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1.
A new method for the radioisotopic assay of neuraminidase activity has been developed. The substrate utilized, α-d-N-acetylneuraminosyl-(2 → 3′)-lactit[3H]ol, was prepared by reduction of α-d-N-acetylneuraminosyl-(2 → 3′)-lactose with tritiated borohydride and purified by ion-exchange chromatography. After incubation with neuraminidase, the reaction mixtures were applied to small columns of AG 1-X2 (formate) in order to remove free sialic acid and unhydrolyzed substrate. The lactit[3H]ol released by neuraminidase action was then recovered by washing the columns with distilled water and quantitated by utilizing a liquid scintillation spectrometer. Studies with bacterial, avian, and mammalian neuraminidases are described. 相似文献
2.
Andrea van der Meer Johannis P. Kamerling Johannes F.G. Vliegenthart Karl Schmid Roland Schauer 《Biochimica et Biophysica Acta (BBA)/General Subjects》1983,757(3):371-376
Analysis of the sialic acid fraction obtained by mild acid hydrolysis of the Cuvierian tubules of the sea cucumber Holothuria forskali Della Chiaje revealed the presence of F-glycolylneuraminic acid and fucopyranosyl-(1 → 4)-N-glycolylneuraminic acid. Furthermore, methylation analysis of a pronase-digest of the tubules demonstrated that all N-glycolylneuraminic acid residues are substituted at C-4, explaining the earlier reported resistance of sialic acid in tubules to cleavage by neuraminidase. 相似文献
3.
Highly microheterogeneous polyglycosylceramides (PGCs) of human erythrocytes with an average composition of about 25 monosaccharides linked to ceramide were analyzed by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). The human gastric pathogen Helicobacter pylori was earlier shown to bind this glycosphingolipid mixture by thin-layer chromatogram binding assay. The receptor activity was present along the whole nonresolved chromatographic interval. Mass spectra of intact PGCs were compared with corresponding spectra of oligosaccharides enzymatically released from the ceramides. Two subfractions of PGCs containing less than one and more than one sialic acid residue per molecule were used. MALDI-MS spectra were recorded in both linear and reflectron mode with the accuracies of =0.08% and =0.02%, respectively, which allowed determination of the constituent parts of the detected ions in form of ceramide and number of hexoses, N-acetylhexosamines, fucoses and sialic acids. Molecular species were found based on ceramide with mainly sphingosine and fatty acids 24:0 and 24:1 (with less amounts of 22:0), and with a total number of monosaccharides ranging from 11 (neutral, m/z = 2605 for [M+Na](+)) to 41 (one sialic acid, m/z = 8057 for [M-H](-)). The saccharide composition obtained supported a successively extended and branched N -acetyllactosamine core with substitutions of fucoses (0 up to 8) and sialic acid (0 to 1). The reliable molecular analysis of large oligosaccharides linked to ceramide using this approach will be of great help for the further structure analysis in order to define the epitope for the sialic acid-dependent binding by the bacterium. 相似文献
4.
Cytochalasin B and the sialic acids of Ehrlich ascites cells 总被引:3,自引:0,他引:3
The effect of cytochalasin B (CB) on the electrophoretic mobility and density of ionized sialic acid groups at the surface of Ehrlich ascites cells was examined together with a biochemical assay of the total sialic acid content of treated and control cells. Sialic acid assays indicated that CB-treated cells had a greater amount of total sialic acid and sialic acid sensitive to neuraminidase than control cells/cell. Equal amounts of sialic acid were removable by neuraminidase treatment from control cells and cells pretreated with neuraminidase and subsequently cultured with CB. The electrophoresis results showed a decrease in electrophoretic mobility in the presence of CB which could be reversed by growth in CB-free medium. Neuraminidase treatment did not make a significant additional reduction in the mobility of CB-treated cells. CB also prevented the recovery of electrophoretic mobility of neuraminidase treated cells. The results suggest that while CB does not inhibit sialic acid synthesis, it does alter the expression of ionized sialic acid groups at the electrokinetic surface. CB-containing culture media could be re-utilized several times suggesting that CB is not significantly bound or metabolized by Ehrlich ascites cells. 相似文献
5.
