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1.
A lectin from fruiting bodies of Lactarius rufus (Scop.: Fr.)Fr. has been purified by affinity chromatography on copolymer of polyvinyl alcohol with a blood group B specific substance. The lectin gives a single band at disk-electrophoresis in acidic (pH 4.3) and alkaline (pH 8.6) buffer systems. Under electrophoresis in 10-20% SDS-PAGE, the lectin consists of identical subunits with molecular weight 17 +/- 1 kDa. Molecular weight of the lectin is 98 kDa according to gel-chromatography on Tojopearl HW-55. It is supposed that the lectin contains six subunits. The lectin is quite enough stable in pH 4.0-10.0, its activity does not depend upon bivalent metal ions. When heating the lectin solution to 65 degrees C it lost more than 85% of its activity. The lectin agglutinates human etrythrocytes without any marked group specificity, it agglutinates 2-4 times worse rabbit erythrocytes, very weakly crucian erythrocytes and does not agglutinate sheep erythrocytes. Mono- and disaccharides are not inhibitors of the lectin activity, while alpha-phenyl-N-acethyl-D-glucosaminopyranosid (0.08 mM) and 4-nitrophenyl-beta-D-glucosamin are the best inhibitors of its activity. Among glycoproteins the best inhibitors of the lectin activity are: group-specific substances from human blood erythrocytes, asialosubmaxillary bovine mucin, human and bovine thyroglobulin and more weak inhibitors are fetuin, transferrin and human Ig G. 相似文献
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D. N. Olennikov S. V. Agafonova G. B. Borovskii T. A. Penzina A. V. Rokhin 《Applied Biochemistry and Microbiology》2009,45(6):626-630
Alkali-soluble polysaccharides have been extracted from Laetiporus Sulphureus (Bull.: Fr.) Murr fruit bodies with a yield of 42.7%. The structure of the dominant polymer (16.05% of fruit bodies’ mass),
named latiglucan I, has been determined. It is linear β-1,3-glucan (molecular weight 1.8 × 105 Da, [α]D-17°). 相似文献
4.
Antoniuk VO 《Ukrainski? biokhimicheski? zhurnal》2005,77(3):96-103
A lectin that revealed affinity for lactose, N-acethylactosamine, 4-nitrophenyl-beta-D-gluco- and galactopyranosides from fruiting bodies of Sarcoscypha coccinea (Fr.) Lambette was purified by affinity chromatography on immobilized ovomucoid. According to electrophoresis data in 15% SDS-PAGE the lectin contains two very low-differing components with molecular weight 32 kDa. Molecular weight of the lectin is 128 kDa according to gel-chromatography on sephadex G-200. The lectin agglutinates rabbit erythrocytes and slightly weaker agglutinates human erythrocytes. After dialysis against 1% EDTA sodium salt solution the lectin loses hemaglutinating activity, but after the next dialysis against CaCl2 solution it is restored. 相似文献
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A novel developmental stage-specific lectin of the basidiomycete Pleurotus cornucopiae. 总被引:3,自引:0,他引:3
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A novel lectin was isolated from mycelia of the basidiomycete Pleurotus cornucopiae grown on solid medium. The lectin was purified to homogeneity by mucin-Sepharose affinity chromatography. The molecular mass of the lectin was 40 kDa under reducing conditions, but the subunits were polymerized through disulfide bridges under physiological conditions. Hemagglutinating activity of this lectin was completely inhibited by 2-mercaptoethanol, indicating that the multimer is active. The activity was also inhibited by EDTA, and restored by CaCl2. N-Acetyl-D-galactosamine was the most potent hapten inhibitor. N-terminal amino acid sequence analysis revealed that the mycelial lectin was different from the fruit body lectin of this organism. The mycelial lectin appeared prior to fruit body formation and disappeared during the formation of fruit bodies. The lectin was localized on the surface of solid-medium-grown mycelia, and only dikaryotic, and not monokaryotic, mycelia produced the lectin. These results suggest that the appearance of this lectin is associated with fruit body formation. 相似文献
7.
