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1.
UDP-d-glucose, at a micromolar level in the presence of MgCl2 and oat (Avena sativa) coleoptile particulate enzyme which contains both β-(1 → 3) and β-(1 → 4) glucan synthetases, produces glucan with mainly β-(1 → 4) glucosyl linkages. An activation of β-(1 → 3) glucan synthetase by UDP-d-glucose and a decrease in the formation of β-(1 → 3) glucan in the presence of MgCl2 have been observed. However, at high substrate concentration (≥ 10−4m), the activation of β-(1 → 3) glucan synthetase is so pronounced that the formation of β-(1 → 3) glucosyl linkage predominates in synthesized glucan regardless of the presence of MgCl2. These observations may explain the striking shift in the composition of glucan of particulate enzyme from a β-(1 → 4) to β-(1 → 3) glucosyl linkage when UDP-d-glucose concentration is raised from a low concentration (≤ 10−5m) to a higher concentration (≥ 10−4m). 相似文献
2.
The isolation of oligosaccharides from the cell-wall polysaccharide of Lactobacillus casei, serological group C 总被引:3,自引:3,他引:0
1. A number of disaccharides and oligosaccharides have been isolated from the products of mild acid hydrolysis of the specific substance from Lactobacillus casei, serological group C. 2. The major disaccharide is O-β-d-glucopyranosyl-(1→3)-N-acetyl- d-galactosamine (B4) and evidence is presented for the structure of a tetrasaccharide composed of O-β-d-glucopyranosyl-(1→6)-d-galactose (B1) joined through its reducing end group to B4. 3. Disaccharide B1 is also a component of a trisaccharide O-β-d-glucopyranosyl-(1→6)-O-β- d-galactopyranosyl-(1→6)-N-acetyl-d-glucosamine (A7). 4. A number of other oligosaccharides have been shown to be related structurally. 5. The ability of certain of the oligosaccharides to inhibit the precipitin reaction has been studied. The disaccharide B1 is more effective as an inhibitor than gentiobiose and the trisaccharide A7 is considerably more effective than B1. 6. These results have been compared with those obtained previously for the composition of the cell wall. 相似文献
3.
Nucleoside Diphosphate-sugar 4-Epimerases I. Uridine Diphosphate Glucose 4-Epimerase of Wheat Germ 总被引:3,自引:2,他引:1
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Uridine diphosphate (UDP)-glucose 4-epimerase (EC 5.1.3.2) has been purified over 1000-fold from extracts of wheat germ by MnCl2 treatment, (NH4)2SO4 fractionation, Sephadex column chromatography, and adsorption onto and elution from calcium phosphate gel. The enzyme has a pH optimum of 9.0. Km values are 0.1 mm for UDP-d-galactose and 0.2 mm for UDP-d-glucose. NAD is required for activity; Ka = 0.04 mm. NADH is an inhibitor strictly competitive with NAD; Ki = 2 μm. Wheat germ also contains UDP-l-arabinose 4-epimerase (EC 5.1.3.5) and thymidine diphosphate (TDP)-glucose 4-epimerase which are distinct from UDP-glucose 4-epimerase. 相似文献
4.
Angela Romanow Timothy G. Keys Katharina Stummeyer Friedrich Freiberger Bernard Henrissat Rita Gerardy-Schahn 《The Journal of biological chemistry》2014,289(49):33945-33957
Crucial virulence determinants of disease causing Neisseria meningitidis species are their extracellular polysaccharide capsules. In the serogroups W and Y, these are heteropolymers of the repeating units (→6)-α-d-Gal-(1→4)-α-Neu5Ac-(2→)n in NmW and (→6)-α-d-Glc-(1→4)-α-Neu5Ac-(2→)n in NmY. The capsule polymerases, SiaDW and SiaDY, which synthesize these highly unusual polymers, are composed of two predicted GT-B fold domains separated by a large stretch of amino acids (aa 399–762). We recently showed that residues critical to the hexosyl- and sialyltransferase activity are found in the predicted N-terminal (aa 1–398) and C-terminal (aa 763–1037) GT-B fold domains, respectively. Here we use a mutational approach and synthetic fluorescent substrates to define the boundaries of the hexosyl- and sialyltransferase domains. Our results reveal that the active sialyltransferase domain extends well beyond the predicted C-terminal GT-B domain and defines a new glycosyltransferase family, GT97, in CAZy (Carbohydrate-Active enZYmes Database). 相似文献
5.
