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1.
When α-d-GlcNAc-OC6H4NO2
-p and β-d-(6-sulfo)-GlcNAc-OC6H4NO2-p (2) were used as substrates, β-N-acetylhexosaminidase from Aspergillus oryzae transferred the β-d-(6-sulfo)-GlcNAc(unit from 2 to α-d-GlcNAc-OC6H4NO2
-p to afford β-d-(6-sulfo)-GlcNAc-(1→4)-α-d-GlcNAc-OC6H4NO2-p (3) in a yield of 94% based on the amount of donor, 2, added. β-d-(6-sulfo)-GlcNAc-(1→4)-α-d-Glc-OC6H4NO2-p (4) was obtained with α-d-Glc-OC6H4NO2
-p as acceptor in a similar manner. With a reaction mixture of 2 and β-d-GlcNAc-OC6H4NO2-p (1) in a molar ratio of 6:1, the enzyme mediated the transfer of β-d-GlcNAc from 1 to 2, affording disaccharide β-d-GlcNAc-(1→4)-β-(6-sulfo)-d-GlcNAc-OC6H4NO2-p (5) in a yield of 13% based on the amount of 1 added. 相似文献
2.
Carvalho de Souza A Ganchev DN Snel MM van der Eerden JP Vliegenthart JF Kamerling JP 《Glycoconjugate journal》2009,26(4):457-465
Cell aggregation in the marine sponge Microciona prolifera is mediated by a multimillion molecular-mass aggregation factor, termed MAF. Earlier investigations revealed that the cell
aggregation activity of MAF depends on two functional domains: (i) a Ca2+-independent cell-binding domain and (ii) a Ca2+-dependent proteoglycan self-interaction domain. Structural analysis of involved carbohydrate fragments of the proteoglycan
in the self-association established a sulfated disaccharide β-d-GlcpNAc3S-(1→3)-α-l-Fucp and a pyruvated trisaccharide β-d-Galp4,6(R)Pyr-(1→4)-β-d-GlcpNAc-(1→3)-α-l-Fucp. Recent UV, SPR, and TEM studies, using BSA conjugates and gold nanoparticles of the synthetic sulfated disaccharide, clearly
demonstrated self-recognition on the disaccharide level in the presence of Ca2+-ions. To determine binding forces of the carbohydrate–carbohydrate interactions for both synthetic MAF oligosaccharides,
atomic force microscopy (AFM) studies were carried out. It turned out that, in the presence of Ca2+-ions, the force required to separate the tip and sample coated with a self-assembling monolayer of thiol-spacer-containing
β-d-GlcpNAc-(1→3)-α-l-Fucp-(1→O)(CH2)3S(CH2)6S- was found to be quantized in integer multiples of 30 ± 6 pN. No binding was observed between the two monolayers in the
absence of Ca2+-ions. Cd2+-ions could partially induce the self-interaction. In contrast, similar AFM experiments with thiol-spacer-containing β-d-Galp4,6(R)Pyr-(1→4)-β-d-GlcpNAc-(1→3)-α-l-Fucp-(1→O)(CH2)3S(CH2)6S- did not show a binding in the presence of Ca2+-ions. Also TEM experiments of gold nanoparticles coated with the pyruvated trisaccharide could not make visible aggregation
in the presence of Ca2+-ions. It is suggested that the self-interaction between the sulfated disaccharide fragments is stronger than that between
the pyruvated trisaccharide. 相似文献
3.
