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1.
Lectins are carbohydrate-binding proteins widely used in biochemical, immunochemical, and histochemical studies. Bauhinia purpurea lectin (BPA) is a leguminous lectin with an affinity for galactose and lactose. Nine amino acids, DTWPNTEWS, corresponding to the amino acid sequence from aspartic acid-135 to serine-143 in the primary structure of BPA were replaced with the corresponding amino acid residues from the mannose-binding Lens culinaris lectin (LCA), and the chimeric lectin obtained was expressed in Escherichia coli cells. The carbohydrate-binding specificity of the recombinant chimeric lectin was investigated in detail by comparing the elution profiles of various glycopeptides and oligosaccharides with defined carbohydate structures from immobilized lectin columns. Glycopeptides carrying three constitutive carbohydrate sequences of Galbeta1-3GalNAc-Ser/Thr and a complex-type biantennary glycopeptide, which show a high affinity for BPA or LCA, were shown to have no affinity for the chimeric lectin. In contrast, hybrid-type and high mannose-type glycopeptides with a Manalpha1-6(Manalpha1-3)Manalpha1-6Man sequence were found to have a moderate affinity for the chimeric lectin. This result demonstrates that a novel type of lectin with a unique carbohydrate-binding specificity can be constructed from BPA by substituting several amino acid residues in its metal-binding region with other amino acid residues. Additional lectin(s) with distinctly different carbohydrate-binding specificities will provide a powerful tool for many studies. 相似文献
2.
The aim of the present study was to isolate a laccase from fruiting bodies of the yellow mushroom Cantharellus cibarius. The fruiting body extract was subjected to a purification protocol that involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel and Con A-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. The laccase was unadsorbed on DEAE-cellulose and Affi-gel blue gel and adsorbed on Con A-Sepharose. The laccase was composed of two identical subunits each with a molecular mass of 46 kDa. The laccase exhibited a temperature-dependent rise in activity over the temperature range 20-50 degrees C. When the temperature was raised above 60 degrees C there was a fall in enzyme activity. The enzyme manifested maximal activity at pH 4. At and above pH 6 there was a dramatic reduction in activity. The unique features of this fruiting body laccase compared with previously reported mycelial laccases include homodimeric nature, a distinctive N-terminal sequence, a higher optimal pH, and adsorption on only ConA-Sepharose among the various chromatographic media tested. 相似文献
3.
Oguri S Amano K Nakashita H Nagata Y Momonoki YS 《Bioscience, biotechnology, and biochemistry》2008,72(10):2640-2650
A cDNA encoding tomato fruit lectin was cloned from an unripe cherry-tomato fruit cDNA library. The isolated lectin cDNA contained an open reading frame encoding 365 amino acids, including peptides that were sequenced. The deduced sequence consisted of three distinct domains: (i) an N-terminal short extensin-like domain; (ii) a Cys-rich carbohydrate binding domain composed of four almost identical chitin-binding domains; (iii) an internal extensin-like domain of 101 residues containing 15 SerPro(4) motifs inserted between the first and second chitin-binding domains. The molecular weight of the lectin was 65,633 and that of the deglycosylated lectin was 32,948, as determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). This correlated with the estimated molecular weight of the deduced sequence. Recombinant tomato lectin expressed in Pichia pastoris possessed chitin-binding but not hemagglutinating activity. These findings confirmed that the cDNA encoded tomato lectin. 相似文献
4.
N E Fink de Cabutti M Caron R Joubert M T Elola D Bladier J Herkovitz 《FEBS letters》1987,223(2):330-334
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. 相似文献
5.
