共查询到20条相似文献,搜索用时 15 毫秒
1.
Gabriel Gachelin Marine-Hlne Buc-caron Halina Lis Nathan Sharon 《生物化学与生物物理学报:生物膜》1976,436(4):825-832
We have studied the interaction of five lectins differing in their sugar specificity, with the surface of clonal cell lines derived from transplantable murine teratocarcinoma. The results show that the differentiation from primitive embryonal carcinoma cells into parietal yolk sac cells is accompanied by changes in cell surface saccharides. These changes consist of a marked decrease in the total number of binding sites for the l-fucose-specific lectin of Lotus tetragonolobus and a large increase in the total number of binding sites for wax bean agglutinin. It is suggested that these differences can be used as markers in the study of this early embryonic differentiation. No agglutination of primitive embryonal carcinoma cells or of parietal yolk sac cells by low concentrations (10 μg/ml) of concanavalin A, soybean agglutinin or the fucose binding proteins was observed. 相似文献
2.
The lectin receptors of confluently grown hamster BHK, wild type polyoma virus transformed PyBHK, and temperature-sensitive polyoma transformed ts3-PyBHK fibroblasts were investigated using cell agglutination, quantitative (125I)lectin binding, and ferritin-lectin labeling. PyBHK and permissively grown ts3-PyBHK cells agglutinated more strongly with Ricinus communis I agglutinin (RCA-I)compared to BHK and nonpermissively grown ts3-PyBHK, although saturation binding of (125I)RCA-I to these cells at 4 degrees resulted in a twofold difference in lectin-binding sites on BHK and nonpermissively grown ts3-PyBHK cells (1.0-1.3 x 10 7 sites/cell) compared to PyBHK and permissively grown ts3-PyBHK (0.4-0.6 x 10 7 sites/cell). These cells bound equivalent amounts of (125I)concanavalin A (0.8-1 x 10 7 sites/cell) and (125I)wheat germ agglutinin (1-2.2 x 10 7 sites/cell). Under these binding conditions little endocytosis occurred, as judged by the subsequent release of greater than 90% cell-bound (125I)RCA-I by the RCA-I inhibitor lactose and localization of ferritin-RCA-I exclusively to the extracellular plasma membrane surface. However, if the binding is performed at 22 degrees, only 50% of the bound lectin can be removed by lactose, and ferritin-RCA-I is localized inside the cell within endocytotic vesicles. The relative mobility of RCA-I receptors was examined on ts3-PyBHK cells by the ability of ferritin-RCA-I to induce clustering of its receptors at 22 degrees. RCA-I receptors on permissively grown ts3-PyBHK cells appeared to be more mobile than on nonpermissively grown cells. BHK and PyBHK cells were treated with neuraminidase, and the resulting enzyme-treated cells were assayed for lectin agglutinability and quantitative binding of RCA-I, concanavalin A, and wheat germ agglutinin. Neuraminidase treatment resulted in decreased concanavalin A and wheat germ agglutinability and a slight increase in RCA-I agglutinability. The enzyme-treated BHK and PyBHK cells bound less (125I)wheat germ agglutinin (2.8 x 10 6 and 2.2 x 10 6 sites/cell, respectively) and 2.5 and 6.2 times more (125I)RCA-I (2.5-3 x 10 7) and 3.5-4 x 10 7 sites/per cell, respectively). There was no change in the number of concanavalin A binding sites after neuraminidase treatment. The increase in RCA-I binding sites approximated the decrease in wheat germ agglutinin binding sites indicating that the predominant penultimate oligosaccharide residue to sialic acid on these cells is D-Gal. 相似文献
3.