Fabry's disease as an -galactosidosis: evidence for an -configuration in trihexosyl ceramide 总被引:2,自引:0,他引:2
I Bensaude J Callahan M Philippart 《Biochemical and biophysical research communications》1971,43(4):913-918
Pure α-galactosidases, devoid of β-galactosidase activity, were purified from coffee beans, ficin (a crude extract from figs), rat and rabbit small intestine. With the exception of the coffee bean enzyme, all α-galactosidase preparations released galactose from 3H- or 14C-labeled trihexosyl ceramide obtained from patients with Fabry's disease. Galactose liberation was specifically inhibited by α-galactosides, such as melibiose and stachyose, while lactose had no effect. Our results corroborate the α-galactosidase deficiency reported in Fabry's disease and establish that the terminal galactosyl residue of the trihexosyl ceramide stored in this condition has an α-configuration. 相似文献
6.
《Bioorganic & medicinal chemistry letters》2020,30(2):126883
Artificial substrates for probing neuraminidase activity are powerful tools for studying the physiological and pathological roles of neuraminidases. Most of the substrates are α-O-linked sialosides involving hydroxyl-containing reporters for visualization, and neuraminidase-catalyzed cleavage of the sialic acid residues directly activates the reporters. However, the use of amine-containing reporters has been avoided because α-N-linked sialosides are marginal substrates for neuraminidases. To expand the applicability of reporters to amine-containing compounds, we have focused on prodrug design. Herein we describe the synthesis and enzymatic study of a model substrate involving 4-nitroaniline as an amine-containing chromogenic reporter. The substrate can respond to neuraminidase from Clostridium perfringens. Neuraminidase-mediated hydrolysis of the sialic acid moiety of the substrate initiates self-immolative elimination of the linker moiety, leading the liberation of yellow-colored reporter 4-nitroaniline. The elimination process involves generation of quinone methide intermediate, which causes to neutralize neuraminidase. The substrate, thus, works as not only a chromogenic substrate but also a suicide inactivator. 相似文献
7.
The plant gum isolated from sap of the lac tree, Rhus vernicifera (China), was separated into two fractions having mol. wt. 84,000 and 27,700 by aqueous-phase gel-permeation chromatography. The fractions contain d-galactose (65 mol%), 4-O-methyl-d-glucuronic acid (24 mol%), d-glucuronic acid (3 mol%), l-arabinose (4 mol%), and l-rhamnose (3 mol%). Smith degradation of the carboxyl-reduced polysaccharides gives products of halved molecular weight, and these consist of a β-(1→3)-linked galactopyranan main chain and side chains made up of galactopyranose residues. Peripheral groups, such as α-d-Galp-, α-d-Galp-(1→6)-β-d-Galp-, 4-O-methyl-β-d-GlcpA-, and 4-O-methyl-β-d-GlcpA-(1→6)-β-d-Galp-, are attached to this interior core through β-(1→3)- or β-(1→6)-linkages. 相似文献
8.
DL-Phenylalanine-[3-14C] and cinnamic acid-[3-14C] were fed to this plant and the label from cinnamic acid was incorporated into gallic acid, phyllodulcin and quercetin. By feeding p- coumaric acid-[U-3H], caffeic acid-[U-3H] and hydrangea glucoside A-[U-3H], it was possible to show that hydroxylation at C-3′in phyllodulcin occurs after the ring closure of dihydroisocoumarin. The biosynthetic pathway of phyllodulcin in this plant is thus: phenylalanine → cinnamic acid → p- coumaric acid → hydrangenol → phyllodulcin. 相似文献
9.
