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1.
Samreen Amani Afshin Iram Anwar Shahzad Km Neelofar Aabgeena Naeem 《International journal of peptide research and therapeutics》2011,17(4):317-324
A well-organized protocol has been developed for high frequency root germination from the seed of Canavalia ensiformis on Murashige and Skoog (MS) medium. Surprisingly, the seeds that were grown on the MS medium having no growth hormone showed
the best response. Roots of 30 days old aseptic seedling were homogenized and a lectin from them was purified on Sephadex
G-50 affinity column. The finding that final product is a pure lectin was confirmed by specific hemagglutinating property.
The final root lectin yield was 0.6% and eluted as a single peak. Root lectin specific activity was 50 times more than the
seed lectin. Sugar specificity activity by hemagglutination-inhibition assay indicated that lectin belongs to glucose/mannose-specific
group. Interestingly, the lectin was found to be 25 kDa, similar to molecular mass of Concanavalin A purified from seed of
C. ensiformis, as revealed by SDS–PAGE. Thus, Concanavalin A from either source can be used for development of transgenic crops that are
capable of expressing lectin gene and hence can efficiently perform biological nitrogen fixation by giving rise to nodules
in their root. The advantage of this method is that purification of Concanavalin A in tissue culture conditions is easier,
handy and is less time consuming. 相似文献
2.
A lectin present in seeds of Clitoria ternatea agglutinated trypsin-treated human B erythrocytes. The sugar specificity assay indicated that lectin belongs to Gal/Gal NAc-specific
group. Hence the lectin, designated C. ternatea agglutinin (CTA), was purified by the combination of acetic acid precipitation, salt fractionation and affinity chromatography.
HPLC gel filtration, SDS-polyacrylamide gel electrophoresis and mass spectrometry indicated that the native lectin is composed
of two identical subunits of molecular weight 34.7 kDa associated by non covalent bonds. The N-terminal sequence of CTA shared
homology with Glycine max and Pisum sativum. Complete sequence was also found to be homologous to S-64 protein of Glycine max, suggesting that CTA probably exhibits both hemagglutination and probably sugar uptake activity. The carbohydrate binding
specificity of the lectin was investigated by quantitative turbidity measurements, and percent inhibition assays. Based on
these assays, we conclude that CTA binds β-d-galactosides, and also may has an extended specificity towards non-reducing terminal Neu5Acα2,6Gal. 相似文献
3.
C. L. Díaz M. Hosselet G. J. J. Logman E. van Driessche B. J. J. Lugtenberg J. W. Kijne 《Planta》1990,181(4):451-461
We report on the distribution and initial characterization of glucose/mannose-specific isolectins of 4- and 7-d-old pea (Pisum sativum L.) seedlings grown with or without nitrate supply. Particular attention was payed to root lectin, which probably functions as a determinant of host-plant specificity during the infection of pea roots by Rhizobium leguminosarum bv. viciae. A pair of seedling cotyledons yielded 545±49 g of affinity-purified lectin, approx. 25% more lectin than did dry seeds. Shoots and roots of 4-d-old seedlings contained 100-fold less lectin than cotyledons, whereas only traces of lectin could be found in shoots and roots from 7-d-old seedlings. Polypeptides with a subunit structure similar to the precursor of the pea seed lectin could be demonstrated in cotyledons, shoots and roots. Chromatofocusing and isoelectric focusing showed that seed and non-seed isolectin differ in composition. An isolectin with an isoelectric point at pH 7.2 appeared to be a typical pea seed isolectin, whereas an isolectin focusing at pH 6.1 was the major non-seed lectin. The latter isolectin was also found in root cell-wall extracts, detached root hairs and root-surface washings. All non-seed isolectins were cross-reactive with rabbit antiserum raised against the seed isolectin with an isolectric point at pH 6.1. A protein similar to this acidic glucose/mannose-specific seed isolectin possibly represents the major lectin to be encountered by Rhizobium leguminosarum bv. viciae in the pea rhizosphere and at the root surface. Growth of pea seedlings in a nitrate-rich medium neither affected the distribution of isolectins nor their hemagglutination activity; however, the yield of affinity-purified root lectin was significantly reduced whereas shoot lectin yield slightly increased. Agglutination-inhibition tests demonstrated an overall similar sugar-binding specificity for pea seed and non-seed lectin. However root lectin from seedlings grown with or without nitrate supplement, and shoot lectin from nitrate-supplied seedlings showed a slightly different spectrum of sugar binding. The absorption spectra obtained by circular dichroism of seed and root lectin in the presence of a hapten also differed. These data indicate that nutritional conditions may affect the sugar-binding activity of non-seed isolectin, and that despite their similarities, seed and non-seed isolectins have different properties that may reflect tissue-specialization.Abbreviations IEF
isoelectric focusing
- MW
molecular weight
- pI
isoelectric point
- Psl1, Psl2 and Psl3
pea isolectins
- SDSPAGE
sodium dodecyl sulfate polyacrylamide gel electrophoresis
The authors wish to thank Professors L. Kanarek and M. van Poucke for helpful discussions. 相似文献
4.