Hydrolytic Activity and Substrate Specificity of an Endoglucanase from Zea mays Seedling Cell Walls 总被引:1,自引:1,他引:0
An endoglucanase was isolated from cell walls of Zea mays seedlings. Characterization of the hydrolytic activity of this glucanase using model substrates indicated a high specificity for molecules containing intramolecular (1→3),(1→4)-β-d-glucosyl sequences. Substrates with (1→4)-β-glucosyl linkages, such as carboxymethylcellulose and xyloglucan were, degraded to a limited extent by the enzyme, whereas (1→3)-β-glucans such as laminarin were not hydrolyzed. When (1→3),(1→4)-β-d-glucan from Avena endosperm was used as a model substrate a rapid decrease in vicosity was observed concomitant with the formation of a glucosyl polymer (molecular weight of 1-1.5 × 104). Activity against a water soluble (1→3),(1→4)-β-d-glucan extracted from Zea seedling cell walls revealed the same depolymerization pattern. The size of the limit products would indicate that a unique recognition site exists at regular intervals within the (1→3),(1→4)-β-d-glucan molecule. Unique oligosaccharides isolated from the Zea (1→3),(1→4)-β-d-glucan that contained blocks of (1→4) linkages and/or more than a single contiguous (1→3) linkage were hydrolyzed by the endoglucanase. The unique regions of the (1→3),(1→4)-β-d-glucan may be the recognition-hydrolytic site of the Zea endoglucanase. 相似文献
6.
Erin L. Westman David J. McNally Armen Charchoglyan Dyanne Brewer Robert A. Field Joseph S. Lam 《The Journal of biological chemistry》2009,284(18):11854-11862
The lipopolysaccharide of Pseudomonas aeruginosa PAO1 contains an
unusual sugar, 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid
(d-ManNAc3NAcA). wbpB, wbpE, and wbpD
are thought to encode oxidase, transaminase, and N-acetyltransferase
enzymes. To characterize their functions, recombinant proteins were
overexpressed and purified from heterologous hosts. Activities of
His6-WbpB and His6-WbpE were detected only when both
proteins were combined in the same reaction. Using a direct MALDI-TOF mass
spectrometry approach, we identified ions that corresponded to the predicted
products of WbpB (UDP-3-keto-d-GlcNAcA) and WbpE
(UDP-d-GlcNAc3NA) in the coupled enzyme-substrate reaction.
Additionally, in reactions involving WbpB, WbpE, and WbpD, an ion consistent
with the expected product of WbpD (UDP-d-GlcNAc3NAcA) was
identified. Preparative quantities of UDP-d-GlcNAc3NA and
UDP-d-GlcNAc3NAcA were enzymatically synthesized. These compounds
were purified by high-performance liquid chromatography, and their structures
were elucidated by NMR spectroscopy. This is the first report of the
functional characterization of these proteins, and the enzymatic synthesis of
UDP-d-GlcNAc3NA and UDP-d-GlcNAc3NAcA.Gram-negative organisms such as Pseudomonas aeruginosa produce
lipopolysaccharide
(LPS)4 as an essential
component of the outer leaflet of the outer membrane. LPS can be conceptually
divided into three parts: lipid A, which anchors LPS into the membrane; core
oligosaccharide, which contributes to membrane stability; and the O-antigen,
which is a polysaccharide that extends away from the cell surface. In P.
aeruginosa, two types of O-antigen are observed: A-band O-antigen, which
is common to most strains, and B-band O-antigen, which is variable and
therefore used as the basis of the International Antigenic Typing Scheme
(1). P. aeruginosa
serotypes O2, O5, O16, O18, and O20 collectively belong to serogroup O2,
because they all share common backbone sugar structures in their O-antigen
repeat units consisting of two di-N-acetylated uronic acids and one
2-acetamido-2,6-dideoxy-d-galactose
(N-acetyl-d-fucosamine). The minor structural variations
in the O-antigen repeat units that differentiate this serogroup into five
serotypes are: the type of glycosidic linkage between O-units (alpha
versus beta) that is formed by the O-antigen polymerase (Wzy),
isomers present (d-mannuronic or l-guluronic acid), and
acetyl group substituents
(2–4).
