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1.
Julia E. Lever Colleen E. Sari 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1983,762(2):265-271
We have explored the relationship between glycoprotein biosynthesis, cell proliferation and function of a differentiated polarized membrane assessed by dome formation in the MDCK epithelial cell line. At 0.1 μg/ml tunicamycin, complete inhibition of cell proliferation was observed in either serum-containing or serum-free, hormone-supplemented growth medium. By contrast, no inhibition of either spontaneous dome formation or that triggered by inducers of cell differentiation such as hexamethylene bisacetamide was observed at 0.5 μg/ml tunicamycin, although total glycosylation of cellular proteins was inhibited by 75%. Our results suggest that the polarized sorting out of epithelial membrane proteins to apical and basolateral surfaces and their functions related to vectorial transepithelial fluid transport, monitored by dome formation, can persist umimpaired despite considerable underglycosylation of cellular glycoproteins and inhibition of cell proliferation. 相似文献
2.
Plasma membranes have been purified from an established cell line, Mos 20A of Aedes aegypti, and analysed for glycoprotein and polypeptide constituents by isoelectric focusing and sodium dodecyl sulphate polyacrylamide gel electrophoresis. A major glycoprotein of molecular weight 110 000 carrying binding sites for concanavalin A and soybean agglutinin has been purified to homogeneity. Although located on the cell surface, the 110 kdalton glycoprotein is not labelled by lactoperoxidase-catalysed radioactive iodination of whole cells. Analysis indicates the presence of N-glycans, containing on average nine mannose residues, and the N-acetylglucosaminyl-β1,4-N-acetylglucosamine sequence. In addition, O-glycosidically linked N-acetylgalactosamine residues are present. 相似文献
3.
To assess the role in cell-cell adhesion of gp64, a putative cell-cell adhesion molecule ofPolysphondylium pallidum, we treated the cells with tunicamycin (TM), a known inhibitor of the synthesis of the N-linked oligosaccharide precursor,
and examined TM's effect on cell-cell adhesion. The vegetative growth ofPolysphondylium cells was inhibited with TM in a dose-dependent manner. When cells were treated with TM (2.0 μg/ml) during only the first
4 hr of starvation and further starved for 8 hr without TM, the cells dissociated considerably, although even the growth phase
cells ofPolysphondylium normally show EDTA-resistant (Ca2+-independent) cell adhesions. In parallel with the above effects, the amounts of intact gp64 decreased considerably in time
with the lengths of incubation (0 hr>4 hr >8 hr). When TM-treated cells were washed free of TM, and shaken for a further 12
hr, the cells began to aggregate again, accompanied by an increase of gp64. In conclusion, TM affected cell-cell adhesion
ofPolysphondylium cells, but we were not able to distinguish whether the inhibition of cell aggregation was due to defects in glycosylation
on glycoproteins and/or due to reduced levels of glycoproteins themselves. 相似文献
4.
Dr. John E. Michaels 《Cell and tissue research》1980,210(1):121-132
Summary Glycoproteins are associated with several structures of colonic absorptive cells of the mouse. These include the cell coat, Golgi apparatus and vesicles that transport the glycoproteins from the apparatus to the cell surface (Michaels and Leblond 1976). In many in vitro systems, the antibiotic tunicamycin inhibits the glycosylation of asparagine residues yielding carbohydrate-poor glycoproteins. In the present in vivo study, tunicamycin was injected into mice. The murine colonic epithelial cells were prepared routinely for electron microscopy and cytochemistry. Cells from the experimental and control animals were similar morphologically. However, staining by the periodic acid-chromic acid-silver methenamine technique, revealed differences in the distribution of glycoproteins. In animals that received the higher dosages of tunicamycin there was a substantial reduction in silver staining in both the Golgi apparatus and the vesicles of colonic epithelial cells compared to these structures in cells of identically treated control tissues, whereas the staining over the cell coat was not significantly altered. Possible explanations for the staining of the cell coat in the treated animals were provided in the text. This report demonstrates the feasibility of using tunicamycin in vivo and detection of the changes obtained by the silver methenamine method. 相似文献
5.
