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1.
Chrysobactin (-N-(2,3-dihydroxybenzoyl)-d-lysyl-l-serine), a siderophore that is essential for systemic virulence by plant pathogenic Erwinia chrysanthemi, was synthesized with high diastereomeric purity. Chrysobactin was prepared by coupling the N-hydroxysuccinimide ester of -N-(2,3-dibenzyloxybenzoyl)--N-Cbz-d-lysine with l-serine benzyl ester followed by deprotection via hydrogenolysis. Optically pure chrysobactin was obtained with 98% overall yield. A monoclonal antibody to ferric chrysobactin was developed and characterized as IgM. The antibody reacts with chrysobactin, ferric chrysobactin and less strongly with ferric dihydroxybenzoic acid. The antibody reacts weakly with the siderophores ferrichrome, A, ferric pseudobactin and ferric rhodotorulic acid. This antibody was used in a competitive immunoassay to detect ferric chrysobactin at 10–8 to 10–10 mol. This immunoassay may provide a useful method for the detection of chrysobactin in plant samples. 相似文献
2.
Using the ammonium analogue 14CH3NH
3
+
, ammonium transport was studied in the cyanobiont cells freshly isolated from the root nodules of Cycas revoluta. An L-methionine-dl-sulphoximine (MSX)-insensitive ammonium-transport system, which was dependent on membrane potential (), was found in the cyanobiont. However, the cyanobiont was incapable of metabolizing exogenous 14CH3NH
3
+
or NH
4
+
because of the absence of another ammonium-transport system responsible for the uptake of ammonium for assimilation via glutamine synthetase (EC 6.3.1.2). Such a modification seems to be the result of symbiosis because the free-living cultured isolate, Anabaena cycadeae, has been shown to possess both the ammonium-transport systems.Abbreviations and symbol ATS/ATSs
ammonium transport system/systems
- Chl
chlorophyll
- GS
glutamine synthetase
- MSX
L-methionine-dl-sulphoximine
-
membrane potential 相似文献
3.
-Galactosidase II2 (MW 43 390) from resting Vicia faba L. seeds had been shown to possess d-glucose/d-mannose-specific lectin activity. Inhibition studies with monosaccharides and an examination of the effects of heat and pH on the catalytic and lectin activities of the enzyme indicate that the enzyme substrate and the lectin haptens bind at different sites on the protein. d-Mannosebinding has been investigated by equilibrium dialysis and spectrophotometrically. Both methods yield Ka values of approx. 3·103 M-1 for the interaction and there would appear to be two mannosebinding sites per molecule of enzyme protein. The lectin properties of V. faba -galactosidase II2 have been discussed in relation to both V. faba lectin (favin) and other legume -galactosidases.Abbreviations con A
concanavalin A
- CM-cellulose
carboxymethyl cellulose
- MW
molecular weight
- PNPG
p-nitrophenyl -d-galactoside
- SDS
sodium dodecyl sulphate
- PAGE
polyacrylamide-gel electrophoresis 相似文献
4.
Jonathan A. Lindquist Elisabeth Barofsky Philip N. McFadden 《Journal of Protein Chemistry》1996,15(1):115-122
Protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase (PCM, E.C. 2.1.1.77) was previously shown to be enzymatically methyl esterified in an autocatalytic manner at altered aspartyl residues; methyl esters are observed in a subpopulation of the enzyme termed thePCM fraction [Lindquist and McFadden (1994),J. Protein Chem.
13, 23–30]. The altered aspartyl sites serving as methyl acceptors inPCM have now been localized by using proteolytic enzymes and chemical cleavage techniques in combination with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to identify fragments of the [3H]automethylated enzyme that contain a [3H]methyl ester. Methylation was positively identified at positions Asn188 and Asp217 in the enzyme sequence, a consequence of the spontaneous alteration of these sites tol-isoaspartyl ord-aspartyl sites and their methylation by active PCM molecules. The identification of more than one site of automethylation shows thatPCM is not a homogeneous population of damaged PCM molecules, but rather a complex population of molecules with a variety of age-altered damage sites.Abbreviations PCM
protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase
- EDTA
disodium ethylenediaminetetraacetate
- PMSF
phenylmethylsulfonyl fluoride
- TEA
trifluoroacetic acid
- HPLC
high-pressure liquid chromatography 相似文献
5.