A lectin was isolated from the roots of Sesbania aculeata. This is a glucose specific lectin having 39 kDa subunit molecular weight. The expression of this lectin was found to be
developmentally regulated and observed to be the highest in the second week. The lectin was purified by affinity chromatography
using Sephadex G-50 and found to have 28% homology with Arabidopsis thaliana lectin-like protein (accession No. CAA62665). The lectin binds with lipopolysaccharide isolated from different rhizobial
strains indicating the plants interaction with multiple rhizobial species.
Published in Russian in Biokhimiya, 2009, Vol. 74, No. 3, pp. 404–411. 相似文献
5.
Alexandre H. Sampaio David J. Rogers Clive J. Barwell Silvana Saker-Sampaio Kyria S. Nascimento Celso S. Nagano Wladimir R.L. Farias 《Journal of applied phycology》2002,14(6):489-495
The red marine alga Ptilota plumosa has been shownto contain an anti-human blood group B lectin. We report here a new isolationprocedure by affinity chromatography on Sephadex G-200 and characterisation ofthe isolated lectin. The M
r
, determined by gelfiltration, was 52,500. SDS-PAGE revealed a single protein band withM
r
17,440, indicating the native lectin was atrimer of subunits with the same Mr, as reported for the lectinsfromtwo other Ptilota species, P.filicinaand P. serrata. Analysis of amino acid composition showedslightly more basic than acidic amino acids. This was in contrast to theP. filicina and P. serrata lectinspreviously found to contain a higher proportion of acidic than basic aminoacids. Haemagglutination inhibition tests showed the P.plumosa lectin was inhibited by galactose, glucose and theirderivatives with p-nitrophenyl--D-galactoside moststrongly inhibitory. All glycoproteins tested failed to inhibit the lectin. Theamino acid composition, human blood group-B specificity and lack of inhibitionby glycoproteins indicate the lectin from P. plumosapossesses unique characteristics among marine algal lectins. 相似文献
6.
Various monosaccharides and oligosaccharides were used to define the specificity of theButea frondosa lectin using the hapten inhibition technique of human erythrocyte agglutination. AlthoughB. frondosa lectin exhibited higher affinity forN-acetylgalactosamine, lactose andN-acetyllactosamine appeared to be relatively good inhibitors of haemagglutination. The behaviour ofN-acetyllactosamine-type oligosaccharides and glycopeptides on a column ofB. frondosa lectin immobilized on Sepharose 4B showed that the sugar-binding specificity of the lectin is directed towards unmaskedN-acetyllactosamine sequences. Substitution of theseN-acetyllactosamine sequences by sialic acid residues completely abolished the affinity of the lectin for the saccharides. The presence of one or several Fuc(1-3)GlcNAc groups completely inhibited the interaction between the glycopeptides and the lectin. Substitution of the core -mannose residue by an additional bisecting (1-4)GlcNAc residue decreases the affinity of the lectin for these structures as compared with the unsubstituted ones. 相似文献
7.
