Purification and characterization of a lectin from Annona coriacea seeds

A novel lectin, denominated ACLEC, was isolated from Annona coriacea seeds, belonging to the Annonaceae family. The lectin presented one protein band in SDS-PAGE of 14 kDa. Of the sugars tested, Dglucose and D-mannose were the best inhibitors. A search sequence database showed that ACLEC had homology with other plant lectins, belonging to leguminous lectin family.     

Trypsin-like protease from soybean seeds. Purification and some properties

An enzyme was purified from soybean seeds mainly by repeated ion-exchange chromatography using benzoyl-L-arginine p-nitroanilide (BAPA) as a substrate. The purified enzyme was homogeneous as judged by disc gel electrophoresis. The molecular weight was estimated as 59,000 by gel filtration. The enzyme was most active toward BAPA between pH 8 and 10. The enzyme was inactive toward protein substrates but hydrolyzed synthetic substrates and oligopeptides exclusively at the carboxyl side of L-arginine and L-lysine. Kinetic studies using synthetic substrates showed that, on the basis of Vmax/Km, the enzyme preferentially hydrolyzed amide substrates over ester substrates. Benzoyl-L-arginine 4-methylcoumaryl-7-amide (Bz-Arg-MCA) was the best substrate. The enzyme was strongly inhibited by diisopropylfluorophosphate (DFP), tosyl-L-lysine chloromethyl ketone (Tos-Lys-CH2Cl), leupeptin, and antipain. p-Chloromercuribenzoate (PCMB) was only partially inhibitory. Various protein inhibitors of trypsin such as soybean trypsin inhibitor were ineffective. From the primary specificity and susceptibility to chemicals, the enzyme can be said to be a trypsin-like serine protease. Although the physiological role of the enzyme is unclear, it seems likely that it is involved in limited hydrolysis of certain physiological peptides during processing.     

Purification,some properties of a D-galactose-binding leaf lectin from Erythrina indica and further characterization of seed lectin

Lectin from a leaf of Erythrina indica was isolated by affinity chromatography on Lactamyl-Seralose 4B. Lectin gave a single band in polyacrylamide gel electrophoresis (PAGE). In SDS-gel electrophoresis under reducing and non-reducing conditions Erythrina indica leaf lectin (EiLL) split into two bands with subunit molecular weights of 30 and 33 kDa, whereas 58 kDa was obtained for the intact lectin by gel filtration on Sephadex G-100. EiLL agglutinated all human RBC types, with a slight preference for the O blood group. Lectin was found to be a glycoprotein with a neutral sugar content of 9.5%. The carbohydrate specificity of lectin was directed towards D-galactose and its derivatives with pronounced preference for lactose. EiLL had pH optima at pH 7.0; above and below this pH lectin lost sugar-binding capability rapidly. Lectin showed broad temperature optima from 25 to 50 degrees C; however, at 55 degrees C EiLL lost more than 90% of its activity and at 60 degrees C it was totally inactivated. The pI of EiLL was found to be 7.6. The amino acid analysis of EiLL indicated that the lectin was rich in acidic as well as hydrophobic amino acids and totally lacked cysteine and methionine. The N-terminal amino acids were Val-Glu-Thr-IIe-Ser-Phe-Ser-Phe-Ser-Glu-Phe-Glu-Ala-Gly-Asn-Asp-X-Leu-Thr-Gln-Glu-Gly-Ala-Ala-Leu-. Chemical modification studies of both EiLL and Erythrina indica seed lectin (EiSL) with phenylglyoxal, DEP and DTNB revealed an absence of arginine, histidine and cysteine, respectively, in or near the ligand-binding site of both lectins. Modification of tyrosine with NAI led to partial inactivation of EiLL and EiSL; however, total inactivation was observed upon NBS-modification of two tryptophan residues in EiSL. Despite the apparent importance of these tryptophan residues for lectin activity they did not seem to have a direct role in binding haptenic sugar as D-galactose did not protect lectin from inactivation by NBS.     

