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Hamster liver14C-mannolipid, prepared by DEAE-cellulose chromatography of the organic extract, is shown to be a more efficient donor of mannose to endogenous glycoproteins then guanosine-diphosphate-mannose.Analysis of endogenous acceptors after proteolysis reveals that several compounds separated by DEAE-sephadex chromatography are labeled by both14C-mannolipid and GDP-mannose14C. 相似文献
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Galactose transfer to endogenous acceptors within Golgi fractions of rat liver 总被引:8,自引:15,他引:8
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《The Journal of cell biology》1982,92(1):139-146
The distribution of galactosyl transferase was studied using trans and cis Golgi fractions isolated by a modification of the Ehrenreich et al. procedure (1973. J. Cell Biol. 59:45-72) as well as an intact Golgi fraction isolated by a new one-step procedure. Two methods of assay were used. The first method analyzed the ability of Golgi fractions to transfer galactose (from uridine diphosphogalactose [UDP-gal] substrate) to the defined exogenous acceptor ovomucoid. The second method assessed the transfer of galactose from UDP-gal substrate to endogenous acceptors (endogenous glycosylation). The trans Golgi fraction (Golgi light) was highly active by the first method but revealed only low activity by the second method. Golgi fractions enriched in central and cis elements (the Golgi intermediate, heavy and especially the intact Golgi fraction) were highly active in both methods of assay. The endogenous glycosylation approach was validated by gel fluorography of the endogenous acceptors. For all Golgi fractions, transfer of galactose was revealed to secretory glycopeptides. It is concluded that galactosyl transferase activity in vivo occurs primarily in central and cis Golgi elements but not trans Golgi vesicles. 相似文献
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J Fuentes Mota J Fernández-Bola?os Guzmán J M García Fernández W Moreda C Ortiz M A Pradera I Robina C Welsh 《Carbohydrate research》1992,232(1):47-57
The preparation of 2,3,4-tri-O-benzyl- (3), 2,3,4-tri-O-acetyl- (4), and 2,3,4-tri-O-benzoyl-N-(2,2-diethoxycarbonylvinyl)-6-O-trityl-beta- D-glucopyranosylamine (5) is described. The reaction of 3-5 with 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranosyl bromide yields 2,3,4-tri-O-benzyl- (9), 2,3,4-tri-O-acetyl- (10), and 2,3,4-tri-O-benzoyl-N-(2,2-diethoxycarbonylvinyl)-6-O-(2,3,4,6-tet ra-O- acetyl-beta-D-glucopyranosyl)-beta-D-glucopyranosylamine (11), respectively. 2,3,4-Tri-O-benzyl- (6), 2,3,4-tri-O-acetyl- (7), and 2,3,4-tri-O- benzoyl-N-(2,2-diethoxycarbonylvinyl)-beta-D-glucopyranosylamine (8) are also described. 相似文献
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Decreased transfer of oligosaccharide from oligosaccharide-lipid to protein acceptors in regenerating rat liver 总被引:3,自引:0,他引:3
The transfer of [14C]glucose from UDP-[14C]glucose to lipid intermediates and glycoproteins was decreased in regenerating rat liver microsomes 24 h after partial hepatectomy. In regenerating liver microsomes, the concentration of free dolichyl phosphate (Dol-P) was significantly decreased. However, it was only about 10% of total Dol-P, which was not significantly changed. On the addition of exogenous Dol-P, the transfer of [14C]glucose to glycoproteins was still decreased, while the decrease of the transfer to lipid intermediates was no longer observed. These results suggest that the glycoprotein synthesis is not regulated by the amount of Dol-P in regenerating liver microsomes. Oligosaccharide obtained from [14C]glucosyl-oligosaccharide-lipid was not distinguishable between regenerating liver and control by paper chromatography. The oligosaccharide transfer to protein in microsomes was compared by using [14C]glucosyl-oligosaccharide-lipid as oligosaccharide donor. The transfer of oligosaccharide to endogenous proteins decreased to 77% of control in regenerating liver and the transfer to exogenously added denatured alpha-lactalbumin decreased to 59% of control. Therefore, it is unlikely that the acceptor capacity of endogenous protein is decreased in regenerating liver. Neither the change in oligosaccharide-lipid under the condition for oligosaccharide transfer assay nor the stability of oligosaccharide transferase was different between regenerating liver and control. These results strongly suggest that the decrease in the activity of the oligosaccharide transferase in microsomes causes the decrease of glycoprotein synthesis in regenerating liver, which was shown in our previous studies. 相似文献