T/Tn specificity of Artocarpus lakoocha agglutinin (ALA), isolated from the seeds of A. lakoocha (Moraceae) fruit and a heterodimer (16 kD and 12 kD) of molecular mass 28 kD, was further confirmed by SPR analysis using
T/Tn glycan containing mammalian glycoproteins. N-terminal amino acid sequence analysis of ALA showed homology at 15, 19–21,
24–27, and 29 residues with other lectin members of Moraceae family viz., Artocarpus integrifolia (jacalin) lectin, Artocarpus hirsuta lectin, and Maclura pomifera agglutinin. It is mitogenic to human PBMC and the maximum proliferation was observed at 1 ng/ml. It showed an antiproliferative
effect on leukemic cells, with the highest effect toward Jurkat cells (IC50 13.15 ng/ml). Synthesized CdS quantum dot-ALA nanoconjugate was employed to detect the expression of T/Tn glycans on Jurkat,
U937, and K562 leukemic cells surfaces as well as normal lymphocytes by fluorescence microscopy. No green fluorescence was
observed with normal lymphocytes indicating that T/Tn determinants, which are recognized as human tumor associated structures
were cryptic on normal lymphocyte surfaces, whereas intense green fluorescent dots appeared during imaging of leukemic cells,
where such determinants were present in unmasked form. The above results indicated that QD-ALA nanoconjugate is an efficient
fluorescent marker for identification of leukemic cell lines that gives rise to high quality images. 相似文献
8.
Ponpimol Tipthara Polkit Sangvanich Marcus Macth Amorn Petsom 《Journal of Plant Biology》2007,50(2):167-173
A mannose-binding lectin was isolated from rhizomes of the medicinal plantCurcuma zedoaria. We used extraction with 20 mM phosphate buffer, ammonium sulfate precipitation, ion exchange chromatography on Q-Sepharose,
gel filtration chromatography on Superdex 75, and reverse-phase HPLC. The purified lectin yielded a single band on SDS-PAGE
that corresponded to a molecular mass of 13 kDa. This lectin exhibited hemagglutinating activity toward rabbit erythrocytes,
which could be inhibited by mannose only. The lectin was digested with trypsin and its digests were analyzed using MALDI-TOF/TOF.
Partial amino acid sequences were obtained from tandem mass spectra via automatedde novo sequencing, and were then identified by MS-BLAST homology searches to enable recognition of related proteins in other species.
Inferred peptide sequences exhibited similarity to a mannose-binding lectin fromEpipactis helleborine, a member of the Orchidaceae. 相似文献
9.
Two lectins were purified by affinity chromatography from mature peanut (Arachis hypogaea L.) nodules, and compared with the previously characterised seed lectin of this plant. One of the nodule lectins was similar to the seed lectin in its molecular weight and amino-acid composition and ability to bind derivatives of galactose. However, unlike the seed lectin, this nodule lectin appeared to be a glycoprotein and the two lectins were only partially identical in their reaction with antibodies prepared against the seed lectin. The other nodule lectin also appeared to be a glycoprotein but bound mannose/glucose-like sugar derivatives, and differed from the seed lectin in molecular weight, antigenic properties and amino-acid composition.Abbreviations Gal
galactose
- Gle
glucose
- GNL
galactose-binding nodule lectin
- Fru
fructose
- MNL
mannosebinding nodule lectin
-
M
r
rerative molecular mass
- PBS
phosphate-buffered saline
- PSL
peanut seed lectin
- SDS
sodium dodecyl sulphate
- Sorb
sorbitol 相似文献
10.