Yoshitaka Yoneyama Florence Moog 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1982,696(1):23-30
The effect of thyroxine on biosynthesis of microvillus membrane glycoproteins has been investigated in organ culture of 18-day-old chick embryonic duodenum. Explants incorporate [3H]leucine and [3H]glucosamine continuously, and overall incorporation is enhanced by 10 nM thyroxine during 48 h of labeling; this increase in radioactivity is associated with vesicles released from the microvilli. Light microscope autoradiography, pulse labeling of brush border fragments, and pulse chase experiments reveal that [3H]glucosamine is incorporated into brush border at an increasing rate during culture, and that newly synthesized glycoproteins are discharged into the medium along with brush border enzymes (alkaline phosphatase and maltase). These results suggest that thyroxine stimulates biosynthesis of microvillus membrane glycoproteins, in addition to stimulating vesiculation of the membrane. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 3H-labeled vesicles and brush border fragments show that [3H]leucine and [3H]glucosamine are incorporated into proteins of high molecular weight. Two protein bands are identified as alkaline phosphatase and maltase. Thyroxine stimulates glycosylation of these enzymes, but does not change protein patterns. Radioactivity assay of alkaline phosphatase- and maltase-active gel slices suggests that thyroxine stimulation of these enzyme activities during culture is not correlated with de novo synthesis of these proteins. 相似文献
6.
Summary The effect of tunicamycin, which is known to inhibit the synthesis of N-linked glycoprotein, on the duodenal absorptive epithelial cell of the mouse was studied in thin-section as well as freeze-fracture images. In tunicamycin-treated animals, the apical part of the epithelial cell was almost negative to the PAS reaction. Moreover, microvilli of the epithelial cell became shorter, larger in diameter, and fewer in number in tunicamycin-treated mice. In addition, freeze-fracture images revealed that the population density of membrane particles of the microvillus membrane was lowered by tunicamycin treatment. These results may indicate that the inhibition of synthesis of N-linked glycoprotein causes a decrease of membrane supply from the Golgi apparatus to the apical plasma membrane.This study was supported by grants from the Ministry of Education, Science and Culture. 相似文献
7.
Patrick Cammarata Linda Chan Costante Ceccarini 《Biochimica et Biophysica Acta (BBA)/General Subjects》1981,674(1):128-135
The normal human fibroblast, WI-38, was labelled with radioactive mannose and its incorporation, as well as the accumulation of acidic and neutral glycopeptides on the cell surface, was followed as a function of time. The transit time of newly made Pronase-released cell surface glycopeptides from their intracellular site of synthesis to the cell surface was slower in nongrowing cells than in a rapidly growing culture. When the surface glycopeptides were separated by high-voltage paper electrophoresis into neutral and acidic species, it was observed that the cell surface material was initially enriched with neutral glycopeptides. However, with time the relative proportion of acidic species increased so that by 3 h the ratio between the acidica and neutral species approached a constant value. Our data are consistent with the hypothesis that multiple pathways for asparagine-linked glycoprotein biosynthesis are possible. 相似文献
8.
Aaron Heifetz Alice R. Johnson Mary K. Roberts 《Biochimica et Biophysica Acta (BBA)/General Subjects》1984,798(1):1-7
Human vascular endothelial cells synthesize lactosaminoglycan-type glycoproteins which are found both associated with cells and secreted into the culture medium. Pronase-derived glycopeptides prepared from [3H]glucosamine-labeled glycoproteins were found to contain about 10% of the labeled products as a large size (Mr > 5000) 3H-labeled glycopeptide. Digestion of these 3H-labeled glycopeptides with endo-β-galactosidase resulted in the release of smaller size saccharides, which were characterized as having the structure sialic acid → Gal → GlcNAc → Gal. Treatment of [3H]glucosamine-labeled cells with melittin caused 3H-labeled glycoconjugates to be released from the cells. Separation of released glycoproteins from proteoglycans by DEAE-cellulose chromatography indicated that melittin had released 25% of the total 3H-labeled glycoproteins from the cell and 3% of the 3H-labeled proteoglycans. The 3H-labeled glycoproteins were digested with Pronase and the resulting 3H-labeled glycopeptides were fractionated on Sephadex G-50. The large size fraction (Mr > 5000) now comprised about 30% of these released 3H-labeled glycopeptides. These high molecular weight 3H-labeled glycopeptides were degraded with endo-β-galactosidase but not with testicular hyaluronidase. Analysis of the released 3H-labeled glycoproteins indicated a preferential release of glycoproteins of 70–90 kDa enriched in lactosaminoglycan-type oligosaccharides. 相似文献
9.