The B-band O-antigen of P. aeruginosa PAO1 (serotype O5) contains a
repeating trisaccharide of
2-acetamido-3-acetamidino-2,3-dideoxy-d-mannuronic acid
(d-ManNAc3NAmA),
2,3-diacetamido-2,3-dideoxy-d-mannuronic acid
(d-ManNAc3NAcA), and 2-acetamido-2,6-dideoxy-d-galactose
(3).The biosynthesis of the two mannuronic acid derivatives has yet to be fully
understood and has been the subject of investigation by our group. To produce
UDP-d-ManNAc3NAcA, a five-step pathway has been proposed
(Fig. 1) that requires the
products of five genes localized to the B-band O-antigen biosynthesis cluster
(5). The O-antigen biosynthesis
cluster was shown to be identical for all serotypes within serogroup O2, which
further underscores the high similarity between these serotypes
(5). The five genes, including
wbpA, wbpB, wbpE, wbpD, and wbpI, have been shown to be
essential for B-band LPS biosynthesis, because knockout mutants of each of
these genes are deficient in B-band O-antigen
(6–8).
Homologs of all five of the proteins required for the
UDP-d-ManNAc3NAcA biosynthesis pathway are conserved in other
bacterial pathogens, including Bordetella pertussis, Bordetella
parapertussis, and Bordetella bronchiseptica.
Cross-complementation of P. aeruginosa knockout mutants lacking
wbpA, wbpB, wbpE, wbpD, or wbpI with the homologues from
B. pertussis could fully restore LPS production in the P.
aeruginosa LPS mutants, suggesting that the genes from B.
pertussis are functional homologs of the wbp genes
(7). Homologs of these genes
could be identified in diverse bacterial species, demonstrating the importance
of UDP-d-ManNAc3NAcA biosynthesis beyond its role in P.
aeruginosa (7).Open in a separate windowFIGURE 1.Proposed pathway for the biosynthesis of UDP-d-ManNAc3NAcA in
P. aeruginosa PAO1. The full names of the sugars are as follows:
GlcNAc, 2-acetamido-2-deoxy-d-glucose; GlcNAcA,
2-acetamido-2-deoxy-d-glucuronic acid; 3-keto-d-GlcNAcA,
2-acetamido-2-deoxy-d-ribo-hex-3-uluronic acid; GlcNAc3NA,
2-acetamido-3-amino-2,3-dideoxy-d-glucuronic acid; GlcNAc3NAcA,
2,3-diacetamido-2,3-dideoxy-d-glucuronic acid; ManNAc3NAcA,
2,3-diacetamido-2,3-dideoxy-d-mannuronic acid. Adapted from Ref.
8.The first enzyme of the UDP-d-ManNAc3NAcA biosynthesis pathway,
WbpA, is a 6-dehydrogenase that converts
UDP-2-acetamido-2-deoxy-d-glucose
(N-acetyl-d-glucosamine; UDP-d-GlcNAc) to
UDP-2-acetamido-2-deoxy-d-glucuronic acid
(N-acetyl-d-glucosaminuronic acid,
UDP-d-GlcNAcA) using NAD+ as a coenzyme
(9)
(Fig. 1). Following this, the
second step in UDP-d-ManNAc3NAcA biosynthesis is proposed to be an
oxidation reaction catalyzed by WbpB, forming
UDP-2-acetamido-2-deoxy-d-ribo-hex-3-uluronic acid
(3-keto-d-GlcNAcA), which in turn is used as the substrate for
transamination by WbpE, creating
UDP-2-acetamido-3-amino-2,3-dideoxy-d-glucuronic acid
(d-GlcNAc3NA).This residue is thought to be the substrate for WbpD, a putative
N-acetyltransferase of the hexapeptide acyltransferase superfamily
(10) that requires acetyl-CoA
as a co-substrate (8). WbpD has
been proposed to synthesize
UDP-2,3-diacetamido-2,3-dideoxy-d-glucuronic acid
(UDP-d-GlcNAc-3NAcA), which is utilized in the B-band O-antigen of
P. aeruginosa serotype O1. In P. aeruginosa serogroup O2,
the UDP-d-GlcNAc3NAcA is then epimerized by WbpI to create the
UDP-d-ManNAc3NAcA required for incorporation into B-band LPS
(11). A derivative of
UDP-d-ManNAc3NAcA is also used in the synthesis of B-band O-antigen
of P. aeruginosa serogroup O2. UDP-d-ManNAc3NAmA is
thought to be produced through additional modification of
UDP-d-ManNAc3NAcA via the action of WbpG, an amidotransferase,