An isolectin complex from Trichosanthes anguina seeds

Sepharose 4B affinity chromatography of Trichosanthes anguina seed extract and subsequent elution with galactose resulted in the isolation of an apparently single lectin with molecular weight of 45,000 +/- 700. However, major amount of the hemagglutinating activity was recovered as unadsorbed protein fraction. High affinity matrix Lactamyl Seralose could retain most of the galactose specific lectin activity from fraction 'A' which was eluted with lactose. It is evident from PAGE and SDS-PAGE analysis of the purified protein that T. anguina seeds contains a mixture of isolectins ranging in molecular weight from 30,000 to 50,000 +/- 1300. Periodic Acid Schiff's staining of the gels revealed this lectin complex to be a combination of glycosylated and non-glycosylated lectins. Two Isolectins SLc and IEL from within this complex have been isolated by affinity and ion exchange chromatography respectively. Apparent homology of these two lectins is indicated by their identical molecular weight (45 kDa), sub unit composition, non glycoprotein nature and immunological identity. However, these two lectins show minor differences in their biological and physicochemical properties. The peptide maps of the two lectins obtained after digestion with Trypsin and Pronase E also indicate minor changes in the primary structure.     

Purification and some properties of an aminopeptidase from the seeds of Cannabis sativa

An aminopeptidase (HSA) with a molecular mass of 78 kDa was purified from hemp (Cannabis sativa) seeds. The activity was inhibited by monoiodeacetic acid, p-chloromercuri-phenylsulfonic acid, and Zn2+ ion. The specificity of HSA was similar to that of a leucyl aminopeptidase [EC 3.4.11.1] from mammalian cytosol. However, other enzyme properties were different from these of leucyl aminopeptidase.     

Human placenta beta-galactoside-binding lectin. Purification and some properties

A beta-galactoside-binding lectin was extracted from human placenta homogenate with lactose solution and purified to apparent homogeneity by affinity chromatography on asialofetuin-Sepharose. The apparent subunit molecular weight of the lectin was 13,800 and its isoelectric point was about 5. Several saccharides containing D-galactose inhibited the hemagglutinating activity. The lectin resembles other vertebrate beta-galactoside-binding lectins in various biochemical characteristics.     

Purification and partial characterization of a lectin from the seeds of Trichosanthes kirilowii Maximowicz

A lectin was purified from the seeds of Trichosanthes kirilowii, belonging to the family Cucurbitaceae, growing in China. The lectin is a glycoprotein of 57 kDa, consists of two subunits with apparent molecular masses of 37 and 25 kDa, is specific for galactose, and is not mitogenic for human lymphocytes.     

Purification and some properties of a lectin from the hemolymph of Periplaneta americana (American cockroach)

A lectin showing specificity for human A-type red blood cells was purified to homogeneity from the hemolymph of the American cockroach Periplaneta americana by affinity chromatography on bovine submaxillary gland mucin. This lectin was a huge molecule with molecular mass of about 1500 kDa, with a single subunit of 30-kDa protein, and required Ca2+ for expression of lectin activity. Electron microscopic examination showed that these molecules were rods with helical structure with an average length of 50.5 nm and width of 10 nm. The molecule was suggested to contain tandemly aligned basic units of 10 nm length.     

Purification and properties of a lectin from the seeds of Croton tiglium with hemolytic activity toward rabbit red cells

The pH dependence of the chemical shift of the inorganic phosphate (P_i) inside mitochondria observable by ³¹P nmr has been examined and used for the measurement of the internal pH. The pH gradient and the P_i concentration gradient were in the simple relation expected for the neutral exchange process of H₂PO₄^? and OH^?. This P_i distribution across the mitochondrial membrane was not influenced by the cross-membrane electrical potential. Both the P_i, distribution and the pH titration curve of the internal P_i indicate that the activity of the internal P_i can be well represented by the concentration of P_i measured by ³¹P nmr peak intensity. The present results give a sound base for applying ³¹P nmr to study bioenergetics and cell metabolism.     

Purification and partial characterization of a lectin from Canavalia grandiflora benth. seeds

A D-glucose/D-mannose specific lectin from seeds of Canavalia grandiflora (ConGF) was purified by affinity chromatography on Sephadex G-50. By SDS-PAGE ConGF yielded three protein bands with apparent molecular masses of 29-30 kDa (alpha chain), 16-18 kDa (beta fragment) and 12-13 kDa (gamma fragment), like other related lectins from the genus Canavalia (Leguminosae). ConGF strongly agglutinates rabbit erythrocytes, has a high content of ASP and SER, and its N-terminal sequence (30 residues) is highly similar to the sequences of other related lectins from subtribe Diocleinae.     