An DS Cui CH Sung BH Yang HC Kim SC Lee ST Im WT Kim SG 《Applied microbiology and biotechnology》2012,94(3):673-682
The gene encoding an α-l-arabinofuranosidase that could biotransform ginsenoside Rc {3-O-[β-d-glucopyranosyl-(1–2)-β-d-glucopyranosyl]-20-O-[α-l-arabinofuranosyl-(1–6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol} to ginsenoside Rd {3-O-[β-d-glucopyranosyl-(1–2)-β-d-glucopyranosyl]-20-O-β-d-glucopyranosyl-20(S)-protopanaxadiol} was cloned from a soil bacterium, Rhodanobacter ginsenosidimutans strain Gsoil 3054T, and the recombinant enzyme was characterized. The enzyme (AbfA) hydrolyzed the arabinofuranosyl moiety from ginsenoside
Rc and was classified as a family 51 glycoside hydrolase based on amino acid sequence analysis. Recombinant AbfA expressed
in Escherichia coli hydrolyzed non-reducing arabinofuranoside moieties with apparent K
m values of 0.53 ± 0.07 and 0.30 ± 0.07 mM and V
max values of 27.1 ± 1.7 and 49.6 ± 4.1 μmol min−1 mg−1 of protein for p-nitrophenyl-α-l-arabinofuranoside and ginsenoside Rc, respectively. The enzyme exhibited preferential substrate specificity of the exo-type
mode of action towards polyarabinosides or oligoarabinosides. AbfA demonstrated substrate-specific activity for the bioconversion
of ginsenosides, as it hydrolyzed only arabinofuranoside moieties from ginsenoside Rc and its derivatives, and not other sugar
groups. These results are the first report of a glycoside hydrolase family 51 α-l-arabinofuranosidase that can transform ginsenoside Rc to Rd. 相似文献
4.
Mhenia Haidar Nabila Seddiki Jean Claude Gluckman Liliane Gattegno 《Glycoconjugate journal》1994,11(2):73-79
Envelope glycoproteins of human immunodeficiency virus (gp120 and gp41) occur as oligomers. Here, we show by gel filtration analysis that gp 120 oligomerizationin vitro is calcium- and temperature-dependent. Recombinant gp120 (rgp120) species were recovered as monomers at 20 °C in the absence of calcium, but as tetramers at 37 °C in 10mm CaCl2. Under the latter condition,N-glycanase-deglycosylated rgp120 formed hexamers. Relative to intact rgp120, which has been reported to display carbohydrate-binding properties forN-acetyl--d-glucosaminyl and mannosyl residues, deglycosylation enhanced rgp120 specific binding to mannose-divinylsulfone-agarose, para-aminophenyl--d-GlcNAc-agarose and fetuin-agarose matrices. Taken together, these results rule out the role of homologous lectin-carbohydrate interactions viaN-linked glycans in the rgp120 oligomerization, even though its lectin properties may also be calcium-dependent. Deglycosylation may unmask domains of rgp120 polypeptide backbone that independently play a role either in rgp120 lectin activity or in calcium-dependent oligomerization. 相似文献
5.
Carbohydrate binding properties of the envelope glycoproteins of human immunodeficiency virus type 1
Here, we confirm and extend our previous findings on human immunodeficiency virus type 1 (HIV-1) envelope glycoproteinN-acetylglucosaminyl binding properties. We show the occurrence of saturable, temperature, pH, and calcium dependent carbohydrate-specific interactions between recombinant precursor gp160 (rgp160) and two affinity matrices:d-mannose-divinylsulfone-agarose, and natural glycoprotein, fetuin, also coupled to agarose. Binding of rgp160 to the matrices was inhibited by soluble mannosyl derivatives, -d-Man17-BSA and mannan, by -d-GlcNAc47-BSA and by glycopeptides from Pronase-treated porcine thyroglobulin, which produces oligomannose and complex N-linked glycans. Glycopeptides from Endoglycosidase H-treated thyroglobulin partially inhibited rgp160 binding, as did the asialo-agalacto-tetraantennary precursor oligosaccharide of human 1-acid glycoprotein for binding to fetuin-agarose. -d-Glucan and -d-Gal17-BSA had no or only limited effect. Also, surface unit rgp120 specifically interacted with fetuin-agarose and soluble fetuin, but in the latter case with a twofold reduced affinity relative to rgp160. After affinity chromatography, rgp160 was specifically retained by the two matrices and eluted by mannan in both cases, while rgp120 was not retained by fetuin-agarose but only eluted as a significantly retarded peak, which confirms its specific but weak interaction. Thus, rgp160 interacts with both oligomannose type, and the mannosyl core of complex type N-linked glycans, and its gp120 region plays a role in this interaction. Because fetuin and asialofetuin inhibit to nearly the same extent, the binding of rgp160 or rgp120 to fetuin-agarose, interaction with sialic acid or -d-galactosyl structures of complex N- or O-linked glycans can be ruled out. Specific rgp160 and rgp120 binding to ap-aminophenyl--d-GlcNAc-agarose matrix, which was inhibited by -d-GlcNAc47-BSA and by fetuin, confirms that HIV-1 envelope glycoproteins can also specifically interact with theN-acetylglucosaminyl core of oligosaccharide structures. 相似文献
6.