For the first time, a β-glucosidase gene from the edible straw mushroom, Volvariella volvacea V1-1, has been over-expressed in E. coli. The gene product was purified by chromatography showing a single band on SDS-PAGE. The recombinant enzyme had a molecular
mass of 380 kDa with subunits of 97 kDa. The maximum activity was at pH 6.4 and 50 °C over a 5 min assay. The purified enzyme
was stable from pH 5.6–8.0, had a half life of 1 h at 45 °C. The β-glucosidase had a Km of 0.2 mM for p-nitrophenyl-β-D-glucopyranoside. 相似文献
8.
From the dried fruiting bodies of the mushroom Agrocybe cylindracea a heterodimeric lectin with a molecular weight of 31.5 kDa and displaying high hemagglutinating activity was isolated. The molecular weights of its subunits were 16.1 kDa and 15.3 kDa respectively. The larger and the smaller subunits resembled Agaricus bisporus lectin and fungal immunomodulatory protein from Volvariella volvacea respectively in N-terminal sequence. The lectin was adsorbed on DEAE-cellulose in 10 mM Tris-HCl buffer (pH 7.4) and was eluted by the same buffer containing 150 mM NaCl. It was adsorbed on SP-Sepharose in 10 mM NH4OAc (pH 4.5) and eluted by approximately 0.19 M NaCl in the same buffer. The lectin was obtained in a purified form after the mushroom extract had been subjected to (NH4)2SO4 precipitation and the two aforementioned ion exchange chromatographic steps. The lectin exhibited potent mitogenic activity toward mouse splenocytes. The hemagglutinating activity of the lectin was inhibited by lactose, sialic acid and inulin. 相似文献
9.
Lectins are carbohydrate-binding proteins present in a wide variety of plants and animals, which serve various important physiological
functions. A soluble β-galactoside binding lectin has been isolated and purified to homogeneity from buffalo brain using ammonium
sulphate precipitation (40–70%) and gel permeation chromatography on Sephadex G50–80 column. The molecular weight of buffalo brain lectin (BBL) as determined by SDS-PAGE under reducing and non-reducing conditions
was 14.2 kDa, however, with gel filtration it was 28.5 kDa, revealing the dimeric form of protein. The neutral sugar content
of the soluble lectin was estimated to be 3.3%. The BBL showed highest affinity for lactose and other sugar moieties in glycosidic
form, suggesting it to be a β-galactoside binding lectin. The association constant for lactose binding as evidenced by Scatchard
analysis was 6.6 × 103 M−1 showing two carbohydrate binding sites per lectin molecule. A total inhibition of lectin activity was observed by denaturants
like guanidine HCl, thiourea and urea at 6 M concentration. The treatment of BBL with oxidizing agent destroyed its agglutination
activity, abolished its fluorescence, and shifted its UV absorption maxima from 282 to 250 nm. The effect of H2O2 was greatly prevented by lactose indicating that BBL is more stable in the presence of its specific ligand. The purified
lectin was investigated for its brain cell aggregation properties by testing its ability to agglutinate cells isolated from
buffalo and goat brains. Rate of aggregation of buffalo brain cells by purified protein was more than the goat brain cells.
The data from above study suggests that the isolated lectin may belong to the galectin-1 family but is glycosylated unlike
those purified till date. 相似文献
10.
Rameshwaram NR Karanam NK Scharf C Völker U Nadimpalli SK 《Glycoconjugate journal》2009,26(2):161-172
A new unique lectin (galactose-specific) purified from the seeds of Dolichos lablab, designated as DLL-II is a heterodimer composed of closely related subunits α and β. These were separated by SDS-PAGE and
isolated by electroelution. By ESI-MS analysis their molecular masses were found to be 30.746 kDa (α) and 28.815 kDa (β) respectively.