J. Zhang V. K. Tiwari T. J. Golds N. W. Blackhall E. C. Cocking B. J. Mulligan J. B. Power M. R. Davey 《Plant Cell, Tissue and Organ Culture》1995,41(2):125-138
Cat gene expression has been investigated following PEG-mediated plasmid uptake into barley protoplasts. The uptake conditions optimised for transient expression were employed for stable transformation. Transformed protoplast-derived calli of the cvs. Dissa and Igri, were selected on medium containing G418 at 40 g ml–1 or kanamycin sulphate at 250 g ml–1. Absolute transformation frequencies of 28.9×10–5 and 21.3×10–5 were recorded for Dissa with kanamycin sulphate and G418 selection, respectively. The frequency for Igri was 11.5×10–5 with G418 selection. Antibiotic resistant protoplast-derived colonies expressed NPTII activity; Southern hybridisation confirmed integration of the nptII gene into barley genomic DNA.Abbreviations ABA
abscisic acid
- AC-CAP
acetylated chloramphenicol
- BAP
6-benzylaminopurine
-
cat
chloramphenicol acetyltransferase gene
- CAT
chloramphenicol acetyltransferase activity
- CaMV
cauliflower mosaic virus
- CAP
chloramphenicol, 2,4-d-2,4-dichlorophenoxyacetic acid
- GA3
gibberellic acid
- G418
Geneticin
-
gus
-glucuronidase gene
- HEPES
(N[2-hydroxyethyl] piperazine-N-[2-ethanesulphonic acid])
- IAA
indole acetic acid
- MES
2-N-morpholinoethane sulphonic acid
- NAA
-naphthaleneacetic acid
-
npt II
neomycin phosphotransferase gene
- NPTH
neomycin phosphotransferase activity
- PEG
polyethylene glycol
- SCV
settled cell volume 相似文献
6.
Hiroaki Nakagawa Noriko Takahashi Kazuhisa Fujikawa Yoshiya Kawamura Masaki Iino Hiroyuki Takeya Hiroyuki Ogawa Koji Suzuki 《Glycoconjugate journal》1995,12(2):173-181
Human blood coagulation factor X has two N-linked oligosaccharides at Asn39 and Asn49 residues and two O-linked oligosaccharides at Thr17 and Thr29 residues in the region of the factorX activationpeptide (XAP) which is cleaved off during its activation by factor IXa. We determined the structure of oligosaccharides in the XAP region of human factor X. Four glycopeptides each containing a glycosylation site were isolated by digestion of XAP with endoproteinase Asp-N followed by reversed-phase HPLC. N-linked oligosaccharides released from the glycopeptides by glycoamidase A digestion were derivatized with 2-aminopyridine. Pyridylamino(PA)-oligosaccharides were separated by HPLC into neutral and sialyl oligosaccharides using an anion-exchange column. Structures of oligosaccharides and their contents at each glycosylation site were determined by a two-dimensional sugar mapping method. The contents of the neutral oligosaccharides at Asn39 and Asn49 residues were 32.5% and 30.0%, respectively. Six neutral and twelve monosialyl oligosaccharides isolated from both N-linked glycosylation sites showed similar elution profiles composed of bi-, tri-and tetra-antennary complex type oligosaccharides. The predominant component in neutral oligosaccharides was biantennary without a fucose residue. Two major monosialyl oligosaccharides were also biantennary without fucose and with a Neu5Ac-26 residue. In addition, the structures of O-linked oligosaccharides at Thr17 and Thr29 residues were suggested to be disialylated Gal/3GalNAc sequences by their component analyses.Abbreviations Gal
d-galactose
- GlcNAc
N-acetyl-d-glucosamine
- Man
d-mannose
- HPLC
high-performance liquid chromatography
- NDV
Newcastle disease virus
- Neu5Ac
5-N-acetylneuraminic acid
- ODS
octadecylsilyl
- PA
pyridylamino
- RVV-X
Russell's viper venom factor X activator
- TBS
Tris-buffered saline
- XAP
factor X activation peptide. 相似文献
7.