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Homogenates of embryonic chick neural retina prepared in 1% Triton X-100 have the ability to transfer N-acetyl[32P]galactosamine [( 32P]GalNAc) from beta-32P-labeled uridine diphosphate N-acetylgalactosamine [( beta-32P]UDP-GalNAc) to endogenous macromolecular acceptors. The phosphotransferase activity sediments as three distinct peaks upon centrifugation on sucrose gradients. These peaks are coincident with the transferase/acceptor complexes previously described [Balsamo, J., & Lilien, J. (1982) J. Biol. Chem. 257, 345-354]. The parameters of the 32P transfer reaction closely parallel those observed with UDP-[3H]GalNAc as substrate when the densest particles, H, are used as a source of transferase/acceptors. Treatment of 3H- and 32P-labeled products with alpha-N-acetylgalactosaminidase removes [3H]GalNAc residues and exposes 32P-labeled groups. These data suggest that the sugar-phosphate is transferred intact, resulting in a terminal phosphodiester linkage. The resistance of the macromolecular products to digestion by endoglycosidase F and its sensitivity to hydrolysis under mild alkaline conditions suggest that the alpha-linked sugar is transferred to an oligosaccharide chain attached to the protein core via an O-serine or threonine residue. Characterization of the 32P- and 3H-labeled H particle products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals a series of coincident high molecular weight polypeptides. 相似文献
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P Bellemin-Magninot G Azzar R Létoublon R Got 《The International journal of biochemistry》1992,24(5):827-830
1. Nuclei were prepared from rat hepatocytes. A biochemical analysis of marker enzymes showed that the nuclei are not contaminated by other subcellular fractions, especially endoplasmic reticulum. 2. The transfer of [14C]N-acetylglucosamine to endogenous acceptors were studied comparatively in the nuclei and in the other subcellular fractions of rat hepatocytes. 3. In this report we describe the presence of the transfer of N-acetylglucosamine within the nucleus of rat hepatocytes. We found 21% of this transfer in the nucleus fraction with an enrichment of 26 in comparison to homogenate. 相似文献
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Endogenous protein phosphorylation has been studied in extracts from rat cells transformed by temperature-sensitive derivatives of Rous sarcoma virus which can reversibly express a transformed behavior at 33 degrees C and reacquire "normal" properties at 39 degrees C. The expression of transformation appeared associated with marked alterations in the type of phosphoprotein acceptors and with an increased protein kinase activity, particularly in detergent solubilized fractions. A comparison of the effect of dibutyryl cyclic AMP on protein phosphorylation revealed a transformation-dependent response, as well as a different effect of the cycle nucleotide in cytoplasmic and detergent solubilized fractions. Our studies suggest that the processes leading to malignant transformation are accompanied by altered response of the phosphorylating system to cyclic nucleotide-mediated modulation and by marked alteration in phosphoprotein acceptors. 相似文献