Jong Won Han Kang Sup Yoon Tatyana A. Klochkova Mi-Sook Hwang Gwang Hoon Kim 《Journal of applied phycology》2011,23(4):745-753
A novel lectin was isolated and characterized from Bryopsis plumosa (Hudson) Agardh and named BPL-3. This lectin showed specificity to N-acetyl-d-galactosamine as well as N-acetyl-d-glucosamine and agglutinated human erythrocytes of all blood types, showing slight preference to the type A. SDS-PAGE and
MALDI-TOF MS data showed that BPL-3 was a monomeric protein with molecular weight of 11.5 kDa. BPL-3 was a non-glycoprotein
with pI value of ∼7.0. It was stable in high temperatures up to 70°C and exhibited optimum activity in pH 5.5–10. The N-terminal
and internal amino acid sequences of the lectin were determined by Edman degradation and enzymatic digestion, which showed
no sequence homology to any other reported proteins. The full sequence of the cDNA encoding this lectin was obtained from
PCR using cDNA library, and the degenerate primers were designed from the N-terminal amino acid sequence. The size of the
cDNA was 622 bp containing single ORF encoding the lectin precursor. This lectin showed the same sugar specificity to previously
reported lectin, Bryohealin, involved in protoplast regeneration of B. plumosa. However, the amino acid sequences of the two lectins were completely different. The homology analysis of the full cDNA sequence
of BPL-3 showed that it might belong to H lectin group, which was originally isolated from Roman snails. 相似文献
11.
Sclerotium rolfsii lectin (SRL), a secretory protein from the soil borne phytopathogenic fungus Sclerotium rolfsii, has shown in our previous studies to bind strongly to the oncofetal Thomson-Friedenreich carbohydrate (Galβ1-3GalNAc-ser/thr,
T or TF) antigen. TF antigen is widely expressed in many types of human cancers and the strong binding of SRL toward such
a cancer-associated carbohydrate structure led us to characterize the carbohydrate binding specificity of SRL. Glycan array
analysis, which included 285 glycans, shows exclusive binding of SRL to the O-linked mucin type but not N-linked glycans and
amongst the mucin type O-glycans, lectin recognizes only mucin core 1, core 2 and weakly core 8 but not to other mucin core
structures. It binds with high specificity to “α-anomers” but not the “β-anomers” of the TF structure. The axial C4-OH group
of GalNAc and C2-OH group of Gal is both essential for SRL interaction with TF disaccharide, and substitution on C3 of galactose
by sulfate or sialic acid or N-acetylglucosamine, significantly enhances the avidity of the lectin. SRL differs in its binding to TF structures compared
to other known TF-binding lectins such as the Arachis hypogea (peanut) agglutinin, Agaricus bisporus (mushroom) lectin, Jackfruit, Artocarpus integrifolia (jacalin) and Amaranthus caudatus (Amaranthin) lectin. Thus, SRL has unique carbohydrate-binding specificity toward TF-related O-linked carbohydrate structures.
Such a binding specificity will make this lectin a very useful tool in future structural as well as functional analysis of
the cellular glycans in cancer studies. 相似文献
12.
Luciano S. Pinto Celso S. Nagano Taianá M. Oliveira Tales R. Moura Alexandre H. Sampaio Henri Debray Vicente P. Pinto Odir A. Dellagostin Benildo S. Cavada 《Journal of biosciences》2008,33(3):355-363
A new galactose-specific lectin was purified from seeds of a Caesalpinoideae plant, Bauhinia variegata, by affinity chromatography on lactose-agarose. Protein extracts haemagglutinated rabbit and human erythrocytes (native and
treated with proteolytic enzymes), showing preference for rabbit blood treated with papain and trypsin. Among various carbohydrates
tested, the lectin was best inhibited by D-galactose and its derivatives, especially lactose. SDS-PAGE showed that the lectin,
named BVL, has a pattern similar to other lectins isolated from the same genus, Bauhinia purpurea agglutinin (BPA). The molecular mass of BVL subunit is 32 871 Da, determined by MALDI-TOF spectrometry. DNA extracted from
B. variegata young leaves and primers designed according to the B. purpurea lectin were used to generate specific fragments which were cloned and sequenced, revealing two distinct isoforms. The bvl gene sequence comprised an open reading frame of 876 base pairs which encodes a protein of 291 amino acids. The protein carried
a putative signal peptide. The mature protein was predicted to have 263 amino acid residues and 28 963 Da in size. 相似文献
13.
Potato lectin (Solanum tuberosum agglutinin, STA) is an unusual glycoprotein containing approximately 50% carbohydrates by weight. Of the total carbohydrates,
92% is contributed by L-arabinose, which are O-linked to hydroxyproline residues. The ferric chloride-orcinol assay (Bial’s test), which is specific for pentoses has so
far been used only for the determination of free pentoses in biological samples. However, this colorimetric assay has not
been used for the detection of pentoses in bound form as it occurs in Solanaceae lectins (potato, tomato, and Datura lectins).