Lectins are used extensively as research tools to detect and target specific oligosaccharide sequences. Ricinus communis agglutinin I (RCA120) recognizes non-reducing terminal β-d-galactose (Galβ) and its specificities of interactions with neutral and sialylated oligosaccharides have been well documented. Here we use carbohydrate arrays of sulfated Galβ-containing oligosaccharide probes, prepared from marine-derived galactans, to investigate their interactions with RCA120. Our results showed that RCA120 binding to Galβ1–4 was enhanced by 2-O- or 6-O-sulfation but abolished by 4-O-sulfation. The results were corroborated with competition experiments. Erythrina cristagalli lectin is also a Galβ-binding protein but it cannot accommodate any sulfation on Galβ. 相似文献
7.
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
8.
A novel phosphorylase from Clostridium phytofermentans belonging to the glycoside hydrolase family (GH) 65 (Cphy1874) was characterized. The recombinant Cphy1874 protein produced
in Escherichia coli showed phosphorolytic activity on nigerose in the presence of inorganic phosphate, resulting in the release of d-glucose and β-d-glucose 1-phosphate (β-G1P) with the inversion of the anomeric configuration. Kinetic parameters of the phosphorolytic activity
on nigerose were k
cat = 67 s−1 and K
m = 1.7 mM. This enzyme did not phosphorolyze substrates for the typical GH65 enzymes such as trehalose, maltose, and trehalose
6-phosphate except for a weak phosphorolytic activity on kojibiose. It showed the highest reverse phosphorolytic activity
in the reverse reaction using d-glucose as the acceptor and β-G1P as the donor, and the product was mostly nigerose at the early stage of the reaction. The
enzyme also showed reverse phosphorolytic activity, in a decreasing order, on d-xylose, 1,5-anhydro-d-glucitol, d-galactose, and methyl-α-d-glucoside. All major products were α-1,3-glucosyl disaccharides, although the reaction with d-xylose and methyl-α-d-glucoside produced significant amounts of α-1,2-glucosides as by-products. We propose 3-α-d-glucosyl-d-glucose:phosphate β-d-glucosyltransferase as the systematic name and nigerose phosphorylase as the short name for this Cphy1874 protein. 相似文献
9.
Summary In this study, the variety of sugar residues in the gut glycoconjugates of Triturus carnifex (Amphibia, Caudata) are investigated by carbohydrate conventional histochemistry and lectin histochemistry. The oesophageal
surface mucous cells contained acidic glycoconjugates, with residues of GalNAc, Gal β1,3 GalNAc and (GlcNAc β1,4)
n
oligomers. The gastric surface cells mainly produced neutral glycoproteins with residues of fucose, Gal β1-3 GalNAc, Gal-αGal,
and (GlcNAc β1,4)
n
oligomers in N- and O-linked glycans, as the glandular mucous neck cells, with residues of mannose/glucose, GalNAc, Gal β1,3
GalNAc, (GlcNAc β1,4)
n
oligomers and fucose linked α1,6 or terminal α1,3 or α1,4 in O-linked glycans. The oxynticopeptic tubulo-vesicular system
contained neutral glycoproteins with N- and O-linked glycans with residues of Gal-αGal, Gal β1-3 GalNAc and (GlcNAc β1,4)
n
oligomers; Fuc linked α1,2 to Gal, α1,3 to GlcNAc in (poly)lactosamine chains and α1,6 to GlcNAc in N-linked glycans. Most
of these glycoproteins probably corresponds to the H+K+-ATPase β-subunit. The intestinal goblet cells contained acidic glycoconjugates, with residues of GalNAc, mannose/ glucose,
(GlcNAc β1,4)
n
oligomers and fucose linked α1,2 to Gal in O-linked oligosaccharides. The different composition of the mucus in the digestive
tracts may be correlated with its different functions. In fact the presence of abundant sulphation of glycoconjugates, mainly
in the oesophagus and intestine, probably confers resistance to bacterial enzymatic degradation of the mucus barrier. 相似文献
10.
E. Vollbrecht R. Heckmann V. Wray M. Nimtz S. Lang 《Applied microbiology and biotechnology》1998,50(5):530-537
The bacterium Tsukamurella sp. nov., isolated from soil, was found to produce novel glycolipids when grown on sunflower oil as the sole carbon source.