Purification, properties, and crystallographic data for a principal nontoxic lectin from seeds of Abrus precatorius.

Nontoxic abrus lectin has been prepared by a new purification procedure. The method is accomplished by 45% saturation ammonium sulfate fractionation from a 5% acetic acid extract of the seeds of Abrus precatorius followed by diethylaminoethyl-Sephadex A-50 and Sepharose 4B affinity chromatography. The abrus lectin appeared homogeneous as judged by electrophoresis, analytical ultracentrifugation, and isoelectric focusing. The lectin molecule has a weight of 126,000 as determined by sedimentation equilibrium. It is composed of four subunits, of which two pairs have either identical or closely similar molecular sizes (33,800 and 32,200 daltons) as revealed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The results of amino acid analyses are given; none of the cysteic acid appears to arise from cysteine. An electrofocusing experiment indicated the isoelectric point to be 5.0. Crystals large enough for x-ray investigation were obtained by a vapor diffusion technique. X-ray precession photographs revealed that abrus lectin crystallizes in a tetragonal unit cell of symmetry P41212 and dimensions a = 140 and c = 210 A. The asymmetric unit contains 2 protein molecules of molecular weight 126,000 and has a solvent content of approximately 41% by volume.     

Purification and characterization of a lectin from seeds and cotyledonary callus of Zizyphus mauritiana

A lectin from Zizyphus mauritiana lamk. has been purified from the 25–50% (NH₄)₂SO₄ fraction of crude seed and cotyledonary leaf callus extracts. The lectins purified from the two sources had the same structure and properties. Upon specific adsorption on Sephadex G-100, the lectin (ZML) could be displaced with 0.1 m d-glucose. ZML yielded a single band corresponding to a M_r of 66 kDa both in the absence and presence of β-mercaptoethanol on native- as well as in SDS-PAGE. It is thermostable but pH sensitive and agglutinates human erythrocytes only. Lectin activity could also be detected in the cotyledons, leaf, and stem, and in their cultures, as well as in in vitro regenerants. Cotyledons, cotyledonary leaf and its callus showed much higher lectin activity than seeds. Received: 5 April 1997 / Revision received: 30 June 1997 / Accepted: 31 October 1997     

Isolation and properties of a lectin from the seeds ofMimosa invisa L.

A lectin has been purified from the seeds ofMimosa invisa L. by gel filtration and preparative Polyacrylamide gel electrophoresis. The purified lectin was homogeneous as judged by analytical Polyacrylamide gel electrophoresis, immunodiffusion and Immunoelectrophoresis. The apparent molecular weight is 100,000; the protein is a tetramer with two types of subunits (molecular weight 35,000 and 15,000). The lectin is a glycoprotein with approximately 21% carbohydrate and interacts with Sephadex and concanavalin A-Sepharose. It agglutinates erthrocytes non-specifically, does not agglutinate leucocytes and is not mitogenic, agglutinates Mimosa-nodulatingRhizobium and is a panagglutinin; the agglutination is not inhibited by several simple sugars. It is thermo-stable and has no metal ions.     

Purification and properties of a beta-galactoside-binding lectin from neonatal marmoset.

A beta-galactoside-binding lectin was extracted from whole neonatal marmoset homogenate with lactose solution and purified to homogeneity by ion-exchange chromatography on Q Sepharose Fast Flow and by affinity adsorption to trypsinized and glutaraldehyde-fixed ghosts of rabbit erythrocytes. The lectin has a dimeric structure composed of two 15K subunits. Its amino acid composition and partial amino acid sequences were quite similar to those of beta-galactoside-binding lectins from human placenta and lung.     

Purification and characterization of a mannose/N-acetyl-D-glucosamine-specific lectin from the seeds of Platymiscium floribundum Vogel

Platymiscium floribundum lectin (PFL), a mannose/N-acetyl-D-glucosamine-specific lectin, was isolated from P. floribundum seeds using Sepharose-mannose affinity media chromatography. PFL is a glycoprotein that is a potent agglutinin for rabbit erythrocytes. In addition, PFL is highly stable because it is able to maintain its hemagglutinating activity after exposure to temperatures of up to 60 °C for 1 h and exposure to a wide pH range. The PFL purification process was monitored using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the results showed that the purified lectin consists of a single band with a molecular mass of approximately 29 kDa in either the presence or the absence of a reducing agent. The analysis of purified PFL by electrospray ionization-mass spectrometry showed that most ions had a molecular weight of 27,053 ± 2 Da, and other less abundant ions had similar molecular weights. Gel filtration shows that the lectin exists as a dimer in solution with mass at approximately 65 kDa. Sixteen peptides were sequenced, and as a result, a total of 130 amino acids were identified and resulted in a coverage of approximately 65% of the PFL sequence. The partial sequence of PFL was aligned with sequences of other lectins from evolutionarily related species, and PFL showed considerable similarity to the other lectins.     