which has also been demonstrated to be essential for the production of B-band
O-antigen (12,
13).In the current study, our aim was to define the function of WbpB, WbpE, and
WbpD, because only genetic evidence has previously been given for the
involvement of wbpB and wbpE
(7), and the reaction catalyzed
by WbpD could not be demonstrated due to the unavailability of its presumed
substrate, UDP-d-GlcNAc3NA
(8). The functional
characterization of these proteins is also important for understanding LPS
biosynthesis in B. pertussis, because the genes in the LPS locus of
this species, wlbA, wlbC, and wlbB, could cross-complement
knockouts of wbpB, wbpE, and wbpD, respectively, when
expressed in P. aeruginosa PAO1
(7). Furthermore, these three
proteins form a cassette for the generation of C-3 N-acetylated
hexoses and may be important for the biosynthesis of a variety of other
sugars. Capillary electrophoresis and MALDI-TOF mass spectrometry were used to
analyze reaction mixtures of WbpB and WbpE and showed that the expected
products were produced only when both enzymes were present together. Achieving
the enzymatic synthesis of the product of both enzymes, which was demonstrated
to be UDP-d-GlcNAc3NA by 1H NMR spectroscopy, was a key
breakthrough, because this rare sugar has never before been produced by any
means. UDP-d-GlcNAc3NA was also essential for use as the substrate
of WbpD, which not only allowed us to determine the enzymatic activity of this
protein but also allowed the enzymatic synthesis of
UDP-d-GlcNAc3NAcA to be achieved as well. Although this sugar had
previously been produced through a 17-step chemical synthesis
(11,
14), the 4-step concurrent
enzymatic reaction demonstrates the advantage of linking chemistry with
biology and represents a significant saving of both time and reagents as
compared with chemical synthesis. Finally, our data also showed the success in
reconstituting in vitro the 5-step pathway for the biosynthesis of
UDP-d-ManNAc3NAcA in P. aeruginosa. 相似文献
7.
K. W. Knox 《The Biochemical journal》1965,94(3):534-535
1. The enzymic synthesis of O-β-d-glucopyranosyl-(1→6)-d-galactose has been described and evidence for the structure presented. 2. It has been shown that the transglycosylase of A. niger provides a convenient means of synthesizing (1→6)-linked disaccharides. 相似文献
8.
d-Glucosamine-6-P N-acetyltransferase (EC 2.3.1.4) from mung bean seeds (Phaseolus aureus) was purified 313-fold by protamine sulfate and isoelectric precipitation, ammonium sulfate and acetone fractionation, and CM Sephadex column chromatography. The partially purified enzyme was highly specific for d-glucosamine-6-P. Neither d-glucosamine nor d-galactosamine could replace this substrate. The partially purified enzyme preparation was inhibited up to 50% by 2 × 10−2m EDTA, indicating the requirement of a divalent cation. Among divalent metal ions tested, Mg2+ was required for maximum activity of the enzyme. Mn2+ and Zn2+ were inhibitory, while Co2+ had no effect on the enzyme activity. The pH optimum of the enzyme in sodium acetate and sodium citrate buffers was found to be 5.2. The effect of Mg2+ on the enzyme in sodium acetate and sodium citrate buffers was particularly noticeable in the range of optimum pH. Km values of 15.1 × 10−4m and 7.1 × 10−4m were obtained for d-glucosamine-6-P and acetyl CoA, respectively. The enzyme was completely inhibited by 1 × 10−4mp-hydroxymercuribenzoate, and this inhibition was partially reversed by l-cysteine; indicating the presence of sulfhydryl groups at or near the active site of the enzyme. 相似文献
9.
