Purification of the lectin from Datura stramonium.
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ABSTRACT:
SUBMITTER: Kilpatrick DC
PROVIDER: S-EPMC1186181 | biostudies-other | 1978 Dec
REPOSITORIES: biostudies-other
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Project description:Plants of Datura stramonium (thorn-apple) were dissected into their component tissues and examined for the presence of the Datura lectin. This lectin was easily detected in seeds and in various parts of the flowers of adult plants. Traces were also found in green (emerged) cotyledons and roots of seedlings. The specific lectin activity in seeds contained within the fruits increased as the seeds matured. Mature seeds were homogenized in sucrose and separated by differential centrifugation into four fractions, three of which were clearly of distinct composition. Most of the lectin activity sedimented with the low-speed (cell-wall/protein-body) pellet, but a similar specific activity was recovered from the other fractions. However, if EDTA was included in the homogenization medium, three or four times more lectin activity was recovered in the soluble fraction. Immunofluorescent staining of formaldehyde-fixed sections showed that the lectin was localized in the cytoplasm, with little associated with cell walls. The possible relevance of these results to the function of the lectin in plant cells is discussed.
| S-EPMC1161754 | biostudies-other
Project description:The lectin from Datura stramonium (thorn-apple; Solanaceae) has been purified by affinity chromatography and shown to be a glycoprotein containing about 40% (w/w) of carbohydrate. The most abundant amino acids are hydroxyproline, cystine, glycine and serine. Results obtained by gel filtration in 6m-guanidinium chloride on Sepharose 4B suggest that it has a subunit mol.wt. of about 30000 and that it probably associates into dimers. The lectin is inhibited specifically by chitin oligosaccharides and bacterial-cell-wall oligosaccharides, but only weakly by N-acetylglucosamine. Glycopeptides from soya-bean (Glycine max) lectin and fetuin are also strong inhibitors of Datura lectin, indicating that it interacts with internal N-acetylglucosamine residues. Its specificity is similar to, but not identical with, that of potato (Solanum tuberosum) lectin. After prolonged proteolytic digestion of reduced and S-carboxymethylated or S-aminoethylated derivatives of the lectin, glycopeptides of mol.wt. of about 18000 were isolated. The glycopeptides contained all the carbohydrate and hydroxyproline of the original glycoprotein, and lesser amounts of serine, S-carboxymethylcysteine and other amino acids. The arabinose residues of the glycoprotein are present as beta-l-arabinofuranosides linked to the polypeptide chain through the hydroxyproline residues, and can be removed by mild acid treatment; the ratio of arabinose to hydroxyproline is 3.4:1. Some of the serine residues of the polypeptide chain are substituted with one or two alpha-galactopyranoside residues, most of which can be removed by the action of alpha-galactosidase. The galactose residues are more easily removed from the acid-treated glycopeptide (from which arabinose has been removed) than from the complete glycopeptide, indicating a steric hindrance of the galactosidase action by the adjacent chains of arabinosides. There is a slow release of galactose residues by a process of beta-elimination in 0.5m-NaOH (pH13.7) from the complete glycopeptide, and a fairly rapid release of galactose by this process from the acid-treated glycopeptide, which lacks arabinose. This is probably due to the inhibitory effect of the negative charge on the adjacent arabinofuranoside residues. The similarities and differences between the lectins from Datura and potato are discussed, as are their structural resemblance to glycopeptides that have been isolated from plant cell walls.
| S-EPMC1163133 | biostudies-other
Project description:BackgroundCancer is a horrific disease relentlessly affecting human population round the globe. Genus Datura encompasses numerous species with reported medicinal uses. However, its potential as a source of natural anticancer agents is yet to be determined. Datura stramonium (DS) and Datura inoxia (DI) are the two species chosen for this study.MethodsTotal phenolic and flavonoid content (TPC and TFC) as well as antioxidant activity were assessed through colorimetric method. Polyphenolic quantification was done by RP-HPLC. Following extract standardization ethyl acetate leaf extracts of both species (DSL-EA and DIL-EA) were chosen for anticancer studies. In vitro cytotoxicity using various models including cancer cell lines was monitored. Following toxicity studies, benzene (0.2 ml) was used to induce leukemia in Sprague-Dawley rats. Extracts were orally administered to preventive (100 and 200 mg/kg) and treatment (200 mg/kg only) groups. The antileukemic potential of extracts was assessed through haematological, biochemical, endogenous antioxidants and histological parameters.ResultsSignificant TPC and TFC were estimated in DSL-EA and DIL-EA. RP-HPLC quantified (μg/mg extract) rutin (0.89 ± 0.03), gallic acid (0.35 ± 0.07), catechin (0.24 ± 0.02) and apigenin (0.29 ± 0.09) in DSL-EA while rutin (0.036 ± 0.004) and caffeic acid (0.27 ± 0.03) in DIL-EA. Both extracts exhibited significant brine shrimp cytotoxicity (LC50 < 12.5 μg/ml). DIL-EA exhibited greater cytotoxicity against PC-3, MDA-MB 231 and MCF-7 cell lines (IC50 < 3 μg/ml in each case) as well as higher protein kinase inhibitory action (MIC: 25 μg/disc) compared to DSL-EA. Leukemia induced in rats was affirmed by elevated serum levels of WBCs (7.78 ± 0.012 (× 103) /μl), bilirubin (7.56 ± 0.97 mg/dl), Thiobarbituric acid reactive substances (TBARs) (133.75 ± 2.61 nM/min/mg protein), decreased RBCs (4.33 ± 0.065 (× 106)/μl), platelets (344 ± 3.19 (× 103)/μl), total proteins (2.14 ± 0.11 g/dl), Glutathione S-transferases (GST) (81.01 ± 0.44 nM/min/ml), endogenous antioxidant enzymes levels and abnormal liver and kidney functionality in disease control rats. Both species revealed almost identical and significant (p < 0.05) alleviative effects in benzene induced leukemia.ConclusionComprehensive screening divulged the tremendous potential of selected species as potent source of natural anticancer agents in a variety of cancers particularly leukemia. Present study might provide useful finger prints in cancer research and mechanistic studies are prerequisite in logical hunt of this goal.