Both subunits were glycosylated and displayed similar amino acid composition. Using advanced mass spectrometry in combination
with de novo sequencing and database searches for the peptides derived by enzymatic and chemical cleavage of these subunits, the primary
sequence was deduced. This revealed DLL-II to be made of two polypeptide chains of 281(α) and 263(β) amino acids respectively.
The β subunit differed from the α subunit by the absence of some amino acids at the carboxy terminal end. This structural
difference suggests that possibly, the β subunit is derived from the α subunit by posttranslational proteolytic modification
at the COOH-terminus. Comparison of the DLL-II sequence to other leguminous seed lectins indicates a high degree of structural
conservation.
Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
11.
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
12.
An α-D-galactose-specific lectin from the seeds of jack fruit (Artocarpus integra) has been isolated in pure form by affinity chromatography on immobilised guar gum (a galactomannan). The lectin is shown
to be a glycoprotein containing 3% carbohydrate and having a molecular weight of 39,500 as determined by gel filtration. Sodium
dodecyl sulphate gel electrophoresis revealed a single polypeptide of 10,500 dalton, indicating that the native lectin is
a tetrarner of identical subunits. The hemagglutinating activity of the lectin towards erythrocytes of all blood groups is
found to be the same. 相似文献
13.
D. N. Olennikov S. V. Agafonova A. V. Rokhin T. A. Penzina G. B. Borovskii 《Applied Biochemistry and Microbiology》2012,48(1):65-70
A new glucan, namely, piptoporane I, with a molecular mass of 270 kDa was isolated from fruiting bodies of Piptoporus betulinis (Bull.:Fr.) Karst. (Fomitopsidacaeae). Using a combination of physicochemical methods, it was established that piptoporane
I was a branched glucan with a backbone consisting of α-(1 → 3)-glucopyranose residues substituted at the C-6 position by
single residues of β-D-glucopyranose by 17.3%. A polysaccharide with such a structure was isolated for the first time from
the fungus genus Piptoporus. 相似文献
14.
D. N. Olennikov S. V. Agafonova G. B. Borovskii T. A. Penzina A. V. Rokhin 《Applied Biochemistry and Microbiology》2009,45(5):536-543
Endopolysaccharides represented by glucans, galactans, and glycoproteins were isolated from the fruiting bodies of the sulfur
shelf (Laetiporus sulphureus (Bull.:Fr.) Murr.), which were obtained by the naturalplantation method. The study of the major 56-kDa polysaccharide, laetiporan
A, the content of which accounted for 0.28% of the fruiting body weight, showed that it is a β-1,3-glucan containing mannose,
galactose, fucose, xylose, and rhamnose residues at position C-6. An antioxidant effect of laetiporan A was discovered. 相似文献
15.
Ghulam Md Ashraf Sabika Rizvi Shoa Naqvi Nida Suhail Nayeem Bilal Shirin Hasan Mohammad Tabish Naheed Banu 《Amino acids》2010,39(5):1321-1332
A soluble β-galactoside-binding lectin was purified by gel filtration chromatography from Bubalus bubalis heart. Its metal-independent nature, molecular weight of 14.5 kDa, preferential affinity for β-d-lactose, and 87–92% identity with carbohydrate recognition domain of previously reported galectin-1 confirmed its inclusion
in galectin-1 subfamily. Stokes radii determination using gel filtration under reducing and non-reducing conditions revealed
its homo-dimeric nature, further confirming its Gal-1 nomenclature. The purified lectin was found to be the most stable mammalian
heart galectin purified till date, suggesting its preferential use in various recognition studies. Treatment of the purified
lectin with oxidizing agent, thiol blocking reagents, denaturants, and detergents resulted in significant changes in UV–VIS,
fluorescence, CD and FTIR spectra, which strongly emphasized the important aspect of regular secondary structure of galectins
for the maintenance of their active conformation. Reduction of the activity of the purified lectin after oxidation by H2O2, with remarkable fluorescence quenching, may suggest potential role for galectin-1 in free radical-induced, oxidative stress-mediated
cardiovascular disorders. The predictions of bioinformatics studies were found to be in accordance with the results obtained
in wet lab. 相似文献
16.