Purification of a lectin from Eugenia uniflora L. seeds and its potential antibacterial activity

Aims: The aim of this work was to analyse the antimicrobial properties of a purified lectin from Eugenia uniflora L. seeds. Methods and Results: The E. uniflora lectin (EuniSL) was isolated from the seed extract and purified by ion‐exchange chromatography in DEAE‐Sephadex with a purification factor of 11·68. The purified lectin showed a single band on denaturing electrophoresis, with a molecular mass of 67 kDa. EuniSL agglutinated rabbit and human erythrocytes with a higher specificity for rabbit erythrocytes. The haemagglutination was not inhibited by the tested carbohydrates but glycoproteins exerted a strong inhibitory action. The lectin proved to be thermo resistant with the highest stability at pH 6·5 and divalent ions did not affect its activity. EuniSL demonstrated a remarkable nonselective antibacterial activity. EuniSL strongly inhibited the growth of Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella sp. with a minimum inhibitory concentration (MIC) of 1·5 μg ml^?1, and moderately inhibited the growth of Bacillus subtilis, Streptococcus sp. and Escherichia coli with a MIC of 16·5 μg ml^?1. Conclusions: EuniSL was found to be effective against bacteria. Significance and Impact of the Study: The strong antibacterial activity of the studied lectin indicates a high potential for clinical microbiology and therapeutic applications.     

Purification and some characteristics of a beta-galactoside binding soluble lectin from amphibian ovary

Soluble extracts of Bufo ovaries agglutinate sialidase-treated rabbit erythrocytes. Unlike other amphibian lectins this agglutination activity does not require the presence of calcium ions. It is specifically inhibited by D-galactose and its derivatives. Thiodi-D-galactoside is the most potent saccharide inhibitor followed by lactose and methyl-beta-D-galactoside, respectively. D-Fucose, D-glucose and D-mannose do not inhibit the activity at concentrations at or above 100 mM. The lectin has been purified 500-fold to apparent homogeneity from the ovaries by salt extraction and affinity chromatography on lactose-aminophenyl-agarose, with a yield of about 0.2%. The molecular mass determined by gel filtration under native conditions was 30 kDa; polyacrylamide gel electrophoresis in SDS gave a molecular mass of 15 kDa, suggesting that the lectin is a dimer. The lectin has an isoelectric point of 40 and contains a high proportion of acidic amino acids.     

Purification,primary structure and potential functions of a novel lectin from Bauhinia forficata seeds

A new lectin, BfL, was purified from Bauhinia forficata seeds by ammonium sulfate fractionation, DEAE-Sephadex ion exchange chromatography, Sepharose-4B and chitin affinity chromatographies and Superdex 75 size exclusion chromatography. The molecular homogeneity and purity of BfL were assessed by reversed-phase HPLC. BfL appeared as a single band of approximately 27.0 kDa on SDS-PAGE under non-reducing and reducing conditions, and its molecular weight was determined to be 27,850 Da by LC/ESI-MS. BfL is a glycoprotein with a carbohydrate content of 6.24% determined by the phenol–sulfuric acid method. Fetuin, asialofetuin, thyroglobulin and azocasein inhibited the hemagglutinating activity of BfL, whereas saccharides did not. BfL hemagglutinating activity was stable at 100 °C for 30 min, pH-dependent, with the highest activity at pH 6.0, and metal-independent. The primary structure of BfL shows similarity with other lectins from the genus Bauhinia. Deconvolution of the BfL circular dichroism (CD) spectrum indicated the presence of α-helix and β structures. BfL increases coagulation time, but this effect is not related to human plasma kallikrein or human factor Xa inhibition. BfL also inhibits ADP- and epinephrine-induced platelet aggregation in a dose-dependent manner and is the only currently described lectin from Bauhinia that exhibits anticoagulant and antiplatelet aggregating properties.