Utilizing the pentose colorimetric assay for monitoring the presence of potato lectin, a simpler and shorter procedure for
the purification of this lectin from potato tubers has been developed. The yield of potato lectin (1.73 mg per 100 g potato
tuber) is twice compared to the yields reported in earlier procedures. Although potato lectin is well known for its specificity
to free trimers and tetramers of N-acetyl-D-glucosamine (GlcNAc), it possesses a similar specificity to the core (GlcNAc)2 of N-linked glycoproteins. The utilization of the pentose assay in the purification of arabinose-rich lectins/agglutinins
obviates the necessity for the use of agglutination assay in the various purification steps. The pentose assay appears to
be a simple and convenient colorimetric assay for detecting any pentose-rich glycoprotein in plant extracts. The utility of
the pentose assay appears to have a significant potential in the detection of hydroxyproline-rich glycoproteins (HRGPs), which
are generally O-arabinosylated. 相似文献
14.
M. A. Bauchrowitz D. G. Barker I. Nadaud P. Rougé B. Lescure 《Plant molecular biology》1992,19(6):1011-1017
We report the cloning and characterization of two lectin genes from Medicago truncatula, designated Mtlec1 and Mtlec2. The two genes show a high degree of homology and apparently belong to a small multigene family. Mtlec1 appears to encode a functional lectin with 277 amino acids, whereas Mtlec2 is probably non-functional, since a frameshift mutation (insertion of two nucleotides) leads to premature translation termination after only 98 amino acids. The deduced amino acid sequence of the polypeptide MtLEC1 suggests that this lectin is a metalloprotein with Glc/Man specificity. 相似文献
15.
Pea lectin is correctly processed,stable and active in leaves of transgenic potato plants 总被引:5,自引:0,他引:5
Glyn A. Edwards Andrew Hepher Stephen P. Clerk Donald Boulter 《Plant molecular biology》1991,17(1):89-100
A gene encoding the preproprotein of the pea (Pisum sativum) lectin was expressed in transgenic potato plants using a cauliflower mosaic virus (CaMV) 35S promoter or a tobacco ribulose bisphosphate carboxylase small subunit (ssRubisco) promoter. Presence of the pea lectin to levels greater than 1% of total soluble leaf protein was detected by radioimmunoassay (RIA). The pattern of expression derived from the two promoters was established using both RIA and a squash-blot immunolocalisation technique. Western blotting demonstrated that the preproprotein was correctly processed, generating and subunits that assembled to give an isolectin form observed in pea seeds and roots. It was also found that the haemagglutination activity and specificity of pea lectin synthesised in transgenic potato leaves was comparable to purified lectin from pea cotyledons. 相似文献
16.
Mahr K van Wezel GP Svensson C Krengel U Bibb MJ Titgemeyer F 《Antonie van Leeuwenhoek》2000,78(3-4):253-261
Glucose kinase of Streptomyces coelicolor A3(2) is essential for glucose utilisation and is required for carbon catabolite repression (CCR) exerted through glucose and other carbon sources. The protein belongs to the ROK-family, which comprises bacterial sugar kinases and regulators. To better understand glucose kinase function, we have monitored the cellular activity and demonstrated that the choice of carbon sources did not significantly change the synthesis and activity of the enzyme. The DNA sequence of the Streptomyces lividans glucose kinase gene glkA was determined. The predicted gene product of 317 amino acids was found to be identical to S. coelicolor glucose kinase, suggesting a similar role for this protein in both organisms. A procedure was developed to produce pure histidine-tagged glucose kinase with a yield of approximately 10 mg/l culture. The protein was stable for several weeks and was used to raise polyclonal antibodies. Purified glucose kinase was used to explore protein-protein interaction by surface plasmon resonance. The experiments revealed the existence of a binding activity present in S. coelicolor cell extracts. This indicated that glucose kinase may interact with (an)other factor(s), most likely of protein nature. A possible cross-talk with proteins of the phosphotransferase system, which are involved in carbon catabolite repression in other bacteria, was investigated. 相似文献
17.