WGA binding to the surface of two autologous human melanoma cell lines: different expression of sialyl and N-acetylglucosaminyl residues 总被引:1,自引:0,他引:1
M Aubery M Reynier M Lopez E Ogier-Denis J Font F Bardin 《Cell biology international reports》1990,14(3):275-286
Two autologous human melanoma cell lines were studied to determine their capacities to bind wheat germ agglutinin (WGA). Both cell lines were derived from the same patient, the first, IGR 39, originated from the primary tumor, the second, IGR 37, was established from a metastatic lymph node. WGA binding sites on the surface of these cell lines were compared before and after sialidase and/or tunicamycin treatments. IGR 39 cells exhibited two classes of WGA binding sites with high and low affinities, whereas IGR 37 cells had only one class of high affinity binding sites. After tunicamycin treatment, the capacity of IGR 39 cells to bind WGA was markedly altered, since only one class of WGA binding sites with high affinity was observed under these conditions, whereas tunicamycin did not induce significant changes in the lectin binding of IGR 37 cells. The low affinity WGA binding sites, which were only found on IGR 39 cells, corresponded to sialyl residues present in N-linked glycoproteins. The high affinity binding sites present on both cell lines probably involved sialyl and N-acetyl-glucosaminyl residues associated with O-linked glycoproteins and/or glycolipids. No direct correlation could be drawn between the number of WGA binding sites and the overall sialic acid levels exposed to sialidase treatment. The 3-fold increase in the amount of cell surface glycopeptides obtained after pronase digestion and specifically binding to WGA-Sepharose was in good agreement with the overall higher number of WGA binding sites on IGR 39 compared to IGR 37 cells. Thus, subtle carbohydrate changes of cell surface glycoconjugates might account for the differences between the biological properties of human melanoma cell lines of low and high tumorigenicity. 相似文献
4.
Retinoids enhance lectin binding to gp130, a glycoprotein of NIH-3T3 cells: Correlation with cell growth and adhesion 总被引:1,自引:0,他引:1
Our previous work has shown that retinoic acid (RA) enhances fibroblast cell attachment to plastic and to laminin. The treatment of NIH-3T3 cells with RA for 2 days also caused a reproducible increase in the binding of the lectin Phaseolus vulgaris leukoagglutinin (PHA-L) to a glycoprotein of molecular weight 130,000 (gp130) as judged by SDS-PAGE analysis. This finding is consistent with an increased number of beta-1,6-linked N-acetylglucosaminyl residues on gp130. Of the 11 additional lectins tested Ricinus communis agglutinin I (RCA), Phaseolus vulgaris erythroagglutinin (PHA-E), soybean agglutinin (SBA), and succinylated wheat germ agglutinin (sWGA) showed a significant increase in binding specifically to gp130. Similar to RA, 13-cis-RA and 3,5-di-tert-butyl-4-chalcone carboxylic acid, a synthetic retinoid, also increased PHA-L binding to gp130; they also enhanced cell adhesiveness and inhibited cell growth. N-(4-Hydroxyphenyl)-all-trans-retinamide and thyroxine failed to influence adhesion and did not increase PHA-L binding to gp130. Moreover these compounds also failed to inhibit cell growth and to alter the morphology of the cultured cells. Since trypsin is utilized to remove cells from the culture dishes before they are used in the attachment assay to laminin, we studied the effect of this trypsinization step on PHA-L binding to gp130. Trypsin reduced PHA-L binding thus suggesting cell surface localization of gp130. After trypsin treatment RA-treated cells still showed enhanced PHA-L binding compared to dimethyl sulfoxide (DMSO) control. In conclusion RA-induced cell adhesiveness and growth inhibition are accompanied by an increase in the PHA-L, PHA-E, SBA, RCA, and sWGA binding to gp130. The sensitivity of gp130 to trypsin suggests that it is a cell surface glycoprotein. 相似文献
5.