An acetylcholinesterase purified from the greenbug (Schizaphis graminum) with some unique enzymological and pharmacological characteristics 总被引:1,自引:0,他引:1
An acetylcholinesterase (AChE, EC 3.1.1.7) was purified from the greenbug, Schizaphis graminum (Rondani). The maximum velocities (Vmax) for hydrolyzing acetylthiocholine (ATC), acetyl-(beta-methyl) thiocholine (AbetaMTC), propionylthiocholine, and S-butyrylthiocholine were 78.0, 67.0, 37.4, and 2.3 micromol/min/mg, and the Michaelis constants (Km) were 57.6, 60.6, 31.3, and 33.4 microM, respectively. More than 98% of AChE activity was inhibited by 10 microM eserine or BW284C51, but only 7% of the activity was inhibited by ethopropazine at the same concentration. Based on the substrate and inhibitor specificities, the purified enzyme appeared to be a true AChE. Nondenaturing polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing of the purified AChE revealed three molecular forms. The isoelectric points were 7.3 for the major form and 6.3 and 7.1 for two minor forms. The major form of purified AChE showed molecular masses of 129 kDa for its native protein and 72 kDa for its subunits on SDS-PAGE. However, the purified AChE exhibited some distinctive characteristics including: (1) lack of affinity to the affinity ligand 3-(carboxyphenyl) ethyldimethyl ammonium, which has been used widely in purification of AChE from various insect species; and (2) 20-200-fold higher substrate-inhibition thresholds for ATC and AbetaMTC than AChE from other insect species. These biochemical properties may reflect structural differences of AChE purified from the greenbug compared with that from other insect species. 相似文献
6.
The complete amino acid sequence of a galactose-specific lectin from the plasma of the ascidian Halocynthia roretzi has been determined by sequential Edman degradation analysis of peptide fragments derived by proteolytic fragmentation and chemical cleavage of the reductive S-pyridylethylated lectin. Peptide fragments were separated by reverse-phase HPLC. The N-terminal and C-terminal amino acid sequences were determined by Edman degradation and enzymatic digestion. The H. roretzi plasma lectin is a single-chain protein consisting of 327 amino acids and four disulfide bonds, one of which was found to be cross-linked intramolecularly. A comparison of the amino acid sequence of the H. roretzi plasma lectin with the sequences of other proteins reveals that the H. roretzi lectin has a structure consisting of a twice-repeated sequence, a fibrinogen-related sequence and a C-type lectin-homologous sequence. The above amino acid sequence was verified by cDNA cloning of this lectin. Three cDNA clones that have single ORFs encoding the lectin precursor were isolated from an H. roretzi hepatopancreas cDNA library. The deduced amino acid sequences in the three cDNA clones contain the same sequence of the mature lectin molecule and the same putative signal sequence. In addition, it was demonstrated that this lectin can enhance phagocytosis by H. roretzi hemocytes. Thus, the plasma lectin is constructed into an oligomer structure via intermolecular disulfide bonds and plays a role in the biological defense of H. roretzi as a defense molecule. 相似文献
7.
A comparison of tomato (Lycopersicon esculentum) lectin with its deglycosylated derivative 总被引:1,自引:0,他引:1
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D C Kilpatrick C Graham S J Urbaniak C E Jeffree A K Allen 《The Biochemical journal》1984,220(3):843-847
A regime is proposed for the design of coupled enzyme assays in which auxiliary enzymes are added at concentrations proportional to their Km values. Under these conditions it is possible to calculate the complete time course of the assay including the time required for the system to approach its steady state. The consequence of increasing the number of coupling enzymes is shown to be a considerable decrease in time required to reach the steady state provided that the overall transient time remains the same. The method is extended to the general consideration of pathways and shows that pathways of the same length exhibit identical temporal responses provided that the units of concentration and time used are based on the steady-state concentration of intermediates and the transient time respectively. An unexpected finding is that increasing the number of intermediates in a pathway can decrease the time required to enter a steady state. 相似文献
8.
S Madadi-Kahkesh E C Duin S Heim S P Albracht M K Johnson R Hedderich 《European journal of biochemistry》2001,268(9):2566-2577
Heterodisulfide reductase (Hdr) from methanogenic archaea is an iron-sulfur protein that catalyses the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic thiol coenzymes, coenzyme M (H-S-CoM) and coenzyme B (H-S-CoB). In EPR spectroscopic studies with the enzyme from Methanothermobacter marburgensis, we have identified a unique paramagnetic species that is formed upon reaction of the oxidized enzyme with H-S-CoM in the absence of H-S-CoB. This paramagnetic species can be reduced in a one-electron step with a midpoint-potential of -185 mV but not further oxidized. A broadening of the EPR signal in the 57Fe-enriched enzyme indicates that it is at least partially iron based. The g values (gxyz = 2.013, 1.991 and 1.938) and the midpoint potential argue against a conventional [2Fe-2S]+, [3Fe-4S]+, [4Fe-4S]+ or [4Fe-4S]3+ cluster. This species reacts with H-S-CoB to form an EPR silent form. Hence, we propose that only a half reaction is catalysed in the presence of H-S-CoM and that a reaction intermediate is trapped. This reaction intermediate is thought to be a [4Fe-4S]3+ cluster that is coordinated by one of the cysteines of a nearby active-site disulfide or by the sulfur of H-S-CoM. A paramagnetic species with similar EPR properties was also identified in Hdr from Methanosarcina barkeri. 相似文献
9.