Giorgos A Fragkiadakis Emmanoel K Stratakis 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1997,117(4):545-552
A lectin that recognized sialic acids and agglutinated mouse erythrocytes was purified from hemolymph of the crab Liocarcinus depurator. It consisted of 38-kDa subunits and had a pI about 6.0. The specificity of the lectin was assayed by hemagglutination inhibition. N-acetylneuraminic acid (Neu5Ac) was a good inhibitor and its N-acetyl group at C-5 was critical for lectin-ligand interaction. Substitution of the C-9 hydroxyl on Neu5Ac with an O-acetyl group (9-O-Ac-Neu5Ac) increased the inhibitory potency of this molecule. Furthermore, O-acetyl substitution of all the hydroxyl groups yielded even better inhibitors (2,4,7,8,9-O-Ac-Neu5Ac and its 1-O-methyl ester). Removal of the hydroxyl or O-acetyl group connected to C-2 reduced the potency of these inhibitors. The lectin agglutinated and stimulated human but not mouse lymphocytes. It was also inhibited by Escherichia coli (O111:B4) lipopolysaccharide and agglutinated specific gram-negative bacteria. In vitro labeling with [35S]methionine indicated that the lectin was synthesized in hepatopangreas of L. depurator. Immunofluorescence showed that among hemocytes it localized mainly in the large-granule population. 相似文献
18.
Iga Steuden Maria Duk Marcin Czerwiński Czeslaw Radzikowski Elwira Lisowska 《Glycoconjugate journal》1985,2(3-4):303-314
The monoclonal antibody 22.19 of IgM class obtained after immunization of BALB/c mice with asialoglycophorin of human erythrocyte membranes is described. The specificity of this antibody for -d-Gal-1-3--d-GalNAc- disaccharide chains (Thomsen-Friedenreich receptors) was established by studying its reactivity against various erythrocytes, glycoproteins and oligosaccharides and by comparison with two lectins, peanut agglutinin andVicia graminea lectin, which recognize these disaccharide chains.Abbreviations PNA
peanut agglutinin
- VgL
Vicia graminea lectin
- TF
Thomsen-Friedenreich
- HSA
human serum albumin
- MoAb
monoclonal antibody 相似文献
19.
A new lectin gene was isolated by using genomic walker technology and revealed to encode a mannose-binding lectin. Analysis of a 2233 bp segment revealed a gene including a 1169 bp 5′ flanking region, a 417 bp open reading frame (ORF) and a 649 bp 3′ flanking region. There are two putative TATA boxes and eight possible CAAT boxes lie in the 5′ flanking region. The ORF encodes a 15.1 kDa precursor, which contains a 24-amino acid signal peptide. One possible polyadenylation signal is found in the 3′-flanking region. No intron was detected within the region of genomic sequence corresponding to zaa (Zantedeschia aethiopica agglutinin) full-length cDNA, which is typical of other mannose-binding lectin gene that have been reported. The deduced amino acid sequence of the lectin gene coding region shares 49–54% homology with other known lectins. The cloning of this new lectin gene will allow us to further study its structure, expression and regulation mechanisms. 相似文献
20.
Using RNA extracted from Pinellia cordata young leaves and primers designed according to the conserved regions of Araceae lectins, the full-length cDNA of Pinellia cordata agglutinin (PCL) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of pcl was 1,182 bp and contained a 768 bp open reading frame (ORF) encoding a lectin precursor of 256 amino acids. Through comparative
analysis of pcl gene and its deduced amino acid sequence with those of other Araceae species, it was found that pcl encoded a precursor lectin with signal peptide. PCL is a mannose-binding lectin with three mannose-binding sites. Semi-quantitative
RT-PCR analysis revealed that pcl is expressed in all tested tissues including leaf, stem and bulbil, but with the highest expression in bulbil. PCL protein
was successfully expressed in Escherichia coli with the molecular weight expected. 相似文献