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1.
Glycolipid anchors are attached to Thy-1 glycoprotein rapidly after translation. 总被引:12,自引:0,他引:12 下载免费PDF全文
The attachment of glycolipid anchors to the Thy-1 glycoprotein during biosynthesis was followed by the change of detergent-binding properties of biosynthetically labelled Thy-1 precursors upon phospholipase C treatment in the murine thymoma lines BW5147 and S1A. In S1A, 80% of the Thy-1 molecules were phospholipase-C-sensitive after a 2 min pulse with [35S]methionine, indicating that these molecules were already anchored via a glycolipid tail. In BW5147, 47% of the Thy-1 molecules had phospholipase-C-sensitive anchors attached after a 1.5 min labelling and, with longer pulses, this percentage rose to 76%. Tunicamycin did not block the addition of glycolipid anchors, and glycolipid attachment also occurred at 21 degrees C. The findings suggest that the attachment of glycolipid anchors occurs in the rough endoplasmic reticulum. 相似文献
2.
Expression of UDP-N-acetylgalactosamine: beta-galactose beta 1,4-N-acetylgalactosaminyltransferase in functionally defined T-cell clones. 总被引:1,自引:0,他引:1 下载免费PDF全文
To measure UDP-N-acetylgalactosamine: beta-galactose beta 1,4-N-acetylgalactosaminyltransferase (beta 1,4-GalNActransferase) in crude cell and tissue extracts we designed an assay containing UDP-[3H]N-acetylgalactosamine as donor and biotinylated human glycophorin A as an acceptor. After incubation the labelled acceptor was separated by the use of avidin-agarose from extract-derived endogenous acceptors. This assay permitted one to measure specifically the beta 1,4-GalNActransferase in crude extracts. This glycosyltransferase has previously been shown to be involved in the biosynthesis of Vicia villosa (hairy winter vetch)-lectin (VV)-binding sites of the murine cytotoxic T-cell line B6.1. Since VV-binding sites are a distinct marker for the cytotoxic subclass of murine T-lymphocytes, we used this assay to determine enzyme levels in a panel of functionally defined murine T-cell clones. Non-cytolytic T-cell lines generally have low activity, whereas most cytotoxic lines have high levels of activity. However, one cytotoxic T-cell line does not express the enzyme, although it has large numbers of VV-binding sites. This suggests the existence of another type of VV-binding sites which is independent of the beta 1,4-GalNActransferase in some cytotoxic-T-lymphocyte lines. The enzyme was also assayed in a variety of other tissues and found to have a very high activity in the intestine but a low activity in most other tissues. This was in considerable contrast with the ubiquitously high expression of UDP-GalNAc:peptide alpha 1-GalNActransferase. Therefore, the beta 1,4-GalNActransferase seems to be regulated during differentiation. 相似文献
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We have reported the presence of N-acetylgalactosamine linked beta 1,4 to galactose on O-linked oligosaccharides of a cloned murine cytotoxic T cell line and the absence of these residues from the O-linked structures of a Vicia villosa lectin-resistant mutant line, VV6, derived from parental B6.1.SF.1 cells (Conzelmann, A., and Kornfeld, S. (1984) J. Biol. Chem. 259, 12528-12535). This study shows that B6.1.SF.1 cells contain an enzyme which transfers N-acetylgalactosamine from UDP-GalNAc onto the O-linked tetrasaccharides of human glycophorin A, giving rise to pentasaccharides which contain beta-glycosidically linked N-acetylgalactosamine. Desialylated glycophorin was inactive as an acceptor. The enzyme also transfers N-acetylgalactosamine to the N-linked oligosaccharides of the Tamm-Horsfall glycoprotein. This glycoprotein is known to contain N-linked oligosaccharides with beta-linked N-acetylgalactosamine residues which constitute the Sda blood group determinant. This N-acetylgalactosaminyltransferase could not be detected in VV6 cells which can account for the lack of beta-linked N-acetylgalactosamine residues on its O-linked oligosaccharides. The two cell lines have comparable levels of UDP-GalNAc:apomucin N-acetylgalactosaminyltransferase, demonstrating that the enzyme deficiency in VV6 cells is selective. Both cell lines have a similar glycolipid content, with the major component being asialo-GM1. Since this glycolipid contains N-acetylgalactosamine linked beta 1,4 to galactose, it would appear that the N-acetylgalactosyltransferase involved in the biosynthesis of glycolipids is different from the UDP-GalNAc:glycoprotein N-acetylgalactosaminyltransferase. An independently derived murine CTL line also contains the UDP-GalNAc:glycoprotein N-acetylgalactosaminyltransferase, suggesting that the expression of this enzyme is a common characteristic of this type of cell line. 相似文献
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Mokola virus glycoprotein and chimeric proteins can replace rabies virus glycoprotein in the rescue of infectious defective rabies virus particles. 总被引:3,自引:1,他引:2 下载免费PDF全文