The glycolipids were isolated by chromatography on silica columns and their structures elucidated using a combination of multidimensional
NMR and MS techniques. The three main components are 2,3-di-O-acyl-α-d-glucopyranosyl-(1-1)-α-d-glucopyranose, 2,3-di-O-acyl-β-d-glucopyranosyl-(1-2)-4,6-di-O-acyl-α-d-glucopyranosyl-(1-1)-α-d-glucopyranose and 2,3-di-O-acyl-β-d-glucopyranosyl-(1-2)-β-d-galactopyranosyl-(1-6)-4,6-di-O-acyl-α-d-glucopyranosyl-(1-1)-α-d-glucopyranosl which are linked to fatty acids varying in chain length from C4 to C18. The glycolipids are mainly extracellular but are also found attached to the cell walls. During the cultivation the composition
of the glycolipids changed from disaccharide- to tri- and tetrasaccharide lipids. The glycolipids show good surface-active
behaviour and have antimicrobial properties.
Received: 22 May 1998 / Received revision: 24 August 1998 / Accepted: 26 August 1998 相似文献
11.
Biosynthesis of six saponins (ginsenosides) in suspension culture of P. quinquefolium Z5 was investigated. Ginsenoside content in biomass reached the highest level, nearly 30 mg g−1 d.w., between 25 and 30 days of the culture. Saponins were synthesized simultaneously with cell growth but their synthesis
rate was not proportional to the growth rate. During the phase of rapid biomass multiplication, after which biomass reached
90% of its maximum yield, only half examined ginsenosides was produced. The second half of the final saponins yield was produced
during the slow growth phase, in which only 10% of biomass was grown. During the intensive growth phase the productivity of
six saponins examined per biomass (dry weight) unit was 3.4 μg mg−1 d.w. day−1, however, this parameter calculated for slow growth phase reached nearly 30 μg mg−1 d.w. day−1. There were differences in increase of the contents of six saponins determined in biomass, and it was the highest for saponins
Re (20(S)-protopanaxatriol-6-[O-α-l-rhamnopyranosyl(1 → 2)-β-d-glucopyranoside]-20-O-β-d-glucopyranoside) and Rg1 (20(S)-protopanaxatriol-6,20-di-O-β-d-glucoside). 相似文献
12.
M.L. Tierney B. Birnir N.P. Pillai J.D. Clements S. M. Howitt G. B. Cox P.W. Gage 《The Journal of membrane biology》1996,154(1):11-21
The conserved leucine residues at the 9′ positions in the M2 segments of α1 (L264) and β1 (L259) subunits of the human GABAA receptor were replaced with threonine. Normal or mutant α1 subunits were co-expressed with normal or mutant β1 subunits in Sf9 cells using the baculovirus/Sf9 expression system. Cells in which one or both subunits were mutated had a
higher ``resting' chloride conductance than cells expressing wild-type α1β1 receptors. This chloride conductance was blocked by 10 mm penicillin, a recognized blocker of GABAA channels, but not by bicuculline (100 μm) or picrotoxin (100 μm) which normally inhibit the chloride current activated by GABA: nor was it potentiated by pentobarbitone (100 μm). In cells expressing wild-type β1 with mutated α1 subunits, an additional chloride current could be elicited by GABA but the rise time and decay were slower than for wild-type
α1β1 receptors. In cells expressing mutated β1 subunits with wild-type or mutated α1 subunits (αβ(L9′T) and α(L9′T)β(L9′T)), no response to GABA could be elicited: this was not due to an absence of GABAA receptors in the plasmalemma because the cells bound [3H]-muscimol. It was concluded that in GABAA channels containing the L9′T mutation in the β1 subunit, GABA-binding does not cause opening of channels, and that the L9′T mutation in either or both subunits gives an
open-channel state of the GABAA receptor in the absence of ligand.
Received: 17 April 1996/Revised: 5 July 1996 相似文献
13.
A kinetic study of the interaction of bivalent and monovalent sugar ligands with a lectin was undertaken with the aid of surface
plasmon resonance (SPR) method. The study involved a series of bivalent α-d-mannopyranoside containing sugar ligands, with systematic variation in the distance between the sugar ligands. The detailed
kinetic studies showed that bivalent ligands underwent a faster association (k
on) and a slower dissociation (k
off) of the ligand–lectin complexes, in comparison to the monovalent ligand–lectin complexes. The kinetic constants were complemented
further by assessing the thermodynamic parameters with the aid of isothermal titration calorimetry (ITC). The initiation of
cross-linking of ligand–lectin interactions emerge from the early stages of the complexation. The dynamic light scattering
(DLS) and the transmission electron microscopy (TEM) techniques allowed judging the sizes and morphologies of the complex
in the solution and solid states, respectively. 相似文献
14.