Mouse blastocysts were exposed to a series of ferritin-conjugated lectins during Day 5 (preadhesive) and Day 6 (adhesive; collected Day 5, 24 hr in vitro) of embryogenesis to determine whether there were any changes in lectin binding characteristics that coincided with the acquisition of adhesiveness. After exposure to lectin, the blastocysts were processed for electron microscopy and lectin binding sites were determined by visualization of ferritin particles with the electron microscope. No binding sites were observed for either Dolichos biflorus agglutinin or soybean agglutinin on blastocysts from either stage examined. Binding sites for Ulex europaeus agglutinin, Con A, and wheat germ agglutinin were seen on blastocysts from both stages without apparent increase or reduction in binding sites from either stage. Ricinus communis agglutinin-I (RCA-I) bound heavily to the surface of Day 5 blastocysts and did not bind at all to Day 6 blastocysts and did bind, though with apparent diminution, to Day 6 blastocysts, as compared with the binding observed on Day 5 blastocysts. Peanut agglutinin (PNA) did not bind at all to Day 5 blastocysts but did bind heavily to the surface of Day 6 blastocysts. Both RCA-I and PNA bound to the surface of embryos during Day 5 of delayed implantation, thus indicating that neither the appearance of PNA binding sites on Day 6 blastocysts nor the apparent reduction of RCA-I binding sites on Day 6 blastocysts could be solely implicated in the acquisition of adhesiveness. PNA binding sites were abolished from the surface of Day 6 blastocysts by treatment with Pronase, indicating that the PNA binding molecule was associated with a glycoprotein rather than a glycolipid. 相似文献
6.
Neuraminidase treatment of blood type A and B human erythrocytes, which is required for the agglutination of these cells by peanut (Arachis hypogaea) lectin, increased the number of receptor sites for the lectin from about 5 × 104 to 1.8 × 106 sites/ cell for both blood types. The same treatment also increased the agglutinability of type A cells by the blood group A-specific Dolichos biflorus lectin, but the number of receptor sites for this lectin (~6 × 105 sites/cell) did not change. D. biflorus lectin binding and agglutination of blood type B cells were negligible both before and after neuraminidase treatment. To isolate the peanut agglutinin receptor from the membrane of these cells, washed type A erythrocytes were incubated with neuraminidase and galactose oxidase and then treated with NaB3H4, thus labeling the galactose residues on the membrane. For measuring peanut agglutinin receptor activity, a radioaffinity assay was developed based on the displacement of [14C]asialofetuin from peanut agglutinin by receptor and precipitation of the complex in the presence of polyethyleneglycol. Membranes were isolated by hypotonic lysis and were solubilized in 0.5% Empigen BB, a zwitterionic detergent, which was found to be superior to Triton X-100 for this purpose. The cell extract, after centrifugation, was subjected to affinity chromatography on peanut agglutinin-polyacrylhydrazido-Sepharose. Elution with lactose afforded a peak of radioactivity (32% yield) containing 70% of the applied receptor activity. The eluting sugar and the receptor were separated by chromatography on Bio-Gel P-2 with subsequent dialysis against 80% acetone to remove the detergent. The bulk of the isolated receptor radioactivity (91%) precipitated with peanut agglutinin. The amino acid composition, the glucosamine and galactosamine content and the electrophoretic mobility, on polyacrylamide gel electrophoresis in sodium dodecyl sulfate of the peanut receptor were similar to those of asialoglycophorin. In addition, the peanut receptor coprecipitated with asialoglycophorin and with isolated erythrocyte T antigen on Ouchterlony double-diffusion plates against peanut agglutinin and the Ricinus communis lectin, but not with D. biflorus lectin, suggesting that the receptor for the latter lectin is distinct from the peanut agglutinin receptor. 相似文献
7.
Membrane binding sites for peanut lectin or peanut agglutinin (PNA) were investigated in the established mammary carcinoma cell lines MCF-7, 734-B, ZR-75.1 and BT-20. The determination of PNA binding sites was performed in a flow cytometer after staining with fluorescein(FITC)-labeled PNA. It appeared that only the estrogen-sensitive cell lines exhibited PNA binding sites, whereas the hormone-insensitive cell line BT-20 was clearly negative. Steroid hormones, when administered singly to the cells in physiological concentrations (10(-9)-10(-8) M) had no effect on PNA binding expression. Only the combination of estradiol and progesterone together increased PNA binding sites. Pharmacological doses (10(-6) M) of medroxyprogesteroneacetate (MPA) and dexamethasone increased the number of binding sites, whereas retinoic acid decreased them. A preliminary characterization of the binding sites revealed that they have high capacity and moderate affinity for PNA (KD greater than 10(-7) M). FITC-PNA binding could be inhibited selectively by fetuin (greater than 10(-5) M) and by galactose (greater than 10(-2) M). Cytosol from MCF-7 cells and from some primary breast cancer specimens were able to decrease PNA binding to the surface of 734-B cells. 相似文献
8.