A new ganglioside, provisionally named GLIVa, was isolated in pure form from pig cerebellum. Ganglioside GLIVa is a disialoganglioside containing fucose. Its basic neutral glycosphingolipid core is the gangliotetraose ceramide: Gal, β 1 → 3 GalNAc, β 1 → 4 Gal, β 1 → 4 Glc, β 1 → Cer. Fucose is α-glycosidically linked to the 2-position of external galactose and one N-acetylneuraminic acid is linked to the other one by an α, 2 → 8 linkage. Thus the total structure of ganglioside GLIVa is the following: Fuc, α 1 → 2 Gal, β 1 → 3 GalNAc, β 1 → 4 (NeuAc, α 2 48 NeuAc, α 2 → 3) Gal, β 1 → 4 Glc, β 1 → Ceramide. According to the IUPAC-IUB Commission on Biochemical Nomenclature is indicated as II3α(NeuAc)2 IV2αFuc-GgOse4Cer. 相似文献
10.
Convallaria majalis plants were fed dl-methionine-[1-14C]. [1-14C, 4-3H], and [1-14C, 2-3H], S-adenosyl-l-methionine-[1-14C], and dl-homoserine-[1-14C], resulting in the formation of labeled azetidine-2-carboxylic acid (A-2-C). The complete retention of tritium relative to carbon-14 in the feeding experiment involving methionine-[1-14C, 4-3H] indicates that aspartic acid or aspartic-β-semialdehyde are not intermediates between methionine and A-2-C. However, since the A-2-C derived from methionine-[1-14C, 2-3H] had lost 95% of the tritium relative to the C-14, it is not considered that methionine or its S-adenosyl derivative are the immediate precursors of A-2-C. Our data and that of others is consistent with the intermediate formation of γ-amino-α-ketobutyric acid which on cyclization yields 1-azetine-2-carboxylic acid, A-2-C then being formed on reduction. 相似文献
11.
αD -N-acetyl neuraminic acid (Neu5Ac, sialic acid) is a commonly occurring carbohydrate residue in various cell surface glycolipids and glycoproteins. This residue is linked terminally or internally to Gal residues via an α(2 → 3) or α(2 → 6) linkage. In the cell surface receptor, sialyl-LewisX, a terminal α(2 → 3) linkage is present. Previous studies from our laboratory have shown that in solution LewisX adopts a relatively rigid structure. In order to model the Neu5Ac residue, vacuum molecular dynamics of this monosaccharide were compared with simulations that explicitly include solvent water. The dynamical average of the monosaccharide conformation obtained from the two simulations was similar. Vacuum calculations for the disaccharide Neu5Ac α(2 → 3) Gal β-O-methyl show that a number of low energy minima are accessible to this disaccharide. Molecular dynamics simulations starting from the low energy minima show conformational transitions with a time scale of 10–50 ps among several of the minima while large barriers between other minima prevent transitions on the time scale studied. Simulations of this disaccharide in the presence of solvent show fewer conformational transitions, illustrating a dampening effect of the solvent that has been observed in some other studies. Our results are most consistent with an equilibrium among multiple conformations for the Neu5Ac α(2 → 3) Gal β linkage. © 1994 John Wiley & Sons, Inc. 相似文献
12.