The rattlesnake (Crotalus atrox) venom lectin is a readily-prepared decameric C-type lectin, specific for Gal and GalNAc. Glycan microarray analysis showed
it reacted with a wide range of glycans, chiefly recognizing sets of compounds with Galβ1-4GlcNAc (LacNAc), α-Gal or α-GalNAc
non-reducing termini. Its array profile was therefore distinctly different from those of four previously studied mammalian
C-type lectins with the same Gal/GalNAc monosaccharide specificity, and it was more broadly reactive than several Gal- or
GalNAc-specific plant lectins commonly used for glycan blotting. Though a general reactivity towards glycoproteins might be
expected from the avidity conferred by its high valence, it showed a marked preference for glycoproteins with multiple glycans,
terminated by Gal or GalNAc. Thus its ten closely-spaced sites each with a KD for GalNAc of ~2 mM appeared to make RSVL more selective than the four more widely-spaced sites of soybean agglutinin, with
a ten-fold better KD for GalNAc. 相似文献
15.
Marine Bacterial Sulfated Fucoglucuronomannan (SFGM) Lyase Digests Brown Algal SFGM into Trisaccharides 总被引:2,自引:0,他引:2
Sakai T Kimura H Kojima K Shimanaka K Ikai K Kato I 《Marine biotechnology (New York, N.Y.)》2003,5(1):70-78
Abstract
Three kinds of trisaccharides were prepared by digesting fucoidan from the brown alga Kjellmaniella crassifolia, with the extracellular enzymes of the marine bacterium Fucobacter marina. Their structures were determined as Δ4,5GlcpUA1-2(L-Fucp(3-O-sulfate)α1-3)D-Manp, Δ4,5GlcpUA1-2(L-Fucp(3-O-sulfate)α1-3)D-Manp(6-O-sulfate), and Δ4,5GlcpUA1-2(L-Fucp(2,4-O-disulfate)α1-3)D-Manp(6-O-sulfate), which indicated the existence of a novel polysaccharide in the fucoidan and a novel glycosidase in the extracellular
enzymes. In order to determine the complete structure of the polysaccharide and the reaction mechanism of the glycosidase,
the fucoidan was partially hydrolyzed to obtain glucuronomannan, which is the putative backbone of the polysaccharide, and
its sugar sequence was determined as (-4-D-GlcpUAβ1-2D-Manpα1-)n, which disclosed that the main structure of the polysaccharide is (-4-D-GlcpUAβ1-2(L-Fucp(3-O-sulfate)α1-3)D-Manpα1-)n. Consequently, the glycosidase was deduced to be an endo-α-D-mannosidase that eliminatively cleaves the α-D-mannosyl linkage between D-Manp and D-GlcpUA residues in the polysaccharide and produces the above trisaccharides. The novel polysaccharide and glycosidase were tentatively
named as sulfated fucoglucuronomannan (SFGM) and SFGM lyase, respectively. 相似文献
16.