Inaccessibility of certain Ricinus lectin binding sites due to the increase in hyaluronic acid during chick embryo development 总被引:1,自引:0,他引:1
Chick embryo fibroblasts constitute a useful model for investigating cell surface differentiation using Ricinus lectin as a marker. Fibroblasts from 8-day chick embryos had two classes of Ricinus lectin binding sites, whereas those from 16-day embryos displayed only one class. Hyaluronidase treatment of fibroblasts from 8-day embryos had no effect on their capacity to bind Ricinus lectin; however after this treatment, 16-day cells resembled 8-day cells since the former also exhibited two classes of lectin-binding sites. Treatment with hyaluronidase released 2-5 times more hyaluronic acid from the older cells than from the younger cells. The same hyaluronidase treatment did not change the number of 8-day cells detached by trypsin from the substrate, but increased the number of detached 16-day cells. These observations suggest (i) that the greater adhesiveness to the substrate of the 16-day cells might be due to the presence on the cell surface of a larger amount of glycosaminoglycans at 16 days than at 8 days, and (ii) that the increased accumulation of hyaluronic acid on the cell surface might be involved in an alteration in the cell membrane during differentiation. 相似文献
9.
Jacqueline Font Michèle Aubery 《Differentiation; research in biological diversity》1984,25(1-3):23-26
Abstract. Chick embryo fibre-blasts constitute a useful model for investigating cell surface differentiation using Ricinus lectin as a marker. Fibroblasts from 8-day chick embryos had two classes of Ricinus lectin binding sites, whereas those from 16-day embryos displayed only one class. Hyaluronidase treatment of fibroblasts from 8-day embryos had no effect on their capacity to bind Ricinus lectin;.however after this treatment, 16-day cells resembled 8-day cells since the former also exhibited two classes of lectin-binding sites. Treatment with hyaluronidase released 2–5 times more hyaluronic acid from the older cells than from the younger cells. The same hyaluronidase treatment did not change the number of 8-day cells detached by trypsin from the substrate, but increased the number of detached 16-day cells.
These observations suggest (i) that the greater adhesiveness to the substrate of the 16-day cells might be due to the presence on the cell surface of a larger amount of glycosaminoglycans at 16 days than at 8 days, and (ii) that the increased accumulation of hyaluronic acid on the cell surface might be involved in an alteration in the cell membrane during differentiation. 相似文献
These observations suggest (i) that the greater adhesiveness to the substrate of the 16-day cells might be due to the presence on the cell surface of a larger amount of glycosaminoglycans at 16 days than at 8 days, and (ii) that the increased accumulation of hyaluronic acid on the cell surface might be involved in an alteration in the cell membrane during differentiation. 相似文献
10.
Specific binding of fluoresceinated succinyl-concanavalin A, wheat germ agglutinin, and ricin to untreated and trypsinized bloodstream forms of Trypanosoma brucei rhodesiense was quantitated by flow cytofluorimetry, and sites of lectin binding were identified by fluorescence microscopy. All three lectins only bound to the flagellar pocket of untreated parasites. When parasites were trypsinized to remove the variant surface glycoprotein coat, new lectin binding sites were exposed, and specific binding of all three lectins increased significantly. New specific binding sites for succinyl-concanavalin A and wheat germ agglutinin were present along both the free flagellum and flagellar adhesion zone and were uniformly distributed on the parasite surface. However, ricin did not bind uniformly on the surface and did not stain the free flagellum of trypsinized cells. Ricin only bound to the flagellar adhesion zone of trypsinized cells and of cells that had been treated with formaldehyde prior to staining. Electron microscopy of cells exposed to ricin-colloidal gold complexes revealed that that ricin binding was restricted to the anterior membrane of the flagellar pocket of untrypsinized cells and to this portion of the flagellar pocket and the cell body membrane in the flagellar adhesion zone of trypsinized cells. Evidence that these membranes constitute a functionally important membrane microdomain is reviewed. 相似文献
11.