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We presented exact expressions for the ensemble averaged decay of the excitation of a donor molecule due to the energy transfer via anisotropic dipolar interactions to acceptors distributed randomly on a surface. The disorder extended both over the positions of the acceptors and over the orientations of their transition dipoles with respect to that of the donor molecule. Several cases were considered explicitly (a) random orientations of the acceptors in space, with the donor being (a1) perpendicular to the plane, (a2) in the plane, (a3) randomly oriented in space; (b) random orientations of both donor and acceptors in the plane; (c) parallel orientations of donor and acceptors (no orientational disorder). For all these cases we evaluated the analytic, Förster-like expressions, valid for long times and low acceptor densities, and obtained their domains of validity by comparison with the exact, numerically calculated decay laws. 相似文献
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The substrate specificity of N-acetylhexosaminidase (E.C. 3.2.1.51) from Aspergillus oryzae was examined using p-nitrophenyl 6-O-sulfo-N-acetyl-beta-D-glucosaminide (6-O-sulfo-GlcNAc-O-pNP) as the glycosyl donor and a series of beta-d-glucopyranosides and N-acetyl-beta-D-glucosaminides with variable aglycons at the anomeric positions as the acceptors. When beta-D-glucopyranosides with methyl (CH(3)), allyl (CH(2)CHCH(2)), and phenyl (C(6)H(5)) groups at the reducing end were used as the acceptors, this enzyme transferred the 6-O-sulfo-GlcNAc moiety in the donor to the location of O-4 in these glycosyl acceptors with a high regioselectivity, producing the corresponding 6-O-sulfo-N-acetylglucosaminyl beta-D-glucopyranosides. However, beta-D-glucopyranose lacking aglycon was a poor substrate for transglycosylation. This A. oryzae enzyme could also accept various N-acetyl-beta-D-glucosaminides carrying hydroxyl (OH), methyl (CH(3)), propyl (CH(2)CH(2)CH(3)), allyl (CH(2)CHCH(2)) and p-nitrophenyl (pNP; C(6)H(4)-NO(2)) groups at their aglycons, yielding 6-O-sulfo-N-acetylglucosaminyl-beta(1-->4)-disaccharide products. 相似文献
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The purpose of this article is to highlight the versatility of nonfluorescent Förster resonance energy transfer (FRET) acceptors in determination of protein equilibrium dissociation constants and kinetic rates. Using a nonfluorescent acceptor eliminates the necessity to spectrally isolate the donor fluorescence when performing binding titrations covering a broad range of reagent concentrations. Moreover, random distribution of the donor and acceptor chromophores on the surface of proteins increases the probability of FRET occurring on their interaction. Three high-affinity antibodies are presented in this study as characteristic protein systems. Monoclonal antibody (mAb) 106.3 binds brain natriuretic peptide (BNP)5–13(C10A) and full-length BNP1–32 with the dissociation constants 0.26 ± 0.01 and 0.05 ± 0.02 nM, respectively, which was confirmed by kinetic measurements. For anti-hCG (human chorionic gonadotropin) mAb 8F11, studied at two incorporation ratios (IRs = 1.9 and 3.8) of the nonfluorescent FRET acceptor, KD values of 0.04 ± 0.02 and , respectively, were obtained. Likewise, the binding of goat anti-hamster immunoglobulin G (IgG) antibody was not affected by conjugation and yielded KD values of 1.26 ± 0.04, 1.25 ± 0.05, and 1.14 ± 0.04 nM at IRs of 1.7, 4.7, and 8.1, respectively. We conclude that this FRET-based method offers high sensitivity, practical simplicity, and versatility in protein binding studies. 相似文献
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Peptide acceptors in the arginine transfer reaction 总被引:2,自引:0,他引:2
R L Soffer 《The Journal of biological chemistry》1973,248(8):2918-2921
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In this report, we describe the main characteristics of the transfer of N-acetylglucosamine within the nucleus of rat hepatocytes. The glycosylation pathway includes the presence of lipids which mediate the nuclear proteins glycosylation. The level of dolichylphosphate seems low and thus could be a regulation factor in the nuclear glycosylations. The discussion deals with the membranous character of the acceptors and the N-acetylglucosaminyltransferase, the N-linkage of the sugar moiety to nuclear proteins and the function of such glycosylation. 相似文献