Fuyi Wang Juraj Bella John A. Parkinson Peter J. Sadler 《Journal of biological inorganic chemistry》2005,10(2):147-155
The ruthenium arene anticancer complex [(6-bip)Ru(en)Cl][PF6] (1) (bip is biphenyl, en is ethylenediamine) reacted slowly with the amino acid L-histidine (L-His) in aqueous solution at 310 K. Two L-His adducts of 1 were separated by high-performance liquid chromatography and identified by electrospray ionisation mass spectrometry and NMR: an imidazole N-bound complex [(6-bip)Ru(en)(N–L-His)]2+, and an N-bound complex [(6-bip)Ru(en)(N–L-His)]2+. At 310 K, after 24 h only about 22% of complex 1 (2 mM) reacted with L-His, and of the unreacted 1, 59% had hydrolysed. In the presence of 100 mM NaCl, approximately 90% of 1 remained unreacted. In aqueous solution or triethylammonium acetate (TEAA) buffer (pH 7.6), 15N-labelled 1 reacted with cytochrome c to give two monoruthenated protein adducts. The reaction reached equilibrium within 2 h by which time approximately 50% of cytochrome c was ruthenated. On the basis of [1H, 15N] NMR data, one adduct may have Ru bound to the N-terminus, and the other to a carboxylate group on the protein. In TEAA buffer and at 310 K, more than 90% of the 14-mer oligonucleotide d(TATGTACCATGTAT) reacted with 2 mol Eq of 1 to give rise to monoruthenated and diruthenated oligonucleotide adducts. The presence of cytochrome c (1 mol Eq) or L-His (4 mol Eq) had little effect on the course of the reaction with the oligonucleotide. In cells, DNA (or RNA) may be a favoured reaction site for this Ru anticancer complex.Electronic supplementary material is available for this article at .
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8.
Philip H. Johnson Alastair S. R. Donald James Feeney Winifred M. Watkins 《Glycoconjugate journal》1992,9(5):251-264
The acceptor specificity and general properties of a Lewis blood-group gene associated -3/4-L-fucosyltransferase isolated from human milk have been examined at the penultimate purification stage involving affinity chromatography on GDP-hexanolamine Sepharose, and after a subsequent gel filtration step on Sephacryl S-200. Both preparations transferred fucose to theO-4 position ofN-acetylglucosamine in Type 1 (Gal1-3GlcNAc-R) acceptors and theO-3 position of glucose in lactose-based (Gal1-4Glc) oligosaccharides, and both used Type 1 sialylated compounds when the terminalN-acetylneuraminic acid was present in -2,3 linkage. The striking difference between the two preparations was in their reactivity with Type 2 (Gal1-4GlcNAc-R) chains; after Sephacryl S-200 chromatography the apparentK
M values for the -3/4- preparation with unsubstituted low-molecular-weight Type 2 oligosaccharides were considerably increased. Substitution of the terminal galactose with sialic acid in -2,3 linkage decreased theK
M values for low-molecular-weight oligosaccharides but no detectable incorporation of fucose was observed intoN-acetyllactosamine end-groups of glycoproteins withN-linked oligosaccharide chains, irrespective of the presence of sialic acid in the terminal sequences.Deceased 25 June 1991. 相似文献
9.
Willy R. Kinzy David M. Belenky Frank G. Loontiens Nathan Sharon 《Glycoconjugate journal》1992,9(5):225-227
The synthesis of the methyl - and -N-dansyl-d-galactosaminides is described using methyl ,-2-azido-2-deoxy-d-galactopyranoside as starting material. This was reduced to the corresponding methyl ,-2-amino-2-deoxy-d-galactopyranoside and then treated with dansyl chloride to yield a mixture of methyl ,-N-dansyl-d-galactosaminides which was separated into individual anomeric forms by flash chromatography on silica gel. Methyl -N-dansyl-d-galactosaminide was used as a fluorescent indicator ligand in continuous substitution titrations to determine the association constants of nonchromophoric carbohydrates with theN-acetyl-d-galactosamine specific lectin fromErythrina corallodendron.Abbreviations ECorL
Erythrina corallodendron lectin
- MeGalNDns
methyl 2-deoxy-2-(5-dimethylamino-1-naphthalenesulfamido)--d-galactopyranoside
- MeGalNDns
methyl 2-deoxy-2-(5-dimethylamino-1-naphthalenesulfamido)--d-galactopyranoside
Dedicated to Hilde De Boeck (1958–1991). 相似文献
10.