Comparative studies of asparagine-linked oligosaccharide structures of rat liver microsomal and lysosomal beta-glucuronidases 总被引:4,自引:0,他引:4
The sugar chains of microsomal and lysosomal β-glucuronidases of rat liver were studied by endo-β-N-acetylglucosaminidase H digestion and by hydrazinolysis. Only a part of the oligosaccharides released from microsomal β-glucuronidase was an acidic component. The acidic component was not hydrolyzed by sialidase and by calf intestinal and Escherichia coli alkaline phosphatases, but was converted to a neutral component by phosphatase digestion after mild acid treatment indicating the presence of a phosphodiester group. The neutral oligosaccharide portion of microsomal enzyme was a mixture of five high mannose-type sugar chains: (Manα1 → 2)0~4 [Manα1 → 6(Manα1 → 3)Manα1 → 6(Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4GlcNAc]. In contrast, lysosomal enzyme contains only Manα1 → 6 (Manα1 → 3) Manα1 → 6(Manα1 → 3) Manβ1 → 4GlcNAcβ1 → 4GlcNAc. The result indicates that removal of α1 → 2-linked mannosyl residues from (Manα1 → 2)4[Manα1 → 6(Manα1 → 3)Manα1 → 6(Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4GlcNAc → Asn] starts already in the endoplasmic reticulum of rat liver. 相似文献
13.
T Kiyohara T Terao K Shioiri-Nakano T Osawa 《Archives of biochemistry and biophysics》1974,164(2):575-582
A neuraminidase was purified from the culture filtrate of Streptococcus 6646 (group K) by means of ammonium sulfate fractionation and successive column chromatographies on N-(p-aminophenyl)oxamic acid-substituted Sepharose derivative and p-aminophenyl-2-acetamido-2-deoxy-1-thio-β-d-glucopyranoside-substituted Sepharose derivative. The former adsorbent was found to bind a β-galactosidase and a β-N-acetylhexosaminidase in addition to the neuraminidase, and the latter adsorbent bound the β-galactosidase in addition to the β-d-N-acetylhexosaminidase. These adsorbents effectively eliminated the contaminating glycosidase activities and a 1,500-fold purification of the neuraminidase was achieved by this procedure.The neuraminidase thus purified was homogeneous by electrophoresis on polyacrylamide gel, and its molecular weight was estimated to be 110,000 by gel filtration on Biogel P-200. The activity of the purified neuraminidase was slightly stimulated by Ca2+, Mg2+, Mn2+, and Co2+, and strongly inhibited by heavy metals. The specificity of the purified neuraminidase was almost the same with Vibrio cholerae or Clostridium perfringens neuraminidase. It completely hydrolyzes sialic acid residues in neuraminyl lactose and porcine thyroglobulin, but it liberates only 50% of sialic acid residues from porcine submaxillary mucin and ganglioside GD1a. 相似文献
14.
Craig D. Fischer Maria Da Costa Sheldon P. Rothenberg 《Biochimica et Biophysica Acta (BBA)/General Subjects》1978,543(3):328-339
Folate-binding protein(s) from chronic myelogenous leukemia cells have been purified using acid dialysis, ammonium sulfate fractionation and affinity chromatography. The purified preparation which migrates as a single band on disc electrophoresis could be separated by DEAE agarose chromatography into two folate-binding proteins (binders I and II) which bind molar equivalents of folic acid. One binder (I) eluted from DEAE at 1 mM sodium phosphate, pH 6.0, and the other (II) at 100 mM sodium phosphate, pH 7.4. Analysis of the purified mixture, which contained more than 90% binder II, by sedimentation equilibrium centrifugation indicated a homogeneous protein with a calculated molecular weight of 44000. Antiserum raised against the purified mixture gave a single precipitin line by immunodiffusion against a preparation of partially purified cell lysate.Hydrolysis of the more acidic binder (II) with neuraminidase converted it to a weakly acidic protein similar to binder I suggesting that these binders are glycoproteins which differ in sialic acid content. With isoelectric focusing, the binding of folic acid would be demonstrated at pH 6.7, 7.3, 7.8 and 8.2 for binder I, and at pH 5.1, 5.8 and 6.5 for binder II. Binders I and II had equally high affinity for folic acid and dihydroflate, lower affinity of N5-methyl-tetrahydrofolate, and no apparent affinity for N5-formytetrahydrofolate or methotrexate. 相似文献
15.