Takanori Nihira Erika Suzuki Motomitsu Kitaoka Mamoru Nishimoto Ken'ichi Ohtsubo Hiroyuki Nakai 《The Journal of biological chemistry》2013,288(38):27366-27374
A gene cluster involved in N-glycan metabolism was identified in the genome of Bacteroides thetaiotaomicron VPI-5482. This gene cluster encodes a major facilitator superfamily transporter, a starch utilization system-like transporter consisting of a TonB-dependent oligosaccharide transporter and an outer membrane lipoprotein, four glycoside hydrolases (α-mannosidase, β-N-acetylhexosaminidase, exo-α-sialidase, and endo-β-N-acetylglucosaminidase), and a phosphorylase (BT1033) with unknown function. It was demonstrated that BT1033 catalyzed the reversible phosphorolysis of β-1,4-d-mannosyl-N-acetyl-d-glucosamine in a typical sequential Bi Bi mechanism. These results indicate that BT1033 plays a crucial role as a key enzyme in the N-glycan catabolism where β-1,4-d-mannosyl-N-acetyl-d-glucosamine is liberated from N-glycans by sequential glycoside hydrolase-catalyzed reactions, transported into the cell, and intracellularly converted into α-d-mannose 1-phosphate and N-acetyl-d-glucosamine. In addition, intestinal anaerobic bacteria such as Bacteroides fragilis, Bacteroides helcogenes, Bacteroides salanitronis, Bacteroides vulgatus, Prevotella denticola, Prevotella dentalis, Prevotella melaninogenica, Parabacteroides distasonis, and Alistipes finegoldii were also suggested to possess the similar metabolic pathway for N-glycans. A notable feature of the new metabolic pathway for N-glycans is the more efficient use of ATP-stored energy, in comparison with the conventional pathway where β-mannosidase and ATP-dependent hexokinase participate, because it is possible to directly phosphorylate the d-mannose residue of β-1,4-d-mannosyl-N-acetyl-d-glucosamine to enter glycolysis. This is the first report of a metabolic pathway for N-glycans that includes a phosphorylase. We propose 4-O-β-d-mannopyranosyl-N-acetyl-d-glucosamine:phosphate α-d-mannosyltransferase as the systematic name and β-1,4-d-mannosyl-N-acetyl-d-glucosamine phosphorylase as the short name for BT1033. 相似文献
10.
Krzysztof Regulski Pascal Courtin Saulius Kulakauskas Marie-Pierre Chapot-Chartier 《The Journal of biological chemistry》2013,288(28):20416-20426
Peptidoglycan hydrolases (PGHs) are responsible for bacterial cell lysis. Most PGHs have a modular structure comprising a catalytic domain and a cell wall-binding domain (CWBD). PGHs of bacteriophage origin, called endolysins, are involved in bacterial lysis at the end of the infection cycle. We have characterized two endolysins, Lc-Lys and Lc-Lys-2, identified in prophages present in the genome of Lactobacillus casei BL23. These two enzymes have different catalytic domains but similar putative C-terminal CWBDs. By analyzing purified peptidoglycan (PG) degradation products, we showed that Lc-Lys is an N-acetylmuramoyl-l-alanine amidase, whereas Lc-Lys-2 is a γ-d-glutamyl-l-lysyl endopeptidase. Remarkably, both lysins were able to lyse only Gram-positive bacterial strains that possess PG with d-Ala4→d-Asx-l-Lys3 in their cross-bridge, such as Lactococcus casei, Lactococcus lactis, and Enterococcus faecium. By testing a panel of L. lactis cell wall mutants, we observed that Lc-Lys and Lc-Lys-2 were not able to lyse mutants with a modified PG cross-bridge, constituting d-Ala4→l-Ala-(l-Ala/l-Ser)-l-Lys3; moreover, they do not lyse the L. lactis mutant containing only the nonamidated d-Asp cross-bridge, i.e.
d-Ala4→d-Asp-l-Lys3. In contrast, Lc-Lys could lyse the ampicillin-resistant E. faecium mutant with 3→3 l-Lys3-d-Asn-l-Lys3 bridges replacing the wild-type 4→3 d-Ala4-d-Asn-l-Lys3 bridges. We showed that the C-terminal CWBD of Lc-Lys binds PG containing mainly d-Asn but not PG with only the nonamidated d-Asp-containing cross-bridge, indicating that the CWBD confers to Lc-Lys its narrow specificity. In conclusion, the CWBD characterized in this study is a novel type of PG-binding domain targeting specifically the d-Asn interpeptide bridge of PG. 相似文献
11.