Summary The in vivo effects of tunicamycin on the glycosylation of proteoglycans and link protein in rat mandibular condylar chondrocytes were studied by ultrastructural lectin histochemistry. The binding of wheatgerm agglutinin was shown by using anti-lectin antibody followed by protein A-gold complex. In normal rats, wheat-germ agglutinin labeling was restricted to trans cisternae and vacuoles of the Golgi complex, whereas it was observed in neither the cis region of the Golgi complex nor in the rough endoplasmic reticulum. By 3 h after the drug administration, wheat-germ agglutinin binding sites on the disorganized Golgi vacuoles were dramatically reduced in number. At 6 h after the drug administration, the lectin binding sites on the Golgi vacuoles were restored. These results demonstrate that the in vivo use of tunicamycin in combination with histochemical analysis using lectin probes is of significant value for the study of protein glycosylation in chondrocytes of the rat mandibular condyle. 相似文献
10.
Summary Repeated injection of rats with tunicamycin over two days induced a 1- to 5-fold increase in glycogen. This accumulation occurred in the stratum intermedium of the enamel organ and in young secretory odontoblasts. In rats injected over 3 days, the number of glycogen particles was at least 10 times larger than in control rats, and large glycogen accumulations were observed in the cytosol of these two groups of cells. These results were obtained by staining with periodic acid-thiocarbohydrazide and silver proteinate, a specific method for the detection of glycoconjugates containing vic-glycol groups. The existence of a relationship between these local cytosolic accumulations of glycogen and the developmental stage of certain groups of cells was shown by the changes that occurred in glycogen distribution. The present results suggest that the stratum intermedium supplies energy for precursor transport. 相似文献
11.
Bina Toor Lilian McGregor John L. McGregor Serge Renaud Kenneth J. Clemetson 《生物化学与生物物理学报:生物膜》1984,770(2):178-182
Female rats were administered oral contraceptives and the levels of sialic acid on platelet membrane and granule glycoproteins were compared to controls using a sialic acid assay and a fluorescein-conjugated wheat germ agglutinin binding assay and also by measuring the binding of 125I-labelled wheat germ agglutinin to glycoprotein bands from platelets separated by polyacrylamide electrophoresis. The contraceptive-treated rats showed increased levels of glycoprotein sialylation which may partly explain the altered physiological function of the platelets. 相似文献
12.
Shirley E. Poduslo 《生物化学与生物物理学报:生物膜》1983,728(1):59-65
Oligodendroglial plasma membranes are complex structures composed of a heterogeneous mixture of proteins and glycoproteins. The Coomassie stained gel patterns showed a maximum of 40 proteins with molecular weights ranging from . Autoradiography was used to detect binding of radioiodinated lectins to glycoproteins. With concanavalin A, 5 major glycoproteins were seen; with wheat germ agglutinin, 2 major glycoproteins with approximate molecular weights of 95 000 and 78 000 were found; with Ulex europaeus, 7 major glycoproteins were observed. Additional minor bands were also seen. The impermeant probe diazodi[125I]iodosulfanilic acid was used to radiolabel intact cells. It was found that 5 major proteins were radiolabeled in the plasma membranes. In all cases, the whorls of membrane lamellae produced in culture by oligodendroglia tend to have a somewhat less complicated pattern with fewer proteins and glycoproteins than the plasma membranes. However, the whorls of membrane lamellae have far more complicated protein patterns than myelin. 相似文献
13.