The step of recognition and (or) binding for the development of the disease of the cultivated mushroom Agaricus bisporus by the mycoparasite Verticillium fungicola was studied by several approaches: agglutination of V. fungicola germinated spores by an A. bisporus extract from fruit body cell walls, immunofluorescence microscopy of A. bisporus hyphae from fruit bodies and vegetative mycelia pretreated with purified V. fungicola cell wall glucogalactomannan, and finally, by hemagglutination experiments carried out with an A. bisporus fruit body lectin in the presence and absence of the same glucogalactomannan. Hemagglutinating activity of the purified A. bisporus fruit body lectin was clearly inhibited by the V. fungicola glucogalactomannan, whereas in the A. bisporus vegetative mycelium such lectin was not encountered. All the results obtained make evident the recognition and binding of the A. bisporus fruit body lectin to the V. fungicola cell wall glucogalactomannan, clarifying why the mushrooms, but not the vegetative mycelium, become diseased. 相似文献
17.
A new mannose-binding lectin was isolated from Sternbergia lutea bulbs by affinity chromatography on an α(1-2)mannobiose-Synsorb column and purified further by gel filtration. This lectin
(S. lutea agglutinin; SLA) appeared homogeneous by native-gel electrophoresis at pH 4.3, gel filtration chromatography on a Sephadex
G-75 column, and SDS-polyacrylamide gel electrophoresis, These data indicate that SLA is a dimeric protein (20 kDa) composed
of two identical subunits of 10 kDa which are linked by non-covalent interactions.
The carbohydrate binding specificity of the lectin was investigated by quantitative precipitation and hapten inhibition assays.
It is an α-D-mannose-specific lectin that interacts to form precipitates with various α-mannans, galactomannan and asialo-thyroglobulin,
but not with α-glucans and thyroglobulin. Of the monosaccharides tested only D-mannose was a hapten inhibitor of the SLA-asialothryroglobulin
precipitation system, whereas D-glucose, D-galactose and L-arabinose were not. The lectin appears to be highly specific for
terminal α(1-3)-mannooligosaccharides. The primary structure of SLA appears to be quite similar to that of the snow drop (Galanthus nivalis) bulb lectin which is a mannose-binding lectin from the same plant family Amaryllidaceae. The N-terminal 46 amino acid sequence
SLA showed 7% homology with that of GNA. Abbreviations: AAA, Allium ascalonicum agglutinin (shallot lectin); ASA, Allium sativum
agglutinin (garlic lectin); AUA, Allium ursinum agglutinin (ramsons lectin); DAP, 1,3-diaminopropane; GNA, Galanthus nivalis
agglutinin (snowdrop lectin); HHA, Hippeastrum hybr. agglutinin (amaryllis lectin); LOA, Listera ovata agglutinin (orchid
twayblade lectin); NPA, Narcissus pseudonarcissus agglutinin (daffodil lectin); PAGE, polyacrylamide gel electrophoresis;
PBS, phosphate-buffered saline, SLA, Sternbergia lutea agglutinin; SDS, sodium dodecyl sulfate; Me, methyl; Bn, benzyl; PNP,
p-nitrophenyl.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
18.
Vellareddy Anantharam Sankhavaram R. Patanjali Avadesha Surolia 《Journal of biosciences》1985,8(1-2):403-411
A chitooligosaccharide specific lectin (Luffa acutangula agglutinin) has been purified from the exudate of ridge gourd fruits by affinity chromatography on soybean agglutinin-glycopeptides
coupled to Sepharose-6B. The affinity purified lectin was found homogeneous by polyacrylamide gel electrophoresis, in sodium
dodecyl sulphate-polyacrylamide gels, by gel filtration on Sephadex G-100 and by sedimentation velocity experiments. The relative
molecular weight of this lectin is determined to be 48,000 ±1,000 by gel chromatography and sedimentation equilibrium experiments.