Antiserum was raised against the Avena sativa L. caryopsis β-d-glucan fraction with an average molecular weight of 1.5 × 104. Polyclonal antibodies recovered from the serum after Protein A-Sepharose column chromatography precipitated when cross-reacted with high molecular weight (1→3), (1→4)-β-d-glucans. These antibodies were effective in suppression of cell wall autohydrolytic reactions and auxin-induced decreases in noncellulosic glucose content of the cell wall of maize (Zea mays L.) coleoptiles. The results indicate antibody-mediated interference with in situ β-d-glucan degradation. The antibodies at a concentration of 200 micrograms per milliliter also suppress auxin-induced elongation by about 40% and cell wall loosening (measured by the minimum stress-relaxation time of the segments) of Zea coleoptiles. The suppression of elongation by antibodies was imposed without a lag period. Auxin-induced elongation, cell wall loosening, and chemical changes in the cell walls were near the levels of control tissues when segments were subjected to antibody preparation precipitated by a pretreatment with Avena caryopsis β-d-glucans. These results support the idea that the degradation of (1→3), (1→4)-β-d-glucans by cell wall enzymes is associated with the cell wall loosening responsible for auxin-induced elongation. 相似文献
12.
Donor Substrate Regeneration for Efficient Synthesis of Globotetraose and Isoglobotetraose
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Here we describe the efficient synthesis of two oligosaccharide moieties of human glycosphingolipids, globotetraose (GalNAcβ1→3Galα1→4Galβ1→4Glc) and isoglobotetraose (GalNAcβ1→3Galα1→3Galβ1→4Glc), with in situ enzymatic regeneration of UDP-N-acetylgalactosamine (UDP-GalNAc). We demonstrate that the recombinant β-1,3-N-acetylgalactosaminyltransferase from Haemophilus influenzae strain Rd can transfer N-acetylgalactosamine to a wide range of acceptor substrates with a terminal galactose residue. The donor substrate UDP-GalNAc can be regenerated by a six-enzyme reaction cycle consisting of phosphoglucosamine mutase, UDP-N-acetylglucosamine pyrophosphorylase, phosphate acetyltransferase, pyruvate kinase, and inorganic pyrophosphatase from Escherichia coli, as well as UDP-N-acetylglucosamine C4 epimerase from Plesiomonas shigelloides. All these enzymes were overexpressed in E. coli with six-histidine tags and were purified by one-step nickel-nitrilotriacetic acid affinity chromatography. Multiple-enzyme synthesis of globotetraose or isoglobotetraose with the purified enzymes was achieved with relatively high yields. 相似文献
13.
The effect of 0.5 millimolar O-acetyl-l-serine added to the nutrient solution on sulfate assimilation of Lemna minor L., cultivated in the light or in the dark, or transferred from light to the dark, was examined. During 24 hours after transfer from light to the dark the extractable activity of adenosine 5′-phosphosulfate sulfotransferase, a key enzyme of sulfate assimilation, decreased to 10% of the light control. Nitrate reductase (EC 1.7.7.1.) activity, measured for comparison, decreased to 40%. Adenosine 5′-triphosphate (ATP) sulfurylase (EC 2.7.7.4.) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8.) activities were not affected by the transfer. When O-acetyl-l-serine was added to the nutrient solution at the time of transfer to the dark, adenosine 5′-phosphosulfate sulfotransferase activity was still at 50% of the light control after 24 hours, ATP sulfurylase and O-acetyl-l-serine sulfhydrylase activity were again not affected, and nitrate reductase activity decreased as before. Addition of O-acetyl-l-serine at the time of the transfer caused a 100% increase in acid-soluble SH compounds after 24 hours in the dark. In continuous light the corresponding increase was 200%. During 24 hours after transfer to the dark the assimilation of 35SO42− into organic compounds decreased by 80% without O-acetyl-l-serine but was comparable to light controls in its presence. The addition of O-acetyl-l-serine to Lemna minor precultivated in the dark for 24 hours induced an increase in adenosine 5′-phosphosulfate sulfotransferase activity so that a constant level of 50% of the light control was reached after an additional 9 hours. Cycloheximide as well as 6-methyl-purine inhibited this effect. In the same type of experiment O-acetyl-l-serine induced a 100-fold increase in the incorporation of label from 35SO42− into cysteine after additional 24 hours in the dark. Taken together, these results show that exogenous O-acetyl-l-serine has a regulatory effect on assimilatory sulfate reduction of L. minor in light and darkness. They are in agreement with the idea that this compound is a limiting factor for sulfate assimilation and seem to be in contrast to the proposed strict light control of sulfate assimilation. 相似文献
14.