S. P. O. Werbrouck P. Redig H. A. Van Onckelen P. C. Debergh 《Journal of Plant Growth Regulation》1996,15(2):87-93
Imidazole fungicides such as imazalil, prochloraz, and triflurnizole and the triazole growth retardant paclobutrazol promote the shoot-inducing effect of exogenous cytokinins in Araceae, such as Spathiphyllum floribundum Schott and Anthurium andreanum Schott. The mechanism of their action could partially be based on the inhibition of gibberellic acid (GA) biosynthesis, because administration of GA3 inhibits the phenomenon completely in S. floribundum. Not only is the suppression of GA biosynthesis involved, but also the metabolism of endogenous cytokinins is significantly altered. Although the balance between isopentenyladenine, zeatin, dihydrozeatin, and their derivatives was shifted to distinguished directions by administration of BA and/or imazalil and/or GA3, no correlation between these changes in metabolic pathways and the number of shoots could be found. The metabolism of BA was not significantly altered by adding imazalil to the micropropagation medium of S. floribundum.Abbreviations 2,4-D
2,4-dichlorophenoxyacetic acid
- [9R-5P]DHZ
9--d-ribofuranosyl-dihydrozeatin-monophosphate
- [9R-5P]iP
6-isopentenyl-9--d-ribofuranosyladenine-monophosphate
- [9R-5P]Z
9--d-ribofuranosyl-zeatin-monophosphate
- [9G]BA
6-benzyl-9--d-glucopyranosyladenine
- [9G]DHZ
9--d-glucopyranosyl-dihydrozeatin
- [9G]iP
6-isopentenyl-9--d-glucopyranosyladenine
- [9G]Z
9--d-glucopyranosyl-zeatin
- [9R]BA
6-benzyl-9--d-ribofuranosyladenine
- [9R]DHZ
9--d-ribofuranosyl-dihydrozeatin
- [9R]iP
6-isopentenyl-9--d-ribofuranosyladenine
- [9R]Z
9--d-ribofuranosyl-zeatin
- BA
6-benzyladenine
- DHZ
dihydrozeatin
- ES+ LC-MS/MS
HPLC coupled Electrospray Tandem Mass Spectrometry
- f.m.
fresh mass
- mT
6-(3-hydroxybenzyl)adenine
- IMA
imazalil
- iP
isopentenyladenine
- NAA
1-naphthalene acetic acid
- NFT
Nutrient Film Technique
- (OG)[9R]DHZ
O--glucopyranosyl-9--d-ribofuranosyl-dihydrozeatin
- (OG)[9R]Z
O--d-glucopyranosyl-9--d-ribofuranosyl-zeatin
- (OG)DHZ
O--d-glucopyranosyl-dihydrozeatin
- (OG)Z
O--d-glucopyranosyl-zeatin
- PAR
Photosynthetic Active Radiation
- PBZ
paclobutrazol
- PRO
prochloraz
- TDZ
thidiazuron
- TRI
triflurnizole
- Z
zeatin 相似文献
11.