A reverse genetics approach which allows the generation of infectious defective rabies virus (RV) particles entirely from plasmid-encoded genomes and proteins (K.-K. Conzelmann and M. Schnell, J. Virol. 68:713-719, 1994) was used to investigate the ability of a heterologous lyssavirus glycoprotein (G) and chimeric G constructs to function in the formation of infectious RV-like particles. Virions containing a chloramphenicol acetyltransferase (CAT) reporter gene (SDI-CAT) were generated in cells simultaneously expressing the genomic RNA analog, the RV N, P, M, and L proteins, and engineered G constructs from transfected plasmids. The infectivity of particles was determined by a CAT assay after passage to helper virus-infected cells. The heterologous G protein from Eth-16 virus (Mokola virus, lyssavirus serotype 3) as well as a construct in which the ectodomain of RV G was fused to the cytoplasmic and transmembrane domains of the Eth-16 virus G rescued infectious SDI-CAT particles. In contrast, a chimeric protein composed of the amino-terminal half of the Eth-16 virus G and the carboxy-terminal half of RV G failed to produce infectious particles. Site-directed mutagenesis was used to convert the antigenic site III of RV G to the corresponding sequence of Eth-16 G. This chimeric protein rescued infectious SDI-CAT particles as efficiently as RV G. Virions containing the chimeric protein were specifically neutralized by an anti-Eth-16 virus serum and escaped neutralization by a monoclonal antibody directed against RV antigenic site III. The results show that entire structural domains as well as short surface epitopes of lyssavirus G proteins may be exchanged without affecting the structure required to mediate infection of cells. 相似文献
7.
Purification and characterization of an activator protein for the degradation of glycolipids GM2 and GA2 by hexosaminidase A 总被引:7,自引:0,他引:7
E Conzelmann K Sandhoff 《Hoppe-Seyler's Zeitschrift für physiologische Chemie》1979,360(12):1837-1849
The activator protein for the degradation of glycolipids GM2 and GA2 by hexosaminidase A was purified some 2 500-fold from normal human kidney. It has a molecular weight of approximately 25 000 is heat-stable up to 60 degrees C, possesses an isoelectric point of pH 4.8 and is digestible by proteases. Enzymic degradation of the lipid substrates in the presence of this activator proceeds optimally at pH 4.2. The mode of action of the activator was also studied: the protein most probably complexes lipid molecules and presents them to the enzyme which otherwise cannot attack the aggregates formed by the lipids in aqueous solution. The hydrolysis of water-soluble synthetic substrates is not affected by the activator protein. The activator is highly specific for hexosaminidase A: hydrolysis of glycolipids GA2 and GM2 by the hexosaminidase B isoenzyme is almost not enhanced by this protein. The isoenzymes' lipid substrate specificity measured in the presence of the activator is entirely different from that obtained with detergents and can satisfactorily account for the lipid storage pattern observed in patients with variant forms of infantile GM2- gangliosidosis. 相似文献
8.
Peter Schemmer Zhi Zhong Uwe Galli Michael D. Wheeler Li Xiangli Blair U. Bradford Lars O. Conzelmann Dow Forman José Boyer Ronald G. Thurman 《Amino acids》2013,44(3):925-931
It has been demonstrated that a wide variety of white blood cells and macrophages (i.e. Kupffer cells, alveolar and peritoneal macrophages and neutrophils) contain glycine-gated chloride channels. Binding of glycine on the receptor stimulates Cl? influx causing membrane hyperpolarization that prevents agonist-induced influx of calcium. Since platelet-aggregation is calcium-dependent, this study was designed to test the hypothesis that glycine would inhibit platelet aggregation. Rats were fed diets rich of glycine for 5 days, while controls received isonitrogenous valine. The bleeding time and ADP- and collagen-induced platelet aggregation were measured. Dietary glycine significantly increased bleeding time about twofold compared to valine-treated controls. Furthermore, the amplitude of platelet aggregation stimulated with ADP or collagen was significantly decreased in whole blood drawn from rats fed 2.5 or 5 % dietary glycine by over 50 %. Addition of glycine in vitro (1–10 mM) also blunted rat platelet aggregation in a dose-dependent manner. Strychnine, a glycine receptor antagonist, abrogated the inhibitory effect of glycine on platelet-aggregation in vitro suggesting the glycine works via a glycine receptor. Glycine also blunted aggregation of human platelets. Further, the glycine receptor was detected in both rat and human platelets by western blotting. Based on these data, it is concluded that glycine prevents aggregation of platelets in a dose-dependent manner via mechanisms involving a glycine receptor. 相似文献
9.
Retrograde neuronal tracing with a deletion-mutant rabies virus 总被引:1,自引:0,他引:1
We have constructed a deletion-mutant rabies virus encoding EGFP and find it to be an excellent tool for studying detailed morphology and physiology of neurons projecting to injection sites within the mammalian brain. The virus cannot spread beyond initially infected cells yet, unlike other viral vectors, replicates its core within them. The cells therefore fluoresce intensely, revealing fine dendritic and axonal structure with no background from partially or faintly labeled cells. 相似文献