1. The cell wall of Fusicoccum amygdali consisted of polysaccharides (85%), protein (4–6%), lipid (5%) and phosphorus (0.1%). 2. The main carbohydrate constituent was d-glucose; smaller amounts of d-glucosamine, d-galactose, d-mannose, l-rhamnose, xylose and arabinose were also identified, and 16 common amino acids were detected. 3. Chitin, which accounted for most of the cell-wall glucosamine, was isolated in an undegraded form by an enzymic method. Chitosan was not detected, but traces of glucosamine were found in alkali-soluble and water-soluble fractions. 4. Cell walls were stained dark blue by iodine and were attacked by α-amylase, with liberation of glucose, maltose and maltotriose, indicating the existence of chains of α-(1→4)-linked glucopyranose residues. 5. Glucose and gentiobiose were liberated from cell walls by the action of an exo-β-(1→3)-glucanase, giving evidence for both β-(1→3)- and β-(1→6)-glucopyranose linkages. 6. Incubation of cell walls with Helix pomatia digestive enzymes released glucose, N-acetyl-d-glucosamine and a non-diffusible fraction, containing most of the cell-wall galactose, mannose and rhamnose. Part of this fraction was released by incubating cell walls with Pronase; acid hydrolysis yielded galactose 6-phosphate and small amounts of mannose 6-phosphate and glucose 6-phosphate as well as other materials. Extracellular polysaccharides of a similar nature were isolated and may be formed by the action of lytic enzymes on the cell wall. 7. About 30% of the cell wall was resistant to the action of the H. pomatia digestive enzymes; the resistant fraction was shown to be a predominantly α-(1→3)-glucan. 8. Fractionation of the cell-wall complex with 1m-sodium hydroxide gave three principal glucan fractions: fraction BB had [α]D +236° (in 1m-sodium hydroxide) and showed two components on sedimentation analysis; fraction AA2 had [α]D −71° (in 1m-sodium hydroxide) and contained predominantly β-linkages; fraction AA1 had [α]D +40° (in 1m-sodium hydroxide) and may contain both α- and β-linkages. 相似文献
15.
A small quantity of (1→3)-β-d-glucan was extracted with a (1→3),(1→4)-β-d-glucan by hot water after treatment of the insoluble fraction of a buffer homogenate of Zea shoots with 3 molar LiCl. An ammonium sulfate precipitation procedure effected a separation of the (1→3)-β-d-glucan from the more prevalent (1→3),(1→4)-β-d-glucan. The minor component polysaccharide precipitated at a concentration of 20% ammonium sulfate (w/v) and was, as a consequence of precipitation, rendered insoluble in water. The insoluble products were dissolved in 1 normal NaOH followed by neutralization with CH3COOH. The purified polysaccharide accounted for approximately 0.3% of total hot water extract. It consisted mostly of glucose and its average mol wt was estimated to be about 7.0 × 104, based on elution from a calibrated Sepharose CL-4B column. Methylation analysis and enzymic hydrolysis or partial acid-hydrolysis of the polysaccharide followed by analysis of the hydrolysate showed that the polysaccharide consisted of (1→3)-β-linked glucose residues. 相似文献
16.
17.
Biosynthesis of Insoluble Glucans From Uridine-Diphosphate-d-Glucose With Enzyme Preparations From Phaseolus aureus and Lupinus albus 总被引:9,自引:6,他引:3
Particulate, and digitonin-solubilized, enzyme systems from Phaseolus aureus and Lupinus albus catalyze the biosynthesis of aqueous-insoluble glucans from UDP-d-glucose. The digitonin treatment greatly increases the enzymic activity of (per unit protein) both the 34,000g pellet and the supernatant liquid as compared with that of the original particles. Most of the polymer produced (90-95%) is soluble in hot, dilute alkali; the interglucosidic linkages of the alkali-soluble and alkali-insoluble polymers are identical. The optimum concentration for the incorporation of radioactivity from UDP-d-glucose-14C into soluble glucan is high; at 10−3 m at least 50% of the added radioactive glucosyl donor is incorporated. 相似文献
18.