| S-EPMC7302377 | biostudies-literature
Project description:A new withanolide, named withawrightolide (1), and four known withanolides (2-5) were isolated from the aerial parts of Datura wrightii. The structure of compound 1 was elucidated through 2D NMR and other spectroscopic techniques. In addition, the structure of withametelin L (2) was confirmed by X-ray crystallographic analysis. Using MTS viability assays, withanolides 1-5 showed antiproliferative activities against human glioblastoma (U251 and U87), head and neck squamous cell carcinoma (MDA-1986), and normal fetal lung fibroblast (MRC-5) cells with IC50 values in the range between 0.56 and 5.6 ?M.
| S-EPMC3606642 | biostudies-literature
Project description:Leishmaniasis, a disease of the developing world affects about 12 million people and has limited therapeutic interventions available. L-type lectins, Endoplasmic Reticulum Golgi Intermediate Compartment/Vesicular Integral Proteins (ERGIC-53/VIP36) are involved in protein sorting in luminal compartments of animal cells and are important for parasite biology. A lectin homologue was identified through a bioinformatics analysis of Leishmania genome and it was found to have N-terminal conserved carbohydrate recognition domain (CRD) and a unique C-terminal region rich in repetitive amino acids and a poly glutamine tract. The N-terminal CRD region was cloned and expressed in Escherichia coli, but gave an insoluble expression which was re-solubilized by on column refolding. The fold integrity was checked through CD, fluorescence and functional assay of hemagglutination activity using rabbit erythrocyte. Bioinformatics analysis identified 15 members from Tritryps (Leishmania spp., Trypanosoma spp.) and they separate out as a distinct clade in the global phylogenetic analysis of all ERGIC-53/VIP36 sequences downloaded from Uniprot. Our analysis shows that the extended C-terminal regions with repeats is unique to Tritryps and this repeat pattern is different in sequences from Leishmania spp. and Trypanosoma spp. and all these features make this protein an interesting candidate for further detailed studies.
| S-EPMC4980737 | biostudies-literature
Project description:Lectins are carbohydrate binding proteins that recognize specific epitopes present on target glycoproteins. Changes in lectin-reactive carbohydrate repertoires are related to many biological signaling pathways and recognized as hallmarks of several pathological processes. Consequently, lectins are valuable probes, commonly used for examining glycoprotein structural and functional microheterogeneity. However, the molecular interactions between a given lectin and its preferred glycoproteoforms are largely unknown due to the inherent complexity and limitations of methods used to investigate intact glycoproteins. Here, we apply a lectin-affinity purification procedure coupled with native mass spectrometry to characterize lectin-reactive glycoproteoforms at the intact protein level. We investigate the interactions between the highly fucosylated and highly branched glycoproteoforms of haptoglobin and α1-acid glycoprotein using two different lectins Aleuria aurantia lectin (AAL) and Phaseolus vulgaris leucoagglutinin (PHA-L), respectively. Firstly we show a co-occurrence of fucosylation and N-glycan branching on haptoglobin, particularly among highly fucosylated glycoproteoforms. Secondly, we analyze the global heterogeneity of highly branched glycoproteoforms of haptoglobin and α1-acid glycoprotein and reveal that while multi-fucosylation attenuates the lectin PHA-L binding to haptoglobin, it has no impact on AGP. Taken together, our lectin affinity purification native MS approach elucidates lectin specificities between intact glycoproteins, not achievable by other methods. Moreover, since aberrant glycosylation of Hp and AGP are potential markers for many diseases, including pancreatic, hepatic and ovarian cancers, understanding their interactions with lectins will help the development of carbohydrate-centric monitoring methods to understand their pathophysiological implications.
| S-EPMC6524569 | biostudies-literature
Project description:Lectins with sugar-binding specificity are widely distributed in higher plants and various other species. The expression of rice lectin from Oryza sativa is up-regulated in the growing coleoptile when anaerobic stress persists. A rice lectin of molecular weight 15.2 kDa has been crystallized using the hanging-drop vapour-diffusion method. From the diffraction of the lectin crystals at 1.93 A resolution, the unit cell belongs to space group P3(1), with unit-cell parameters a = 98.58, b = 98.58, c = 44.72 A. Preliminary analysis indicates that there are two lectin molecules in an asymmetric unit with a large solvent content, 70.1%.
| S-EPMC2150942 | biostudies-literature
Project description:Genomic signatures of the evolution of defence against its natural enemies in the poisonous and medicinal plant Datura stramonium (Solanaceae)
| PRJNA622882 | ENA
Project description:Tropane alkaloid biosynthesis in Datura innoxia Mill. roots and their differential transport to shoots
| PRJNA719703 | ENA