Specific binding of fluoresceinated succinyl-concanavalin A, wheat germ agglutinin, and ricin to untreated and trypsinized bloodstream forms of Trypanosoma brucei rhodesiense was quantitated by flow cytofluorimetry, and sites of lectin binding were identified by fluorescence microscopy. All three lectins only bound to the flagellar pocket of untreated parasites. When parasites were trypsinized to remove the variant surface glycoprotein coat, new lectin binding sites were exposed, and specific binding of all three lectins increased significantly. New specific binding sites for succinyl-concanavalin A and wheat germ agglutinin were present along both the free flagellum and flagellar adhesion zone and were uniformly distributed on the parasite surface. However, ricin did not bind uniformly on the surface and did not stain the free flagellum of trypsinized cells. Ricin only bound to the flagellar adhesion zone of trypsinized cells and of cells that had been treated with formaldehyde prior to staining. Electron microscopy of cells exposed to ricin-colloidal gold complexes revealed that that ricin binding was restricted to the anterior membrane of the flagellar pocket of untrypsinized cells and to this portion of the flagellar pocket and the cell body membrane in the flagellar adhesion zone of trypsinized cells. Evidence that these membranes constitute a functionally important membrane microdomain is reviewed. 相似文献
12.
M Monsigny C Sene A Obrenovitch A C Roche F Delmotte E Boschetti 《European journal of biochemistry》1979,98(1):39-45
The physicochemical and binding properties of succinylated wheat germ agglutinin are described in comparison with these of unmodified wheat germ agglutinin. Succinylated wheat germ agglutinin is an acidic protein with a pI of 4.0 +/- 0.2 while the native lectin is basic, pI of 8.5. The solubility of succinylated wheat germ agglutinin is about 100 times higher than that of the unmodified lectin at neutral pH. Both lectins are dimeric at pH down to 5, and the dissociation occurs at pH lower than 4.5. The binding of oligosaccharides of N-acetylglucosamine to both lectins is very similar on the basis of fluorescence and phosphorescence studies. The minimal concentration required to agglutinate rabbit red blood cells is about 2 microgram/ml with both lectins and the concentrations of N-acetylglucosamine and di-N-acetylchitobiose which inhibit agglutination are similar with both lectins. The number of succinylated wheat germ agglutinin molecules bound to the surface of mouse thymocytes was ten times lower than that of the unmodified lectin although the apparent binding constant was only slightly different between the two lectins. The dramatic decrease of the apparent number of cell surface receptors upon succinylation of the lectin is discussed on the basis of the decrease of the isoelectric point and of the acidic properties of the cell surface. 相似文献
13.
Cell surface alterations occurred during murine erythroleukemia cell (clone 745) differentiation that were detected by both agglutination and lectin binding. Agglutination of erythroleukemia cells was produced by wheat germ agglutinin; whereas, concanavalin A, Ricin, soybean agglutinin and fucose-binding protein were either ineffective or much less efficacious. Treatment of leukemia cells with the inducer of erythroid differentiation dimethylsulfoxide (DMSO) caused a progressive accumulation of hemoglobin-containing cells in culture and a decrease in the rate of agglutination by wheat germ agglutinin, which began at 24 h after exposure to the polar solvent, reached a nadir at 48 h, and remained essentially constant thereafter. The binding of radioactive wheat germ agglutinin by untreated control erythroleukemia cells increased with time in culture, reaching a maximum value at 48 h, and decreased progressively thereafter. Although an increase in 3H-labeled wheat germ agglutinin binding also occurred in DMSO-treated cells, the level bound was significantly lower than that observed in control cells at 24–96 h. The treatment of erythroleukemia cells with various concentrations of DMSO resulted in a decrease in the number of wheat germ agglutinin receptor sites. Other inducers of differentiation (i.e., dimethylformamide, bis(acetyl)diaminopentane) also inhibited the rate of wheat germ agglutinin-induced agglutination of erythroleukemia cells while, in contrast, the inducer tetramethylurea did not. These studies indicate that membrane changes occur during differentiation and suggest that there may be more than one mechanism involved in the initiation of maturation which ultimately leads to the common pathway of erythroid development. 相似文献
14.
