A study of maturation events in jackbeans (Canavalia ensiformis).
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ABSTRACT:
SUBMITTER: Marcus SE
PROVIDER: S-EPMC1144169 | biostudies-other | 1984 Aug
REPOSITORIES: biostudies-other
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Project description:Plant urease is a seed protein that is common in most legumes. It is also common in many bacteria and fungi and several species of yeast. Urease allows organisms to use exogenous and internally generated urea as a nitrogen source by catalyzing the hydrolysis of urea to ammonia and carbon dioxide. Urease from jack bean meal was purified to electrophoretic homogeneity using a series of steps involving acetone precipitation and size-exclusion and ion-exchange chromatography. The jack bean urease was crystallized and the resulting crystals diffracted to 2.05 A resolution using synchrotron radiation. The crystals belonged to the hexagonal space group P6(3)22, with unit-cell parameters a = b = 138.57, c = 198.36 A.
| S-EPMC2795609 | biostudies-literature
Project description:Although the intake of jack bean (Canavalia ensiformis (L.) DC.), an underutilized tropical legume, can potentially decrease the risk of several chronic diseases, not much effort has been directed at profiling the polyphenolics contained therein. Hence, this work aimed to identify and quantify the dominant jack bean polyphenolics, which are believed to have antioxidant and other bioactivities. Four major compounds were detected and identified as kaempferol glycosides with three or four glycoside units. Their structures were established based on UV-visible, 1d, 2D NMR, and HR-ESI-MS analyses. Specifically, kaempferol 3-O-a-l-rhamnopyranosyl (1®6)- b-d-glucopyranosyl (1®2)-b-d-galactopyranosyl-7-O-[3-O-o-anisoyl]-a-l-rhamnopyranoside was detected for the first time, while the other three compounds have already been described in plants other than jack bean. This new compound was found to have a higher a-glucosidase inhibition activity compared to acarbose.
| S-EPMC7321285 | biostudies-literature
Project description:Serine proteases has been vastly reported to be found (and identified) only in seed extracts of Canavalia ensiformis (C. ensiformis), however, our group observed expressive serine protease activities in aqueous extracts from leaves, seeds, roots, and stem, especially in a leaf extract obtained with distillated water (CE-A). Additionally, this extract demonstrated very good stability in high temperatures and in the presence of chemical agents. In order to study the serine protease activity in leaves, CE-A was submitted to benzamidine-sepharose affinity column and an enriched fraction of serine proteases, named CE-ABza was purified 1.65 fold, yielding a total recovery of about 62%. Based upon zymographic analysis in a gelatin-SDS-PAGE-, the CE-ABza fraction presented an enzymatic activity band at approximately 90 kDa molecular mass region under non-reducing conditions and three bands at 17, 32 and also at 90 kDa under reducing condition. This fraction was able to hydrolyze, in a different way, peptidomimetic and protein substrates. The maximal activity was observed at basic pH values (8.0 to 9.5) and temperature about 40°C. These enzymes exhibited important thermal stability and were significantly inhibited by serine protease inhibitors, such as benzamidine and TPCK. Calcium, magnesium, manganese and zinc ions had a negative modulation on the CE-ABza activity. Mass spectrometry-based proteomics approach identified showed homology with 11 plant proteases from different legumes species, indicating their presence or demonstrating that CE-Bza proteases share specific region of the primary structure of theses enzyme, although the C. ensiformis genome and protein databases are yet to be publicly available. These results demonstrated that these molecules found in both the CE-A extract and the CE-ABza fraction presented important properties that could be used as potential pharmacological and biotechnological targets as therapeutic option. Besides, identification and characterization of C. ensiformis proteases contribute to the studies of plant enzymes.