The mode of inhibition of UDP, one of the products of the reaction catalyzed by (1→3)-β-d-glucan synthase in sugar beet (Beta vulgaris L.) was investigated. In the absence of added UDP, the enzyme, in the presence of Ca2+, Mg2+, and cellobiose, exhibited Michaelis-Menten kinetics and had an apparent Km of 260 micromolar for UDP-glucose. Complex effects on the kinetics of the (1→3)-β-d-glucan synthase were observed in the presence of UDP. At high UDP-glucose concentrations, i.e. greater than the apparent Km, UDP behaved as a competitive inhibitor with an apparent Ki of 80 micromolar. However, at low UDP-glucose concentrations, reciprocal plots of enzyme activity versus substrate concentration deviated sharply from linearity. This unusual effect of UDP is similar to that reported for fungal (1→3)-β-d-glucan synthase. However, papulacandin B, a potent inhibitor of this fungal enzyme, had no effect on the plant (1→3)-β-d-glucan synthase isolated from sugar beet petioles. The inhibitory effect of UDP was also compared with other known inhibitors of glucan synthases. 相似文献
19.
Marina Díez-Municio Blanca de las Rivas Maria Luisa Jimeno Rosario Mu?oz F. Javier Moreno Miguel Herrero 《Applied and environmental microbiology》2013,79(13):4129-4140
The ability of an inulosucrase (IS) from Lactobacillus gasseri DSM 20604 to synthesize fructooligosaccharides (FOS) and maltosylfructosides (MFOS) in the presence of sucrose and sucrose-maltose mixtures was investigated after optimization of synthesis conditions, including enzyme concentration, temperature, pH, and reaction time. The maximum formation of FOS, which consist of β-2,1-linked fructose to sucrose, was 45% (in weight with respect to the initial amount of sucrose) and was obtained after 24 h of reaction at 55°C in the presence of sucrose (300 g liter−1) and 1.6 U ml−1 of IS–25 mM sodium acetate buffer–1 mM CaCl2 (pH 5.2). The production of MFOS was also studied as a function of the initial ratios of sucrose to maltose (10:50, 20:40, 30:30, and 40:20, expressed in g 100 ml−1). The highest yield in total MFOS was attained after 24 to 32 h of reaction time and ranged from 13% (10:50 sucrose/maltose) to 52% (30:30 sucrose/maltose) in weight with respect to the initial amount of maltose. Nuclear magnetic resonance (NMR) structural characterization indicated that IS from L. gasseri specifically transferred fructose moieties of sucrose to either C-1 of the reducing end or C-6 of the nonreducing end of maltose. Thus, the trisaccharide erlose [α-d-glucopyranosyl-(1→4)-α-d-glucopyranosyl-(1→2)-β-d-fructofuranoside] was the main synthesized MFOS followed by neo-erlose [β-d-fructofuranosyl-(2→6)-α-d-glucopyranosyl-(1→4)-α-d-glucopyranose]. The formation of MFOS with a higher degree of polymerization was also demonstrated by the transfer of additional fructose residues to C-1 of either the β-2,1-linked fructose or the β-2,6-linked fructose to maltose, revealing the capacity of MFOS to serve as acceptors. 相似文献
20.
Identification and Quantitative Analysis of Indole-3-Acetyl-l-Aspartate from Seeds of Glycine max L 总被引:12,自引:12,他引:0
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Cohen JD 《Plant physiology》1982,70(3):749-753
Indole-3-acetyl-l-aspartate (IAAsp) was isolated from seeds of Glycine max L. cv. Hark and its identity established by its chromatographic performance and its mass spectral fragmentation. Following acid hydrolysis, the aspartate moiety was shown to be the l-enantiomer by reverse phase high performance liquid chromatographic retention time of the bisethyl ester derivatized with 2,3,4,6-tetra-O-acetyl-β-d-glycopyranosyl isothiocyanate. Isotope dilution analysis using [14C]IAAsp as internal standard showed that soybean seed contained 10 μmol/kg IAAsp and this accounted for one-half of the total indoleacetic acid of the seed. 相似文献