hydrophobic sites of the enzyme participate in the interaction. The conformation of the enzyme at pH 5 which favours the interaction can be arrested by immobilization. After this, the enzyme is able to bind the lectin even at pH 8 where no interaction takes place between the dissolved proteins.     

Multiple isotope effect study of the hydrolysis of formamide by urease from jack bean (Canavalia ensiformis)

Multiple kinetic isotope effects have been measured for the urease-catalyzed hydrolysis of formamide at pH 6.0 and 25 degrees C. These kinetic isotope effects include the carbonyl-C ((13)k = 1.0241 +/- 0.0009), the carbonyl-O ((18)k = 0.9960 +/- 0.0009), the formyl-H ((D)k = 0.95 +/- 0.01), the leaving-N ((15)k= 1.0327 +/- 0.0006), and the nucleophile-O ((18)k = 0.9778 +/- 0.0005). In addition, the enzyme does not catalyze the exchange of oxygen from the solvent into the carbonyl-O of formamide or the product, formate ion. The isotope effects are consistent with the rate-determining collapse of the tetrahedral intermediate (i.e., C-N bond cleavage). The pH optimum for formamide is at pH 5.3, whereas for urea, it is near 8.0. This is best accommodated by the mechanism proposed by Hausinger and Karplus, in which an active site cysteine binds to the nonleaving nitrogen in urea. For urea, the preference is for the anionic form of the sulfhydryl; for formamide, the neutral form is preferred, leading to the lower pH optimum.     

Expression and properties of arginyl-tRNA synthetase from jack bean (Canavalia ensiformis)

The coding region for arginyl-tRNA synthetase from jack bean (Canavalia ensiformis) has been sequenced and cloned into the bacterial expression vector pET32a. Transformation of BL21 cells and induction with IPTG results in the high level expression of the protein fused N-terminally with thioredoxin and bearing a His-tag. A substantial proportion of the enzyme is recovered in the soluble fraction of the cell lysate (10 mg per litre cell culture) and can be isolated with metal-affinity technology. The thioredoxin component and the His-tag portion of the fused protein could be removed with thrombin, resulting in a homogeneous product retaining an N-terminal extension of 3.2 kDa compared to the native arginyl-tRNA synthetase. Both full-length fusion and thrombin-treated products proved to be active in aminoacylation, with similar kinetic parameters.     

Identification and characterization of enamel proteinases isolated from developing enamel. Amelogeninolytic serine proteinases are associated with enamel maturation in pig.

During tooth formation nearly all of the protein matrix of enamel is removed before final mineralization. To study this process, enamel proteins and proteinases were extracted from pig enamel at different stages of tooth development. In the enamel maturation zones, the major enamel matrix proteins, the amelogenins, were rapidly processed and removed. Possibly associated with this process in vivo are two groups of proteinases which were identified in the enamel extracts by enzymography using amelogenin-substrate and gelatin-substrate polyacrylamide gels and by the degradation in vitro of guanidinium chloride-extracted amelogenins. One group of proteinases with gelatinolytic activity consisted of several neutral metalloendoproteinases having Mr values from 62,000 to 130,000. These proteinases were inactive against amelogenins, casein and albumin, and were present in approximately equal proportions in enamel at all developmental stages. In the other group, two serine proteinases, with apparent non-reduced Mr of 31,000 and 36,000 exhibited amelogeninolytic activity. The substrate preference of the enamel serine proteinases was indicated by their limited degradation of casein and their inability to degrade gelatin and albumin. Contrasting with the distribution of the metalloendoproteinase enzymes, the serine proteinases were found only in the enamel scrapings taken from late-maturing enamel. The amelogenin degradation patterns in vivo, observed in the enamel scrapings, were similar to those produced in assays in vitro using partially purified fractions of enamel proteinases and amelogenin substrate. Together, these data strongly indicate an important role for the serine proteinases, and possibly the gelatinolytic proteinases, in the organized processing of the enamel protein matrix during enamel formation.     

Formation of L-canavanine in in vitro cultures of Canavalia ensiformis (L.) DC.

In vitro tissue cultures of Canavalia ensiformis (L.) D.C. derived from hypocotyl have been obtained. They were found to accumulate L-canavanine depending on the medium where they were grown. Addition of polyethylenglycol (4%) to the culture medium led to a reduced accumulation of l-canavanine and an increase in the amino acids and the quaternary ammonium compounds contents.