Ten fluorescein isothiocyanate-labeled lectins were tested on the roots of the tropical legume Macroptilium atropurpureum Urb. Four of these (concanavalin A, peanut agglutinin, Ricinis communis agglutinin I [RCA-I], wheat germ agglutinin) were found to bind to the exterior of root cap cells, the root cap slime, and the channels between epidermal cells in the root elongation zone. One of these lectins, RCA-I, bound to the root hair tips in the mature and emerging hair zones and also to sites at which root hairs were only just emerging. There was no RCA-I binding to immature trichoblasts. Preincubation of these lectins with their hapten sugars eliminated all types of root cell binding. By using a microinoculation technique, preincubation of the root surface with RCA-I lectin was found to inhibit infection and nodulation by Rhizobium spp. Preincubation of the root surface with the RCA-I hapten beta-d-galactose or a mixture of RCA-I lectin and its hapten failed to inhibit nodulation. Application of RCA-I lectin to the root surface caused no apparent detrimental effects to the root hair cells and did not prevent the growth of root hairs. The lectin did not prevent Rhizobium sp. motility or viability even after 24 h of incubation. It was concluded that the RCA-I lectin-specific sugar beta-d-galactose may be involved in the recognition or early infection stages, or both, in the Rhizobium sp. infection of M. atropurpureum. 相似文献
15.
Interactions of a mammalian beta-galactoside-binding lectin with hamster fibroblasts 总被引:1,自引:0,他引:1
A beta-galactoside-binding endogenous lectin extracted from bovine heart binds to the surface of baby hamster kidney (BHK) cells. The binding to and agglutination of cells is reduced in certain ricin-resistant mutants (Ric cells) in parallel with the decreased number of binding sites for the selective agent, ricin, a galactose-specific plant lectin. However, clear differences in the binding specificities of bovine lectin and ricin are shown by the effect of neuraminidase. BHK cells and Ric mutant cells treated with neuraminidase bind similar amounts of the bovine lectin compared with untreated cells, and ricin binding is greatly increased. The mammalian lectin immobilised on inert glass mediates the attachment and spreading of normal BHK cells and agglutinates these cells in solution. Ricin-resistant mutant cells respond poorly. These results are consistent with a role of endogenous lectins in cellular adhesiveness and show that cell adhesion may be regulated by the density of specific surface receptors for lectins. 相似文献
16.
The effects of the sequential application of specific glycosidases on surfaces of living mammalian cells were studied with respect to their ability to bind the beta-galactoside-specific lectin, Ricinus communis agglutinin (RCA). Sialidase and beta-galactosidases from different sources were tested for their actions on two strains of mouse lymphoma cells differing markedly in their metastatic potential. Binding studies were performed by quantitative flow cytometry with fluorescent RCA, and numbers of specific binding sites and equilibrium association constants for the lectin on living cells were determined before and after the various enzyme treatments. Although the number of binding sites for native and sialidase-treated cells were almost identical for both cell strains, differences in the apparent affinity constants could be detected. Differences between the two strains became even more pronounced, also with respect to the number of binding sites, after treatment with beta-galactosidases from S. pneumoniae and from bovine testis. It is suggested that such combined strategies provide valuable tools for the differentiation of surface carbohydrate moieties on intact living cells, especially for comparative purposes. 相似文献
17.
Qualitative variations in the glycoconjugates which make up the lectin receptor sites on the membranes of leukemic lymphocytes, compared with those of normal cells, have been studied by the use of three tritiated lectins: Robinia pseudoacacia lectin, Concanavalin A and Ricinus communis (var. Sanquineus) agglutinin (RCA 120). The binding specificity of these lectins has been demonstrated using specific determinants: alpha-methylmannoside and galactose for Concanavalin A and Ricinus communis agglutinin respectively. For the Robinia lectin this specificity was determined by saturation of the receptor sites with the unlabeled Robinia lectin before the addition of isotopically labeled Robinia lectin. The results show a decrease in the number of receptor sites on the leukemia cells, especially in chronic lymphoid leukemia, relative to that on normal cells. The apparent affinity constants of leukemic cells in all cases remain higher than those of normal cells. 相似文献
18.