Boris A. Dmitriev Michail V. Ovchinnikov Elena B. Lapina Galina N. Pluzhnikova Igor V. Lopyrev Anatoly Ya. Chernyak 《Glycoconjugate journal》1992,9(4):168-173
Synthetic lipopeptideN-palmitoyltyrosyl-seryl-seryl-asparaginyl-alanine, an analogue of B-mitogenic tripalmitoyl-pentapeptide fromEscherichia coli lipoprotein, was coupled with an oligosaccharide hapten fromNeisseria meningitidis lipooligosaccharide to give a glycopeptidolipid conjugate — the artificial antigen of a new type possessing the type-specific microbial determinant.Abbreviations iBu
isobutyl
- But
t-butyl
- Boc
t-butoxycarbonyl
- DCC
N,N-dicyclohexylcarbodiimide
- DMF
N,N-dimethylformamide
- DMSO
dimethylsulfoxide
- ONB
N-hydroxy-5-norbornen-2,3-dicarboximide ester
- ONp
4-nitrophenyl ester
- Pal
palmitoyl
- TEMED
N,N,N,N-tetramethylethylenediamine
- Z
benzyloxycarbonyl
- KDO
2-keto-3-deoxyoctonic acid
- Hep
L-glycero-d-manno-heptose
- TPP
S-[2,3-bis(palmitoyloxy)-(2RS)propyl]-N-palmitoylcysteinyl-seryl-asparaginyl-alanine. 相似文献
12.
Volker Magnus Roger P. Hangarter Norman E. Good 《Journal of Plant Growth Regulation》1992,11(2):67-75
The interaction of free IAA and its amino acid conjugates on growth and development of cultured tomato hypocotyl tissue (Lycopersicon esculentum Mill. cv. Marglobe) was studied. In a nutrient medium containing 10 mol/L of benzyladenine, free IAA stimulated shoot and root development with little callus proliferation. In contrast, all IAA-amino acid conjugates tested supported mostly callus growth. Simultaneous application of free IAA and its conjugates resulted in the expression of mixed morphogenetic responses (i.e., both vigorous callus growth and organogenesis resulted). Growth kinetics and the effect of temporal exposure of the tissues to the bound and the free auxin suggest that some IAA-amino acid conjugates may specifically influence plant morphogenesis in ways that cannot be easily explained as simply a function of their slow hydrolysis to release free IAA.Abbreviations IAA
indole-3-acetic acid
- IAA-Ala
N-(indol-3-ylacetyl)-l-alanine
- IAA-Asp
N-(indol-3-ylacetyl)-dl-aspartic acid
- IAA-Lys
N
-(indol-3-ylacetyl)-l-lysine
- IAA-Orn
N
-(indol-3-ylacetyl)-l-ornithine
- IAA-Thr
N-(indol-3-ylaetyl)-l-threonine 相似文献
13.
Henri Debray Danuta Dus Jean-Michel Wieruszeski Gérard Strecker Jean Montreuil 《Glycoconjugate journal》1991,8(1):29-37
Four bi-antennary glycan fractions of theN-acetyllactosamine-type, derived from a Lewis lung carcinoma (LL2) cell subline resistant to theAleuria aurantia agglutinin were studied by 400 MHz1H-NMR spectroscopy. By this method, their antennae were found to be terminated either by (2-3 or 6)-linkedN-acetylneuraminic acid or (1-3)-linked galactose residues. The primary structure of glycans of these four glycopeptide or derived oligosaccharide-alditols has been determined in full detail.Abbreviations NAc
N-acetyl group
- NGc
N-glycolyl group
- GlcNAc
N-acetylglucosamine
- NeuAc
N-acetylneuraminic acid
- NeuGc
N-glycolylneuraminic acid
- Man
mannose
- Gal
galactose
- Fuc
fucose
- Con A
concanavalin A
- LCA
Lens culinaris agglutinin
- AAA
Aleuria aurantia agglutinin
- WGA
Wheat germ agglutinin
- RCA II
Ricinus communis agglutinin II
- PBS
phosphate buffered saline, 0.01m Na2HPO4/0.14m NaCl, pH 7.2
- HPLC
high performance liquid chromatography
- EMEM
Eagle's Minimal Essential Medium
- LecR
lectin resistant
- MG
-methylglycoside 相似文献
14.