《Biochimica et Biophysica Acta (BBA)/General Subjects》1984,797(1):10-19
The binding of platelets to collagen is the first step in hemostasis. We attempted three approaches for elucidation of the chemical nature of receptors of human platelets for collagen. First, we examined the effect of platelet surface alteration by chymotrypsin treatment. On increasing the concentration of chymotrypsin, collagen-induced platelet aggregation and the release reaction decreased, and in parallel with this change, remarkable decrease of membrane glycoproteins IIb and V, as well as 400 kDa and 300 kDa membrane proteins, was observed. Secondly, effects of several lectins on the platelet-collagen interaction were examined. Lens culinaris agglutinin was found to specifically inhibit the platelet aggregation and release reaction induced by collagen. This inhibition appeared to be caused mainly by blocking of the collagen receptors on platelets by Lens culinaris agglutinin. Furthermore, Lens culinaris agglutinin was found to bind preferentially to glycoprotein IIb as identified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of platelet membranes followed by staining with 125I-Lens culinaris agglutinin. In addition, a polymerized preparation of Lens culinaris agglutinin induced platelet aggregation. Thirdly, the membrane component which could bind to collagen-Sepharose 4B was determined. Analysis by SDS-polyacrylamide gel electrophoresis combined with autoradiography or fluorography revealed that glycoprotein IIb was most enriched in the bound fraction to collagen. From these results, glycoprotein IIb is most likely a receptor for collagen on human platelet membranes. 相似文献
14.
J. Thomas LaMont Allen Ventola 《Biochimica et Biophysica Acta (BBA)/General Subjects》1980,629(3):553-565
In vivo glycoprotein synthesis and secretion was studied in rat colonic epithelial cells using precursor labelling with radiolabelled glucosamine. Sepharose 4B gel filtration of radiolabelled glycoproteins obtained from isolated colonic epithelial cells revealed two major fractions: (1) high molecular weight mucus in the excluded fraction and (2) lower molecular weight glycoproteins in the included volume. These glycoproteins were further fractionated by affinity chromatography on concanavalin A-Sepharose. The low molecular weight [3H]glucosamine-labelled glycoproteins contained a major subfraction which specifically adhered to concanavalin A, and could be eluted with 0.2 M α-methylmannoside. Fractionation of the concanavalin A-reactive glycoproteins on Sephadex G-100 revealed a major peak with a molecular weight of 15 000. In contrast, high molecular weight mucus glycoprotein did not adhere appreciably to concanavalin A-Sepharose. Perfusion experiments indicated that colonic secretions contained both mucus and concanavalin A-reactive glycoproteins. The major concanavalin A-reactive glycoprotein in the colonic perfusate was not derived from serum, but was released directly from the colonic membrane into the lumen. 相似文献
15.
Dr. Miriam M. Brysk Joanne Miller Shu-Jen Chen Peter C. Moller Robert W. Stach 《Cell and tissue research》1986,245(1):215-221
Summary Exposure of fibroblasts to tunicamycin has been found to be cytotoxic for transformed cells, but not for nontransformed cells. With two mouse epidermal cell lines of common origin, we observe a contrary pattern: The malignant cells are more resistant to tunicamycin than their nonmalignant counterparts, as measured by growth and viability. With respect to the glycosylation of sugar precursors, the incorporation of mannose is more inhibited than that of glucosamine, while fucose is least impacted. Sugar incorporation is less reduced for the malignant cells, by a factor of two for fucose and more modestly for the other two sugars. There are no significant morphological changes; in particular, the desmosomal junctions are not affected. On polyacrylamide gels, we note intensity variations in several protein bands in response to tunicamycin, but little difference between malignant and nonmalignant cells when using either Coomassie stains or Concanavalin A overlays. 相似文献
16.