Paula Alayón-Luaces Eduardo A. Pagano Luis A. Mroginski Gabriel O. Sozzi 《Plant Cell, Tissue and Organ Culture》2008,95(3):257-263
α-l-Arabinofuranosidase, α- and β-d-xylosidase, and β-d-glucosidase activity was detected in the soluble fraction (S-F) extracted with water and in the NaCl-released fraction (NaCl-F)
extracted with a high-salt concentration buffer from apple callus cultures. The activity was found to be differentially modulated
by the addition of various plant growth regulators (PGRs) to calluses that had lost their requirement for specific PGRs (“habituation”
phenomenon). α-l-Arabinofuranosidase activity was 93%, 130%, 126% and 186% higher in the NaCl-F from IAA-, IBA-, ABA- and GA3-treated callus than in that extracted from untreated callus while S-F α-l-arabinofuranosidase activity was only 71%, 24%, 55% and 66% higher, respectively. α-d-Xylosidase displayed low activity levels in both S-F and NaCl-F but 2iP-treated callus showed higher α-d-xylosidase activity in both fractions than the control. 2,4-D increased α-d-xylosidase activity by 110% in the NaCl-F but decreased it by 40% in the S-F. β-d-Xylosidase activity increased by 99% in S-F from 2iP-treated callus but slightly decreased in the NaCl-F. In GA3-treated callus, NaCl-F β-d-xylosidase activity increased by 188%. S-F and NaCl-F from Picloram-treated callus showed undetectable or only slightly noticeable
α-l-arabinofuranosidase, α-d-xylosidase and β-d-xylosidase activity. Interestingly, β-d-glucosidase activity rose 28-fold in the S-F extracted from Picloram-treated callus. β-d-glucosidase was the only enzyme assayed that greatly increased its NaCl-F activity after 10 subcultures, and the addition
of any PGR to the callus culture –except for Picloram and ABA– decreased its activity, suggesting that this enzyme may be
associated with certain stress conditions, such as PGR starvation or Picloram addition. This is the first report on glycoside
hydrolases from fruit callus as modulated by different PGRs. 相似文献
17.
Juliana Bello Baron Maurer Adaucto Bellarmino Pereira-Netto Filomena Angela Pettolino Yolanda Maria Gaspar Antony Bacic 《Trees - Structure and Function》2010,24(2):391-398
Arabinogalactan-proteins (AGPs) are a family of highly glycosylated hydroxyproline-rich glycoproteins implicated in several
aspects of plant growth and development. (β-d-glucosyl)3 Yariv phenylglycoside (β-GlcY), commonly known as Yariv reagent, selectively binds AGPs. We treated cell suspension cultures
of Araucaria
angustifolia, the Brazilian pine, with β-GlcY and observed inhibition of biomass increase in a culture medium with 50 μM β-GlcY. However,
the growth was not inhibited by (α-d-galactosyl)3 Yariv phenylglycoside (α-GalY) which does not bind AGPs. Fluorescein diacetate staining of cells indicated that β-GlcY severely
affected cell viability. However, cell swelling, bursting and release of cellular contents, all characteristics of necrotic
cell death, were not observed in β-GlcY-treated cells. Instead, programmed cell death (PCD) structural changes such as cytoplasmic
shrinkage and condensation were observed in β-GlcY-treated cells. In addition, callose accumulation, which is another marker
of PCD, was also observed in β-GlcY-treated cells. The use of both, Ac-VEID-CHO, an inhibitor of caspase-like proteolytic
activity related to PCD, and phenyl methyl sulphonyl fluoride (PMSF), a protease inhibitor known to suppress PCD, in the culture
medium did not reverse the growth inhibition caused by β-GlcY. These data indicate that the β-GlcY-induced inhibition of Araucaria cell’s growth is related to AGP perturbation, and also that this growth inhibition is due to increased cell death not driven
by necrosis. 相似文献
18.
This contribution illustrates the advantages of some chromophoric and fluorophoric carbohydrate derivatives such asp-nitrophenyl (pNO2Phe) or 4-methylumbelliferyl (MeUmb) glycosides andN-dansylgalactosamine in studies of the binding equilibrium and kinetics with some plant lectins. The methods used involve
continuous titrations of changes in ligand or protein absorption and ligand fluorescence, including substitution titrations
as well as stopped-flow, temperature-jump or pressure-jump relaxation kinetics.
When monitored by temperature-jump relaxation, binding of MeUmbαGal to the bloodgroup A specific lectin GSAI-A4 fromGriffonia simplicifolia is a simple bimolecular association with parametersk
+
= 9.4 × 104 M-1 s-1 andk
-1 = 5.3 s-1 at 23°C, but binding to the GSAI-B4 lectin is biphasic.
The complementarity of the peanut agglutinin binding site with Galβ1 → 3GalNAc that occurs in manyO-glycoproteins follows from enthalpic considerations and also from the value of the dissociation-rate parameterk
-1 = 0.24 s-1 of the MeUmbβGalβl → 3GalNAc.lectin complex. This value, obtained by stopped-flow kinetics is 100 times smaller than for other mono-and disaccharides
investigated. The binding mechanism is simple and the derivatisation of Galβ1 → 3GalNAc does not affect the affinity to a considerable degree.