Biosynthetic pathways to p-hydroxybenzoic acid in polar lignin were examined by tracer experiments. High incorporation of radioactivity to the acid was observed when shikimic acid-[1-14C], phenylalanine-[3-14C], trans-cinnamic acid-[3-14C], p-coumaric acid-[3-14C] and p-hydroxybenzoic acid-[COOH-14C] were administered, while incorporation was low from shikimic acid-[COOH-14C], phenylalanine-[1-14C], phenylalanine-[2-14C], tyrosine-[3-14C], benzoic acid-[COOH-14C], sodium acetate-[1-14C] and d-glucose-[U-14C]. Thus p-hydroxybenzoic acid in poplar lignin is formed mainly via the pathway: shikimic acid → phenylalanine → trans-cinnamic acid → p-coumaric acid → p-hydroxybenzoic acid. 相似文献
16.
Ronald M. Sans Walter W. Jolly Robert A. Harris 《Archives of biochemistry and biophysics》1980,200(2):336-345
In the absence of any other oxidizable substrate, the perfused rat heart oxidizes [1-14C]leucine to 14CO2 at a rapid rate and releases only small amounts of α-[1-14C]ketoisocaproate into the perfusion medium. The branched-chain α-keto acid dehydrogenase complex, assayed in extracts of mitochondria prepared from such perfused hearts, is very active. Under such perfusion conditions, dichloroacetate has almost no effect on [1-14C]leucine oxidation, α-[1-14C]ketoisocaproate release, or branched-chain α-keto acid dehydrogenase activity. Perfusion of the heart with some other oxidizable substrate, e.g., glucose, pyruvate, ketone bodies, or palmitate, results in an inhibition of [1-14C]leucine oxidation to 14CO2 and the release of large amounts of α-[1-14C]ketoisocaproate into the perfusion medium. The branched-chain α-keto acid dehydrogenase complex, assayed in extracts of mitochondria prepared from such hearts, is almost completely inactivated. The enzyme can be reactivated, however, by incubating the mitochondria at 30 °C without an oxidizable substrate. With hearts perfused with glucose or ketone bodies, dichloroacetate greatly increases [1-14C]leucine oxidation, decreases α-[1-14C]ketoisocaproate release into the perfusion medium, and activates the branched-chain α-keto acid dehydrogenase complex. Pyruvate may block dichloroacetate uptake because dichloroacetate neither stimulates [1-14C]leucine oxidation nor activates the branched-chain α-keto acid dehydrogenase complex of pyruvate-perfused hearts. It is suggested that leucine oxidation by heart is regulated by the activity of the branched-chain α-keto acid dehydrogenase complex which is subject to interconversion between active and inactive forms. Oxidizable substrates establish conditions which inactivate the enzyme. Dichloroacetate, known to activate the pyruvate dehydrogenase complex by inhibition of pyruvate dehydrogenase kinase, causes activation of the branched-chain α-keto acid dehydrogenase complex, suggesting the existence of a kinase for this complex. 相似文献
17.
Cioffi DL Pandey S Alvarez DF Cioffi EA 《American journal of physiology. Lung cellular and molecular physiology》2012,302(10):L1067-L1077
The surface of vascular endothelium bears a glycocalyx comprised, in part, of a complex mixture of oligosaccharide chains attached to cell-surface proteins and membrane lipids. Importantly, understanding of the structure and function of the endothelial glycocalyx is poorly understood. Preliminary studies have demonstrated structural differences in the glycocalyx of pulmonary artery endothelial cells compared with pulmonary microvascular endothelial cells. Herein we begin to probe in more detail structural and functional attributes of endothelial cell-surface carbohydrates. In this study we focus on the expression and function of sialic acids in pulmonary endothelium. We observed that, although pulmonary microvascular endothelial cells express similar amounts of total sialic acids as pulmonary artery endothelial cells, the nature of the sialic acid linkages differs between the two cell types such that pulmonary artery endothelial cells express both α(2,3)- and α(2,6)-linked sialic acids on the surface (i.e., surficially), whereas microvascular endothelial cells principally express α(2,3)-linked sialic acids. To determine whether sialic acids play a role in endothelial barrier function, cells were treated with neuraminidases to hydrolyze sialic acid moieties. Disruption of cell-cell and cell-matrix adhesions was observed following neuraminidase treatment, suggesting that terminal sialic acids promote endothelial barrier integrity. When we measured transendothelial resistance, differential responses of pulmonary artery and microvascular endothelial cells to neuraminidase from Clostridium perfringens suggest that the molecular architecture of the sialic acid glycomes differs between these two cell types. Collectively our observations reveal critical structural and functional differences of terminally linked sialic acids on the pulmonary endothelium. 相似文献
18.