Wannee Rojanapo James Allen Olson Adrian J. Lamb 《Biochimica et Biophysica Acta (BBA)/General Subjects》1980,633(3):386-399
Glycoproteins and proteins were extracted from segments or scrapings of the intestine in tube-fed, vitamin-A-deficient and control rats on the eight day after withdrawal of retinoic acid from the diet by using either 1% sodium dodecyl sulfate (SDS) or aqueous 5 mM EDTA (pH 7.4). They were then fractionated on columns of Sepharose 4B. Water-soluble peak I material contained large (Mr > 106; S20 = 11.7) glycoprotein aggregates which were rich in hexose, fucose and sialic acid. These aggregates dissociated into several non-identical glycoprotein and protein subunits upon treatment with dithiothreitol. The protein matrix was rich in threonine, valine, proline, serine, glutamate and aspartate. Peak II consisted of smaller proteins and glycoproteins, the latter with much lower carbohydrate content. Some peak II glycoproteins also dissociated into subunits in the presence of dithiothreitol. Peak III consisted mainly of a heterogenous assortment of proteins, including some glycoproteins of low carbohydrate content. Antibodies either to peak II or to peak III reacted both with peaks II and III but not with peak I.The total weight, carbohydrate composition of glycoproteins and the ratio of carbohydrate to protein in the total extract or in each of the three fractions were not significantly affected in vitamin A deficiency despite decreased incorporation of all labeled precursors. Rather, the relatively lower incorporation (approx. 0.8) of radioactive sulfate, D-glucosamine and L-fucose into total SDS-soluble duodenal glycoproteins of vitamin-A-deficient rats could be explained on the basis of a reduced prevalence of goblet cells alone. In contrast, the relative incorporation rate of L-fucose into peak I, but not into peaks II and III, ranged from 0.25 to 0.45, less than expected on the basis of fewer goblet cells alone. The incorporation of radioactive threonine into all protein fractions was reduced to 60% of normal in vitamin A deficiency. Thus, the well established observation that intestinal tissue of vitamin-A-deficient rats synthesizes high molecular weight glycoproteins poorly might be due to several interacting factors: (1) a reduced prevalence of goblet cells, (2) a lower rate of protein synthesis, (3) a lack of retinyl phosphate for the formation of mannosyl or other carbohydrate derivatives, and (4) secondary, and as yet undefined, cellular changes which preferentially reduce the rate of synthesis of high molecular weight fucose- and sialic-acid-enriched glycoproteins. 相似文献
17.
18.
Diana L. Blithe H.Fred Clark Leonard Warren 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,719(2):190-198
The carbohydrate groups of the glycoproteins of human, hamster, chick, reptile and fish cells growing in culture have been fractionated in succession according to size (Sephadex G-50), affinity for concanavalin A, charge (DEAE-Sephadex) and by thin-layer chromatography. It was found that despite the complexity of the array of separable glycopeptides in each type of cell, most of these structures seemed to be common to all of the cells. This suggests that they have existed in a relatively stable state for several hundreds of millions of years throughout the evolution of the vertebrates. 相似文献
19.
John L. McGregor Kenneth J. Clemetson Elizabeth James Phillippe Clezardin Marc Dechavanne Ernst F. Lüscher 《生物化学与生物物理学报:生物膜》1982,689(3):513-522
Washed platelets were surface-labelled by lactoperoxidase catalyzed iodination and either the platelets or membranes were solubilized in detergent and applied to a wheat germ agglutinin-Sepharose column and a Lens culinaris lectin Sepharose column coupled sequentially. The glycoproteins eluted from the lectin columns were separated by two-dimensional gel electrophoresis. Alternatively, labelled whole platelets or membranes were solubilized and then directly separated by two-dimensional polyacrylamide gel electrophoresis. Spots corresponding to specific glycoproteins identified by apparent isoelectric point (), apparent molecular weight (), staining and labelling characteristics were cut from the gels and analyzed by tryptic peptide mapping. The maps of the individual glycoproteins (GP) Ia, Ib, IIa, IIb, GP132–1354–4.5 IIIa, IIIb and IIIc were all different. Glycoproteins with the same but different were distinct with the exception of regions of GP Ib. There were minor differences in the maps of glycoproteins separated in the reduced or non-reduced state. Tryptic peptide maps provide a valuable additional parameter for the identification and characterization of platelet glycoproteins. 相似文献
20.
The effect of tunicamycin (TM) on the metabolism of acetylated low-density lipoprotein (AcLDL) was examined to determine whether N-linked glycosylation is required for the proper function of the AcLDL pathway. Proteolytic degradation of [125I]-AcLDL was increased twofold in the presence of TM. This did not occur via an increase in total lysosomal enzyme activity or extracellular proteolysis; rather, the rate of uptake of [125I]-AcLDL was increased. The enhanced degradation of AcLDL did not lead to a commensurate increase in the rate of synthesis of cholesteryl oleate. Conversely, the rate of cholesterol esterification was reduced in the presence of TM. The uptake of [125I]-AcLDL was more sensitive to inhibition by chloroquine in TM-treated cells. However, the presence of TM did not affect the ability of chloroquine to inhibit constitutive recycling of AcLDL binding sites. These results suggest that N-linked glycosylation may be involved in the regulation of AcLDL metabolism in J774 cells. 相似文献