Holger Stephan Stefan Freund Werner Beck Günther Jung Jean-Marie Meyer Günther Winkelmann 《Biometals》1993,6(2):93-100
Novel linear hydroxamate/hydroxycarboxylate siderophores from strains of Pseudomonas cepacia were isolated and named ornibactins. The ornibactins represent modified tetrapeptide siderophores, possessing the sequence l-Orn1(N
-OH, N
-acyl)-d-threo-Asp(-OH)-l-Ser-l-Orn4(N
-OH, N
-formyl)-1,4-diaminobutane. The N
-acyl groups of Orn1(N
-OH, N
-acyl) may vary and represent the three acids 3-hydroxybutanoic acid, 3-hydroxyhexanoic acid and 3-hydroxyoctanoic acid, leading to a mixture of three different ornibactins, designated according to their acyl chain length as ornibactin-C4, ornibactin-C6 and ornibactin-C8. Each of the siderophores is accompanied by a small amount of a more hydrophilic component with a 16 a.m.u. higher mass. The structure elucidation was based on results from gas chromatography amino acid analysis, electrospray mass spectrometry, and one- and two-dimensional nuclear magnetic resonance techniques. 相似文献
15.
Pascal Reboul Pascal George Delphine Miquel Pierre Louisot Pierre Broquet 《Glycoconjugate journal》1996,13(1):69-79
When treated with retinoic acidin vivo, C6 glioma cells show an enhancement of CMP-Neu5Ac:Gal 1–3 GalNAc-R -2,3 sialyltransferase activity. A 300kDa glycoprotein was detected by lectin affinoblotting in retinoic acid-treated C6 cells which stained weakly or not at all in control cells. Comparative studies with different lectins demonstrated that this glycoprotein contains 2,3 Neu5Ac Gal-GalNAc O-glycan moieties. Cultures in the presence of an inhibitor of O-glycan synthesis (N-acetylgalactosaminide -O-benzyl) demonstrated that enhancement of staining of the 300 kDa glycoprotein was not due to the increase of the 2,3 sialyltransferase but to thede novo synthesis of the polypeptide chain of this glycoprotein.Abbreviations RA
retinoic acid
- Neu5Ac
N-acetylneuraminic acid
- CMP-Neu5Ac
cytidine 5 monophosphosialate
- 2,3 ST
CMP-Neu5Ac:Gal 1–3 GalNAc-R -2,3 sialyltransferase
- GalNAc-O-benzyl
N-acetylgalactosaminide -O-benzyl
- Gal1-3GalNAc-O-benzyl
Galactosyl 1-3N-acetylgalactosaminide -O-benzyl
- TBS
Tris-HCl buffer 50mm pH 7.5 containing NaCl 0.15m and Tween 20 0.05%
- B1 buffer
TBS containing MgCl2 1mm, MnCl2 1mm and CaCl2 1mm 相似文献
16.
Ronald L. Hanson Kenneth S. Bembenek Ramesh N. Patel Laszlo J. Szarka 《Applied microbiology and biotechnology》1992,37(5):599-603
Summary Biotransformations were developed to oxidize N-carbobenzoxy(CBZ)-l-lysine and to reduce the product keto acid to l-CBZ-oxylysine. Lysyl oxidase (l-lysine: O2 oxidoreductase, EC 1.4.3.14) from Trichoderma viride was relatively specific for l-lysine and had very low activity with N-substituted derivatives. l-Amino acid oxidase (l-amino acid: O2 oxidoreductase [deaminating], EC 1.4.3.2) from Crotalus adamanteus venom had low activity with l-lysine but high activity with N-formyl-, t-butyoxycarbonyl(BOC)-, acetyl-, trifluoroacetyl-, or CBZ-l-lysine. l-2-Hydroxyisocaproate dehydrogenase (EC 1.1.1.-) from Lactobacillus confusus catalyzed the reduction by NADH of the keto acids from N-acetyl-, trifluoroacetyl-, formyl- and CBZ-l-lysine but was inactive with the products from oxidation of l-lysine, l-lysine methyl ester, l-lysine ethyl ester or N-t-BOC-l-lysine. Providencia alcalifaciens (SC9036, ATCC 13159) was a good microbial substitute for the snake venom oxidase and also provided catalase (H2O2:H2O2 oxidoreductase EC 1.11.1.6). N-CBZ-l-Lysine was converted to CBZ-l-oxylysine in 95% yield with 98.5% optical purity by oxidation using P. alcalifaciens cells followed by reduction of the keto acid using l-2-hydroxyisocaproate dehydrogenase. NADH was regenerated using formate dehydrogenase (formate: NAD oxidoreductase, EC 1.2.1.2) from Candida boidinii. The Providencia oxidase was localized in the particulate fraction and catalase activity was predominantly in the soluble fraction of sonicated cells. The pH optima and kinetic constants were determined for the reactions.