The investigated species are Lycopersicon cheesmanii s.str., L. cheesmanii var. minor, L. chilense, L. hirsutum s. str., L. hirsuturn f. glabratum , L. "parviflorum", L. chmielewskii, L. peruvianum s. str., L. peruvianum var. humifusum, L. pimpinellifolium and Solanum penellii . Wound-emitted leaf volatiles isolated and concentrated by adsorption on Tenax GC were separated by capillary gas chromatography. The different species produce different and rather broad patterns of volatiles especially compared to a modem tomato cultivar. There is also a considerable difference between varieties of the same species. When more than one accession was investigated, the results indicated a chemotype differentiation. The chemotypes are in some cases specialized with one or a few quantitatively dominating main components. L. hirsutum emits the greatest number of components. Three accessions had α-zingeberene, α-santalene, and limonene as main component, respectively. The investigated accession of L. hirsutum f. glabratum has 2-undecanone as main component, instead of 2-tride-canone previously identified as main component in another accession. Both compounds are natural insecticides. The results illustrate the need for basic knowledge of the chemical characters in the ancestors of cultivated plants. 相似文献
10.
Gaidamashvili M Ohizumi Y Iijima S Takayama T Ogawa T Muramoto K 《The Journal of biological chemistry》2004,279(25):26028-26035
Four major proteins designated DB1, DB2, DB3, and DB4 were isolated and characterized from the yam tuber Dioscorea batatas. The ratios of their yields were 20:50:20:10. DB1 was a mannose-binding lectin (20 kDa) consisting of 10-kDa subunits and was classified as the monocot mannose-binding lectin family. DB2, accounting for 50% of the total protein, was the storage protein, commonly called dioscorins consisting of a 31-kDa subunit. On the basis of amino acid sequence, DB2 was classified to be dioscorin A. DB3 was a maltose-binding lectin, having an apparent molecular mass of 120 kDa and composed of a 66-kDa subunit and two 31-kDa subunits (DB3S). The 66-kDa subunit was further composed of two 31-kDa subunits (DB3L) cross-linked by disulfide bonds. DB3L and DB3S (242 and 241 amino acid residues, respectively) were homologous with each other with 72% sequence identity. They showed a sequence homology to dioscorin B and dioscorin A from Dioscorea alata, with 90 and 93% identity, respectively, and to carbonic anhydrase from Arabidopsis thaliana with about 45% identity. DB3S had one intrachain disulfide bond located at Cys(28)-Cys(187), whereas DB3L had one interchain disulfide bond (Cys(40)-Cys(40)') in addition to the intrachain disulfide bond (Cys(28)-Cys(188)) to form a 66-kDa subunit. DB1 and DB3 agglutinated rabbit erythrocytes at 2.7 and 3.9 microg/ml, respectively. Despite the structural homology between DB2 and DB3, DB2 had no lectin activity. The 66-kDa subunit itself revealed the full hemagglutinating activity of DB3, indicating that DB3L but not DB3S was responsible for the activity. The hemagglutinating activity of DB3 required Ca(2+) ions and was exclusively inhibited by maltose and oligomaltoses (e.g. maltopentaose and maltohexaose) but not by d-glucose. DB3 could not be classified into any known plant lectin family. DB4 was a chitinase, homologous to an acidic chitinase from Dioscorea japonica. DB1, DB2, and DB3 did not show any activity of carbonic anhydrase, amylase, or trypsin inhibitor activity. These results show that two of the four major proteins isolated from the yam tubers D. batatas have unique lectin activities. 相似文献
11.
Carbohydrate binding specificity of the galactose-specific, major lectin of mistletoe extract (ML-1) was studied by an inhibition assay using monosaccharides, monosaccharide derivatives, disaccharides, and compounds containing multiple galactosyl terminals. The results indicate that 1) both alpha- and beta-galactosyl residues are recognized equally well; 2) each of the hydroxyl groups of galactose contributes to varying degrees to the binding process, the 4-OH being the most important and the 6-OH the least important hydroxyl group; 3) disaccharide sequences of Gal beta 2Gal and Gal beta 3Gal have much higher affinity than galactose, whereas affinity of all other Gal-disaccharides is only slightly better than galactose; 4) macromolecular ligands having 10 or more terminal galactosyl residues have 500-fold higher affinity than Gal; and 5) a group on ML-1 with pK alpha of 4.8 appears to be involved in the binding of ligand. 相似文献
12.