The sedimentation coefficient (S20, w) was obtained to be 4.06 S. The Stokes’ radius of the protein was found to be 2.9 nm by gel filtration. In sodium dodecyl
sulphate-polyacrylamide gel electrophoresis the lectin gave a molecular weight of 24,000 in the presence as well as absence
of 2-mercaptoethanol. The subunits in this dimeric lectin are therefore held by non-covalent interactions alone. The lectin
is not a glycoprotein and circular dichroism spectral studies indicate that this lectin has 31% α-helix and no β-sheet. The
lectin is found to bind specifically to chitooligosaccharides and the affinity of the lectin increases with increasing oligosaccharide
chain length as monitored by near ultra-violet-circular dichroism and intrinsic fluorescence titration. The values of ΔG,
ΔH and ΔS for the binding process showed a pronounced dependence on the size of the oligosaccharide. The values for both ΔH
and ΔS show a significant increase with increase in the oligosaccharide chain length showing that the binding of higher oligomers
is progressively more favoured thermodynamically than chitobiose itself. The thermodynamic data is consistent with an extended
binding site in the lectin which accommodates a tetrasaccharide. Based on the thermodynamic data, blue shifts and fluorescence
enhancement, spatial orientation of chitooligosaccharides in the combining site of the lectin is assigned. 相似文献
19.
An acid α-galactosidase from the seeds of the jack fruit seed (Artocarpus integrifolia) has been purified to homogeneity by affinity chromatography on a matrix formed by cross-linking the soluble α-galactose-bearing
guar seed galactomannan. The 35kDa enzyme was a homotetramer of 9.5kDa subunits. Its carbohydrate part (5.5%) was composed
of galactose and arabinose. TheK
m withp-nitrophenyl α-D-galactoside as substrate was 0.35 mM. TheK
i values indicated inhibition by galactose, 1-O-methyl α-galactose and melibiose in the decreasing order. Among α-galactosides,
the enzyme liberated galactose from melibiose, but not from raffinose or stachyose at its pH optimum (5.2). The guar seed
galactomannan was however efficiently degalactosidated; limited enzyme treatment abolished the precipitability of the polysaccharide
by the α-galactose-specific jack fruit seed lectin, and complete hydrolysis yielded insoluble polysaccharide. Though similar
in sugar specificity and subunit assembly, α-galactosidase and the lectin coexisting in the jack fruit seed gave no indication
of immunological identity. 相似文献
20.
A new lectin with highly potent antihepatoma and antisarcoma activities from the oyster mushroom Pleurotus ostreatus 总被引:5,自引:0,他引:5
A dimeric lectin, composed of subunits with a molecular weight of 40 and 41 kDa, respectively, and demonstrating similarity in N-terminal sequence to each other and to Aleuria aurantia lectin, was isolated from fresh fruiting bodies of the edible mushroom Pleurotus ostreatus. The lectin was unadsorbed on DEAE-cellulose in 10 mmol/L phosphate buffer (pH 7.5), subsequently adsorbed on CM-cellulose in 10 mmol/L NH(4)OAc (pH 5.4), and came off in the first peak from a Superose 12 column during fast protein liquid chromatography. The lectin was acid-labile, alkali-labile, and heat-labile. Its hemagglutinating activity was sensitive to inhibition by CaCl(2), MgCl(2), MnCL(2) and FeCl(3) and potentiation by AlCl(3). Melibiose, lactose, d-galactose, alpha-methyl-d-galactopyranoside, N-acetylneuraminic acid, raffinose, and inulin were capable of inhibiting its hemagglutinating activity, with melibiose being the most potent. The lectin exerted potent antitumor activity in mice bearing sarcoma S-180 and hepatoma H-22. Survival in these mice was prolonged and body weight increase reduced after lectin treatment. 相似文献