Correspondence to: R. L. Hanson 相似文献
17.
Lars-Åke Fransson Birgitta Havsmark Katsukiyo Sakurai Sakaru Suzuki 《Glycoconjugate journal》1992,9(1):45-55
To generate xyloside-primed dermatan sulfate suitable for sequence analysis, skin fibroblasts were incubated withp-hydroxyphenyl--d-xylopyranoside and [3H]galactose, and free [3H]glycosaminoglycan chains were isolated from the culture medium by ion exchange and gel chromatography. After125I labelling of their reducing-terminal hydroxyphenyl groups, chains were subjected to various chemical and enzymatic degradations, both partial and complete, followed by gradient polyacrylamide gel electrophoresis and autoradiographic identification of fragments extending from the labelled reducing-end to the point of cleavage. Results of periodate oxidation-alkaline scission indicated that the xylose moiety remained unsubstituted at C-2/C-3; exhaustive treatment with chondroitin AC-I lyase afforded the fragment HexA-Gal-Gal-Xyl-R (R = radio-iodinated hydroxyphenyl group), and complete degradations with chondroitin ABC lyase as well as testicular hyaluronidase yielded the fragments HexA/HexA-GalNAc-GlcA-Gal-Gal-Xyl-R with or without sulfate on theN-acetylgalactosamine. Partial digestions with testicular hyaluronidase or chondroitin B lyase indicated that glucuronic acid was common in the first three repeats after the linkage region and that iduronic acid could occupy any position thereafter. Hence, there were no indications of a repeated, periodic appearance of the clustered GlcA-GalNAc repeats which was previously observed in proteoglycan derived dermatan sulfate [Fransson L-Å, Havsmark B, Silverberg I (1990)Biochem J
269:381–8], suggesting a role for the protein part in controlling the formation of particular copolymeric features during glycosaminoglycan assembly.Abbreviations GAG
glycosaminoglycans
- CS
chondroitin sulfate
- DS
dermatan sulfate
- Ser
serine
- Xyl
d-xylose
- Gal
d-galactose
- GlcA
d-glucuronic acid
- IdoA
l-iduronic acid
- GalNAc
N-acetyl-d-galactosamine
- GlcNAc
N-acetyl-d-glucosamine
- HexA
4-deoxy-l-threo-hex-4-enopyranosyluronic acid
- HO-Phe
p-hydroxyphenyl group
- HO-Phe-Xyl
p-hydroxyphenyl-O--d-xylopyranoside
- O2N-Phe-Xyl
p-nitrophenyl--d-xylopyranoside
- OSO3
ester sulfate
- PAGE
polyacrylamide gel electrophoresis
- HPLC
high performance liquid chromatography
- FPLC
fast performance liquid chromatography
- LC
standard liquid chromatography 相似文献
18.
Wu AM 《Journal of biomedical science》2005,12(1):135-152
Summary The agglutinin isolated from the seeds of Maclura pomifera (MPA) recognizes a mucin-type disaccharide sequence, Gal13GalNAc (T) on a human erythrocyte membrane. We have utilized the enzyme-linked lectinosorbent assay (ELLSA) and inhibition assay to more systematically analyze the carbohydrate specificity of MPA with glyco-recognition factors and mammalian Gal/GalNAc structural units in lectin–glycoform interactions. From the results, it is concluded that the high densities of polyvalent GalNAc1Ser/Thr (Tn) and Gal13GalNAc1Ser/Thr (T) glycotopes in macromolecules are the most critical factors for MPA binding, being on a nanogram basis 2.0 × 105, 4.6 × 104 and 3.9 × 104 more active than monovalent Gal, monomeric T and Tn glycotope, respectively. Other carbohydrate structural units in mammalian glycoconjugates, such as human blood group Sd (a+) related disaccharide (GalNAc14Gal) and Pk/P1 active disaccharide (Gal14Gal) were inactive. These results demonstrate that the configurations of carbon-4 and carbon-2 are essential for MPA binding and establish the importance of affinity enhancement by high-density polyvalencies of Tn/T glycotopes in MPA–glycan interactions. The overall binding profile of MPA can be defined in decreasing order as high density of polyvalent Tn/T (M.W. > 4.0 × 104) >> Tn-containing glycopeptides (M.W. < 3.0 × 103) > monomeric T/Tn and P (GalNAc13Gal) > GalNAc > Gal >> Man, LAra, DFuc and Glc (inactive). Our findings should aid in the selection of this lectin for elucidating functions of carbohydrate chains in life processes and for applications in the biomedical sciences. 相似文献
19.