Flow cytometry was used to quantify the binding of fluorescein isothiocyanate (FITC)-labeled lectins to testis cells from ICR and T/t6 mice before and after trypsin treatment. Soybean agglutinin, wheat germ agglutinin, and concanavalin A bound well to testis cells of both mouse strains. Limax flavus agglutinin (LFA) bound very slightly and Ulex europeas agglutinin (UEA) did not bind at all. Trypsinization increased binding of soybean agglutinin and decreased binding of wheat germ agglutinin in both mouse strains, providing evidence for masked carbohydrate-binding sites on the surface of germ cells. It did not affect binding of the other lectins. Trypsin treatment was an attempt to increase lectin binding, particularly the binding of LFA and UEA to the reported T/t-specific carbohydrates, sialic acid, and L-fucose, respectively. These studies indicate that the T/t6 locus alleles do not alter the surface carbohydrate content of testis cells sufficiently to be detected by lectin-binding differences. 相似文献
19.
Concanavalin A (Con A), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA) bound with either 125I, fluorescent dyes, or fluorescent polymeric microspheres were used to quantitate and visualize the distribution of lectin binding sites on mouse neuroblastoma cells. As viewed by fluorescent light and scanning electron microscopy, over 107 binding sites for Con A, WGA, and RCA appeared to be distributed randomly over the surface of differentiated and undifferentiated cells. An energy-dependent redistribution of labeled sites into a central spot occurred when the cells were labeled with a saturating dose of fluorescent lectin and maintained at 37°C for 60 min. Reversible labeling using appropriate saccharide inhibitors indicated that the labeled sites had undergone endocytosis by the cell. A difference in the mode of redistribution of WGA or RCA and Con A binding sites was observed in double labeling experiments. When less than 10% of the WGA or RCA lectin binding sites were labeled, only these labeled sites appeared to be removed from the cell surface. In contrast, when less than 10% of the Con A sites were labeled, both labeled and unlabeled Con A binding sites were removed from the cell surface. Cytochalasin B uncoupled the coordinate redistribution of labeled and unlabeled Con A sites, suggesting the involvement of microfilaments. Finally, double labeling experiments employing fluorescein-tagged Con A and rhodamine-tagged WGA indicate that most Con A and WGA binding sites reside on different membrane components and redistribute independenty of each other. 相似文献
20.
Cell surface glycoproteins of mitotic neuroblastoma cells and cells differentiated by prostaglandin cyclic adenosine monophosphate treatment were quantified by flow cytometric analysis and specific fluorescent lectins. No differences in fluorescent lectin binding were seen between suspensions of mitotically active and differentiated N2AB-1 cells following exposure to either fluorescein (FL)-labeled soy bean agglutinin (FL-SBA) specific for N acetyl galactosamine or FL-concanavalin A (FL-CON A) which binds to mannose residues. These lectins, however, were shown to bind specifically to these cells as revealed by competitive blocking studies with hapten sugars. When FL Ulex europaeus (FL-UEA) specific for fucose was reacted with control or differentiated cells, no binding was seen even with an increased dose of lectin before or after enzyme treatment. However, differentiated N2AB-1 cells, reacted with FL-wheat germ agglutinin (FL-WGA) specific for N acetyl glucosamine, bound more FL-WGA than that seen for control cultures. Furthermore, specific sites for FL-WGA were shown to be saturable and were lost upon pretreatment of cells with neuraminidase. Neuraminidase pretreatment revealed masked sites for FL-CON A and FL-SBA since binding was increased at least twofold for these lectins on mitotic and differentiated cells. These data indicate that single cell measurements of surface glycoproteins can be made on living neural cells and that differentiation induces an increase in cell surface N-acetyl glucosamine residues. 相似文献