Galactosyl (alpha 1--4)galactosylceramide: galactosyl hydrolase activity in normal and Fabry plasma 总被引:1,自引:0,他引:1
An α-galactosidase fraction which hydrolyzes galactosyl(α1→4)galactosylceramide and 4-methylumbelliferyl-α-galactoside has been isolated from normal human plasma by affinity chromaography. It was partially separated into two enzymatically active proteins by isoelectric focusing or cellulose acetate electrophoresis. The protein fraction obtained by affinity chromatography of Fabry plasma also was divided into two proteins, but only the protein of slower electrophoretic mobility had detectable enzymatic activity. These results indicate that the accumulation of galactosyl(α1→4)galactosylceramide in certain organs in Fabry's disease is due to an alteration of a specific α-galactosidase. 相似文献
19.
LEILA M. ODA CELUTA S. ALVIANO FERNANDO C. SILVA FILHO JAYME ANGLUSTER ISAAC ROITMAN WANDERLEY DE SOUZA 《The Journal of eukaryotic microbiology》1984,31(1):131-134
The surface anionic groups of symbiote-bearing and symbiote-free strains of Crithidia deanei were compared by determining cellular electrophoretic mobility, by ultrastructural cytochemistry, and by identification of sialic acids by thin-layer and gasliquid chromatography. Symbiote-free Crithidia deanei has a highly negative surface charge (-0.9984 μm?s-1? V-1? cm), which is slightly reduced (-0.8527 μm?s-1? V-1? cm) by the presence of the endosymbiote. Treatment of both strains of C. deanei with neuraminidase decreased significantly the electrophoretic mobility of cells toward the cathodic pole, indicating the existence of exposed sialic acid residues responsible for the negative charge on the protozoan cell surface. Thin-layer and gas-liquid chromatography showed that N-glycolyl- and N-acetylneuraminic acids were present in both strains of C. deanei. 相似文献
20.
Abstract: N′-Acetyl-d -[6-3H]mannosamine was administered to 13- and 28-day-old rats by intraventricular injection. At various time intervals following the injection, synaptic membranes were prepared and the incorporation of radiolabel into sialic acid residues released from endogenous glycoproteins and gangliosides by intrinsic sialidase determined. Radiolabel was incorporated into synaptic membrane gangliosides and glycoproteins, and at all times tested, >90% of the label was associated with sialic acid. Sialic acid released from endogenous glycoproteins by intrinsic sialidase present in 28-day membranes incorporated only 20–25% as much radiolabel per nmole as sialic acid released by mild acid hydrolysis or by exogenous neuraminidase. In contrast, sialic acid released from glycoproteins present in 13-day-old membranes by intrinsic sialidase, mild acid hydrolysis, or exogenous neuraminidase all were similarly labelled. At both ages the specific radioactivity (cpm/nmol) of sialic acid released from gangliosides by the intrinsic enzyme was similar to the total ganglioside sialic acid released by mild acid hydrolysis. The results identify glycoprotein substrates for intrinsic synaptic membrane sialidase as a distinct metabolic class in the mature brain and suggest the occurrence of a developmentally related change in the metabolism of these glycoproteins. 相似文献