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. 相似文献
13.
A galactose-binding lectin was isolated in electrophoretically pure form from the seeds of the snake gourd,Trichosanthes anguina, by affinity chromatography on an immobilised lactose column, as well as on a cross-linkedGuar Gum column. The lectin agglutinates native erythrocytes of human A, B and 0 phenotypes and of rabbit, rat and mouse. The molecular mass of the lectin, as estimated bySephadex G-200 gel chromatography, is 49 kDa. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis, after reduction with β-mercaptoethanol, revealed two polypeptide chains linked by disulphide bonds in the lectin molecule. It contains no covalently linked sugars. Amino acid analysis of the lectin revealed a high content of acidic amino acids, relatively lower proportion of basic amino acids and traces of cysteine and methionine. The lectin has good thermal stability, and is inactivated when oxidised by metaperiodate. 相似文献
14.
Hybrid glycoprotein and neoproteoglycan probes were prepared by coupling various glycoproteins or polysaccharides to peroxidase or biotinyl bovine serum albumin, respectively. Lectins recognizable by the neoglycoconjugate probes were extracted from 16 cultivable mushrooms. Dot-blot assay revealed five extracts to be reactive with only hybrid glycoprotein probes, but others also reacted with neoproteoglycan probes. According to the reactivity pattern with probe screening, the one lectin from Oudemansiella platyphylla extract (OPL) bound best with asialotransferrin-- and asialoagalactotransferrin--peroxidase probes and was isolated using an asialotransferrin column, but it did not bind with other hybrid glycoprotein or neoproteoglycan probes. OPL, consisting of two polypeptides with high homology in the N-terminal amino acid sequences, exhibited weak hemagglutinating activity. Purified OPL specifically bound the beta-GlcNAc probe among various biotinylated polymeric sugar probes, while it exhibited essentially the same binding specificity toward neoglycoconjugate probes as that of the crude extract, showing a preference for the asialobiantennary complex type of N-linked glycans. These results indicate that the neoglycoconjugate probes are valuable in lectin screening. 相似文献
15.
16.
Identification and some properties of a unique DNA polymerase from cells infected with human B-lymphotropic virus. 总被引:1,自引:0,他引:1
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A new DNA polymerase and DNase activity were identified from cells infected with human B-lymphotropic herpesvirus (HBLV). DNA polymerase associated with HBLV infection was similar in its sensitivity to inhibition by ppi analogs as other herpesvirus-specific DNA polymerases but was dissimilar in its inhibition by certain nucleoside triphosphates. 相似文献
17.
Bhagat Yogesh S. Bhat Ramesh S. Kolekar Rohini M. Patil Ashlesha C. Lingaraju S. Patil R. V. Udikeri S. S. 《Transgenic research》2019,28(3-4):299-315
Transgenic Research - Root knot nematodes are serious threats to growth and yield of solaneous crops including tomato. In this study, a binary vector carrying Remusatia vivipara... 相似文献
18.
19.
Two genes involved in iron utilization in Campylobacter coli VC167 T1 have been characterized. The cfrA gene encodes a protein with a predicted Mr of 77,653 which, after processing of the leader sequence, has a predicted Mr of 75,635. This protein has significant sequence identity to siderophore receptors of several bacteria, and site-specific mutants defective in cfrA do not synthesize one of two major iron-repressible outer membrane proteins. An adjacent gene encodes a TonB-like protein; a mutant in this gene lost the ability to utilize hemin, ferrichrome, and enterochelin as iron sources. The cfrA and tonB genes of VC167 T1 hybridized to all strains of C. coli and most strains of C. jejuni examined but did not hybridize to several other strains of C. jejuni, suggesting that the thermophilic campylobacters can be separated into two categories based on the presence of these two iron utilization genes. 相似文献
20.
The lectin from Datura stramonium can be inhibited by oligomers of N-acetylglucosamine. This property was exploited to purify the lectin by affinity chromatography on Sepharosefetuin. The purified lectin is a glycoprotein in having subunits of 40 000 and 45 000 mol.wt. 相似文献