Dorothee Heuermann Peter Roggentin Reinhard G. Kleineidam Roland Schauer 《Glycoconjugate journal》1991,8(2):95-101
The sialidase secreted byClostridium chauvoei NC08596 was purified to apparent homogeneity by ion-exchange chromatography, gel filtration, hydrophobic interaction-chromatography, FPLC ion-exchange chromatography, and FPLC gel filtration. The enzyme was enriched about 10 200-fold, reaching a final specific activity of 24.4 U mg–1. It has a relatively high molecular mass of 300 kDa and consists of two subunits each of 150 kDa. The cations Mn2+, Mg2+, and Ca2+ and bovine serum albumin have a positive effect on the sialidase activity, while Hg2+, Cu2+, and Zn2+, chelating agents and salt decrease enzyme activity. The substrate specificity, kinetic data, and pH optimum of the enzyme are similar to those of other bacterial sialidases.Abbreviations FPLC
fast protein liquid chromatography
- NCTC
National Collection of Type Cultures
- ATCC
American Type Culture Collection
- MU-Neu5Ac
4-methylumbelliferyl--d-N-acetylneuraminic acid
- buffer A
0.02m piperazine, 0.01m CaCl2, pH 5.5
- buffer B
0.02m piperazine, 0.01m CaCl2, 1.0m NaCl, pH 5.5
- buffer C
0.1m sodium acetate, 0.01m CaCl2, pH 5.5
- SDS
sodium dodecyl sulfate
- PAGE
polyacrylamide gel electrophoresis
- Neu5Ac
N-acetylneuraminic acid
- BSM
bovine submandibular gland mucin
- GD1a
IV3Neu5Ac, II3Neu5Ac-GgOse4Cer
- GM1
II3Neu5Ac-GgOse4Cer
- MU-Neu4,5Ac2
4-methylumbelliferyl--d-N-acetyl-4-O-acetylneuraminic acid
- TLC
thin-layer chromatography
- HPTLC
high performance thin-layer chromatography
- EDTA
ethylenediamine tetraacetic acid
- EGTA
ethylene glycol bis(2-aminoethyl-ethen)-N,N,N,N-tetraacetic acid
- BSA
bovine serum albumin
- Neu5Ac2en
2-deoxy-2,3-didehydro-N-acetylneuraminic acid
- IEF
isoelectric focusing
- IEP
isoelectric point 相似文献
20.
Primary structure of the majorO-glycosidically linked carbohydrate unit of human von Willebrand factor 总被引:5,自引:0,他引:5
Bruno Samor Jean-Claude Michalski Claudine Mazurier Maurice Goudemand Pieter De Waard Johannes F G Vliegenthart Gérard Strecker Jean Montreuil 《Glycoconjugate journal》1989,6(3):263-270
A reduced tetrasaccharide chain was obtained from human von Willebrand factor (vWF) by mild alkaline borohydride treatment. The purification of thisO-glycosidically-linked oligosaccharide was achieved by serial affinity chromatography on immobilized concanavalin A andLens culinaris agglutinin and finally gel filtration. Its structure was determined by a combination of methylation studies and 500 MHz1H-NMR spectroscopy to be: NeuAc(2-3)Gal(1-3)[NeuAc(2-6)]GalNAc-ol.Abbreviations ConA
concanavalin A
- LCA
Lens culinaris agglutinin
- vWF
von Willebrand factor
- NeuAc
N-acetylneuraminic acid
- Gal
d-galactose
- GalNAc-ol
N-acetyl-d-galactosaminitol
- HMW
high molecular weight
- LMW
low molecular weight 相似文献