2020-08-28 | PXD010569 | Pride
Project description:1. Argininosuccinate lyase (EC 4.3.2.1) from jack bean [Canavalia ensiformis (L.) DC] seeds was purified 532-fold from an acetone-butanol-dried powder. 2. The enzyme functions reversibly and exhibits maximum stability at 16 degrees . 3. At 16 degrees it has a half-life (t((1/2))) of 263min. 4. The enzyme is both cold-labile (t((1/2)) 131min. at 0 degrees ) and heat-inactivated (t((1/2)) 74min. at 38 degrees ); inactivation appears to be irreversible. 5. Treatment of the acetone-butanol-extracted powder with sodium dodecyl sulphate increased the sensitivity of the enzyme to temperature (t((1/2)) 70min. at 0 degrees ; t((1/2)) 23min. at 38 degrees ). 6. Addition, to the purified enzyme, of a fraction containing lipid from the seed increased the half-life to about 510min. at either 0 degrees or 38 degrees . 7. Arginine or homoarginine, and to a smaller extent some other amino acids or fumarate, protected the enzyme from cold-inactivation. 8. Reactivation attempts with both the cold- and heat-inactivated enzyme failed. 9. The K(m) value for argininosuccinate at pH7.5 is 1.3x10(-4). 10. The enzyme was inactivated completely within 15min. at 16 degrees by 0.5mm-p-hydroxymercuribenzoate, and subsequent exposure to 5mm-cysteine had no restorative effect.
| S-EPMC1187728 | biostudies-other
Project description:Removal of the N-glycan from the concanavalin A (Con A) glycoprotein precursor is a key step in its conversion into an active lectin. N-Glycanase (EC 3.5.1.52), the enzyme from jackbean catalysing this process, has been purified to homogeneity as judged by native PAGE. One of the purification steps is binding of the enzymic activity to Con A-Sepharose and its elution by methyl alpha-mannoside. On SDS/PAGE the principal components were found to be 78 kDa, 74 kDa, 54 kDa, 32 kDa and 30 kDa polypeptides. These did not react with Con A on an affinity blot. Cleveland mapping indicated that some of these polypeptides had related primary structures. The enzyme has a broad pH optimum in the region of 5.0.
| S-EPMC1219101 | biostudies-other
Project description:BackgroundCanavalia ensiformis is a legume native to Central and South America that has historically been a source of protein. Its main proteins, urease, and lectin have been extensively studied and are examples of bioactive compounds. In this work, the effect of pH and light effects on the growth of C. ensiformis were analyzed. Also, the bioactive compounds such as phenols, carotenoids, chlorophyll a/b, and the growth of callus biomass of C. ensiformis from the effect of different types of light treatments (red, blue and mixture) were evaluated. Likewise, the antioxidative activity of C. ensiformis extracts were studied and related to the production of bioactive compounds. For this, a culture of calluses obtained from seeds were carried out. For the light experiments, polypropylene boxes with red, blue, combination (1/3, 3/1 and 1/1 R-B, respectively) lights and white LED were used as control. In each treatment, three glass containers with 25 ml of MS salts containing 0.25 g of fresh callus were seeded.ResultsThe results have shown that the pH of the culture medium notably affects the increase in callogenic biomass. It shows that the pH of 5.5 shows better results in the callogenic growth of C. ensiformis with an average increase of 1.3051 g (198.04%), regarding the initial weight. It was found that the pH 5.5 and the 1/3 R-B LED combination had higher production of bioactive compounds and better antioxidant activity. At the same time, the red-light treatment was least effective.ConclusionsIt was possible to find the ideal conditions of important growth under conditions of pH and light of C. ensiformis. Likewise, it is evaluated whether the production of compounds of interest, such as phenolic compounds and carotenoids, occurs under these conditions. The highest production of calluses occurs in the 1/3 R-B LED combined light treatment, which showed a significant increase in biomass, followed by B. From this study, it could be demonstrated that C. ensiformis produces compounds such as phenols and carotenoids in vitro culture that are essential for the antioxidant activity of the plant.
| S-EPMC7488093 | biostudies-literature
Project description: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.
| S-EPMC1222220 | biostudies-other
Project description:Endosomes are intracellular vesicles that mediate the communication of the cell with its extracellular environment. They are an essential part of the cell's machinery regulating intracellular trafficking via the endocytic pathway. Many viruses, which in order to replicate require a host cell, attach themselves to the cellular membrane; an event which usually initiates uptake of a viral particle through the endocytic pathway. In this way viruses hijack endosomes for their journey towards intracellular sites of replication and avoid degradation without host detection by escaping the endosomal compartment. Recent experimental techniques have defined the role of endosomal maturation in the ability of enveloped viruses to release their genetic material into the cytoplasm. Endosome maturation depends on a family of small hydrolase enzymes (or GTPases) called Rab proteins, arranged on the cytoplasmic surface of its membrane. Here, we model endosomes as intracellular compartments described by two variables (its levels of active Rab5 and Rab7 proteins) and which can undergo coagulation (or fusion) and fragmentation (or fission). The key element in our approach is the "per-cell endosomal distribution" and its dynamical (Boltzmann) equation. The Boltzmann equation allows us to derive the dynamics of the total number of endosomes in a cell, as well as the mean and the standard deviation of its active Rab5 and Rab7 levels. We compare our mathematical results with experiments of Dengue viral escape from endosomes. The relationship between endosomal active Rab levels and pH suggests a mechanism that can account for the observed variability in viral escape times, which in turn regulate the viability of a viral intracellular infection.