The binding preference of tetravalentsoybean agglutinin for MeαGalNAc over MeαGal by a factor of 25 is mainly of enthalpic origin with an additional 7 kJ mol-1; the NAc group causes perturbation of a tryptophanyl residue, evidenced by protein difference absorption spectrometry. In
the glycosides, a large aglycon likeβpNO2 Phe orβMeUmb hardly affects the affinity of SBA but a largeN-dansyl group increases the affinity by a factor 20 as compared to GalNAc. The 10-fold increase in carbohydrate-specificN-dansylgalactosamine fluorescence, together with a very favourable entropic contribution point at the presence of a hydrophobic
region in the vicinity of the carbohydrate-binding site. The dissociation-rate parameter of the MeUmbβGalNAc SBA complex is slower than for any reported monosaccharide-lectin complex: 0.4 s-1.
The divalent lectin fromErythrina cristagalli preferentially binds the Galβ1 → 4GlcNAc structure that occurs in manyN-glycoproteins. The combining site was mapped thermodynamically with carbohydrates ranging from mono-to pentasaccharides as
derived fromN-glycoproteins. Here, N-dansylgalactosamine was used as a fluorescent indicator ligand in substitution titrations. When Galβ1 → 4GlcNAc was linkedα1 → 2 orα1 → 6 to Man, the binding enthalpy and entropy remained practically constant. Application of stopped flow kinetics and pressure-jump
relaxation withN-dansylgalactosamine gave mono-exponential signal changes with a concentration dependence corresponding tok
+ = 4.8 x 104 M-1 s-1
k
- = 0.4 to 0.66 s-1 and a change in reaction volume of+7ml/mol. 相似文献
19.
To develop xylosidases as tools for the hydrolysis of wheat bran arabinoxylans, two β-xylosidases from Bacillus halodurans C-125 have been cloned and expressed in Escherichia coli. The recombinant (His)6-tagged enzymes, designated as XylBH39 and XylBH43, were efficiently purified using Ni2+-affinity chromatography. Determination of native molecular masses indicated that XylBH43 is dimeric in solution, whereas a similar analysis of XylBH39 did not allow differentiation between the dimeric and trimeric states. Both enzymes had similar pH and temperature optima (pH 7.5 and 55 °C for XylBH39 and pH 8 and 60 °C for XylBH43) and were relatively stable over the pH range of 3.5–8.5. In contrast, XylBH39 was more thermostable. At 60 °C, XylBH39 and XylBH43 displayed approximate half-life values of 2.40 and 0.05 h, respectively. The comparison of the ratio k
cat/K
M revealed that XylBH43 hydrolyzed p-nitrophenyl-β-d-xyloside more efficiently (4.6-fold) than XylBH39. Similarly, while XylBH43 was 18-fold less active on p-nitrophenyl-α-l-arabinofuranoside, XylBH39 was essentially inactive on this substrate. Using either p-nitrophenyl-β-d-xyloside or xylotriose, XylBH39 performed transglycosylation, while xylobiose proved to be a poor substrate for both hydrolysis and transglycosylation. The use of XylBH39 and XylBH43 for the posttreatment of endoxylanase-generated wheat bran hydrolysates revealed that XylBH43 efficiently produced xylose monomers (385 μg/ml after 330 min incubation). Its activity was improved by the simultaneous deployment of an α-l-arabinofuranosidase. Together, these enzymes were able to release 521 μg/ml of xylose after 330 min. This constitutes an approximate yield improvement of 35%. 相似文献
20.
Birichevskaya LL Kvach SV Sivets GG Kalinichenko EN Zinchenko AI Mikhailopulo IA 《Biotechnology letters》2007,29(4):585-591
Enzymatic 5′-monophosphorylation and 5′-phosphatidylation of a number of β-l- and β-d-nucleosides was investigated. The first reaction, catalyzed by nucleoside phosphotransferase (NPT) from Erwinia herbicola, consisted of the transfer of the phosphate residue from p-nitrophenylphosphate (p-NPP) to the 5′-hydroxyl group of nucleoside; the second was the phospholipase d (PLD)-catalyzed transphosphatidylation of l-α-lecithin with a series of β-l- and β-d-nucleosides as the phosphatidyl acceptor resulted in the formation of the respective phospholipid-nucleoside conjugates.
Some β-l-nucleosides displayed similar or even higher substrate activity compared to the β-d-enantiomers. 相似文献