| S-EPMC8041880 | biostudies-literature
Project description:1. A convenient method of preparation of jack-bean urease (EC3.5.1.5) involving covalent chromatography by thiol-disulphide interchange is described. 2. Urease thus prepared has specific activity comparable with the highest value yet reported (44.5 +/- 1.47 kat/kg, Km = 3.32 +/- 0.05 mM; kcat. = 2.15 X 10(4) +/- 0.05 X 10(4)s-1 at pH7.0 and 38 degrees C). 3. Titration of the urease thiol groups with 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py) and application of the method of Tsou Chen-Lu [(1962) Sci. Sin. 11, 1535-1558] suggests that the urease molecule (assumed to have mol.wt. 483000 and epsilon280 = 2.84 X 10(5) litre-mol-1-cm-1) contains 24 inessential thiol groups of relatively high reactivity (class-I), six 'essential' thiol groups of low reactivity (class-II) and 54 buried thiol groups (class-III) which are exposed in 6M-guanidinium chloride. 4. The reaction of the class-I thiol groups with 2-Py-S-S-2-Py was studied in the pH range 6-11 at 25 degrees C(I = 0.1 mol/l) by stopped-flow spectrophotometry, and the analogous reaction of the class-II thiol groups by conventional spectrophotometry. 5. The class-I thiol groups consist of at least two sub-classes whose reactions with 2-Py-S-S-2-Py are characterized by (a) pKa = 9.1, k = 1.56 X 10(4)M-1-s-1 and (b) pKa = 8.1, k = 8.05 X 10(2)M-1-s-1 respectively. The reaction of the class-II thiol groups is characterized by pKa = 9.15 and k = 1.60 X 10(2)M-1-s-1. 6. At pH values 7-8 the class-I thiol groups consist of approx. 50% class-Ia groups and 50% class-Ib groups. The ratio class Ia/class Ib decreases an or equal to approx. 9.5, and at high pH the class-I thiol groups consist of at most 25% class-Ia groups and at least 75% class-Ib groups. 7. The reactivity of the class-II thiol groups towards 2-Py-S-S-2-Py is insensitive to the nature of the group used to block the class-I thiols. 8. All the 'essential' thiol groups in urease appear to be eeactive only as uncomplicated thiolate ions. The implications of this for the active-centre chemistry of urease relative to that of the thiol proteinases are discussed.
| S-EPMC1164111 | biostudies-other
Project description:The endoplasmic reticulum (ER) quality control factor EDEM1 associates with a number of ER proteins and ER-associated degradation (ERAD) substrates; however, an understanding of its role in ERAD is unclear. The early maturation events for EDEM1 including signal sequence cleavage and glycosylation were analyzed, and their relationship to the function of EDEM1 was determined. EDEM1 has five N-linked glycosylation sites with the most C-terminal site recognized poorly cotranslationally, resulting in the accumulation of EDEM1 containing four or five glycans. The fifth site was modified post-translationally when bypassed cotranslationally. Signal sequence cleavage of EDEM1 was found to be a slow and inefficient process. Signal sequence cleavage produced a soluble form of EDEM1 that efficiently associated with the oxidoreductase ERdj5 and most effectively accelerated the turnover of a soluble ERAD substrate. In contrast, a type-II membrane form of EDEM1 was generated when the signal sequence was uncleaved, creating an N-terminal transmembrane segment. The membrane form of EDEM1 efficiently associated with the ER membrane protein SEL1L and accelerated the turnover of a membrane-associated ERAD substrate. Together, these results demonstrated that signal sequence cleavage functionally regulated the association of EDEM1-soluble and membrane-integrated isoforms with distinct ERAD machinery and substrates.
| S-EPMC3137065 | biostudies-literature