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1.
Membrane halves of boar sperm flagella were produced by freeze-fracture and labeled in situ with concanavalin A and wheat germ agglutinin; the lectins were visualized with protein-gold complexes. Concanavalin A and wheat germ agglutinin binding sites partition with both protoplasmic and exoplasmic halves of the membrane. A high density of lectin marking was found on protoplasmic membrane halves; we conclude that the label corresponds to transmembrane glycoproteins that, on freeze-fracture, are dragged across the outer (exoplasmic) half of the phospholipid bilayer. Our demonstration of numerous transmembrane proteins in sperm flagella offers the structural setting for previous models on flagellar surface motility that postulate accessibility of motile membrane components to the submembranous cytoskeleton.  相似文献   

2.
《The Journal of cell biology》1983,97(5):1356-1364
We used fracture-label and surface labeling techniques to characterize the distribution and topology of wheat germ agglutinin (WGA) receptors in the plasma membrane of boar sperm heads. We show that freeze- fracture results in preferential, but not exclusive, partition of WGA- binding sites with the outer (exoplasmic) half of the plasma membrane. Labeling of the inner (protoplasmic) half of the membrane is significant, and is denser over the areas that overlie the acrosome. Exoplasmic membrane halves are uniformly labeled. Analysis of freeze- fracture replicas revealed that the distribution of intramembrane particles over protoplasmic faces parallels that of WGA-binding sites as observed by fracture-label. Coating of intact spermatozoa with cationized ferritin results in drastic reduction of the labeling of both protoplasmic and exoplasmic membrane halves. Labeling of sperm cells lysed by short hypotonic shock fails to reveal the presence of WGA-binding sites at the inner surface of the plasma membrane. We conclude that: (a) all WGA-binding glycoconjugates are exposed at the outer surface of the membrane; (b) some of these glycoconjugates correspond to transmembrane glycoproteins that, on fracture, partition with the inner half of the membrane; (c) these transmembrane proteins are accumulated in the region of the plasma membrane that overlies the acrosome; and (d) parallel distribution of intramembrane particles and WGA-binding glycoproteins provides renewed support for the view of particles as the morphological counterpart of integral membrane proteins.  相似文献   

3.
Sindbis virus-infected baby hamster kidney cells were analyzed by thin section fracture-label. Specific immunolabel with antiviral glycoprotein antibodies or with conventional lectin label (wheat germ agglutinin) were used in conjunction with colloidal gold-conjugated protein A or ovomucoid, respectively. In addition, intact infected cells were analyzed with both labeling procedures. Experiments with Sindbis infected-chick embryo fibroblast cells were carried out as controls. Viral transmembrane glycoproteins appeared present in freeze-fractured inner and outer nuclear membrane, endoplasmic reticulum, Golgi stacks and vesicles, and plasma membranes; a clear preferential partition with the exoplasmic faces of all intracellular membranes was observed. By contrast, at the plasma membrane level, Sindbis glycoproteins were found to partition preferentially with the protoplasmic face. It seems likely that this protoplasmic partition is related to the binding with the nucleocapsid that takes place during the budding of the virus. At the cell surface, viral glycoproteins always appeared clustered and were predominantly associated with budding figures: moreover, large portions of the plasma membrane were devoid of both glycoproteins and budding viruses.  相似文献   

4.
It is now generally accepted that ADH-induced increase in water permeability in responsive epithelia is associated with the insertion of specific structures in the apical membrane of epithelial cells. Up to now, these structures have only been recognized in freeze-fractured preparations and their chemical nature is still unknown. In this study, we used the label-fracture method (Pinto da Silva and Kan, J. Cell Biol., 99, 1156-1161, 1984) to investigate the distribution of wheat germ agglutinin (WGA) on the luminal plasma membrane of freeze-fractured frog urinary bladder epithelial cells. With label-fracture, the cytochemical markers are seen superimposed with the conventional high resolution image of the E face. Label-fracture of tissue treated for 15 min with WGA and subsequently labeled with colloidal gold coated with ovomucoid showed uniform distribution of gold particles along the exoplasmic fracture face. Stereomicrographs show that the gold label is under the fracture face as it is attached to the outer surface of the membrane. Preincubation of the bladder with WGA for 3 hr induced a segregation of the intramembranous particles of the apical plasma membrane. In this condition, we observed a co-distribution of WGA-gold complexes with the segregated particles on the E face. This indicates that WGA-binding sites are located on glycoproteins which probably comprise the large intramembranous particles dispersed on the exoplasmic faces of freeze-fractured luminal membranes. In contrast, the numerous small intramembrane particles observed on P faces remained evenly distributed even after exposure to WGA and are, therefore, unrelated to WGA receptor sites. After WGA treatment, ADH still induced the formation of aggregates inside the smooth domains. A few WGA-binding sites appeared to be associated to these aggregates.  相似文献   

5.
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   

6.
The binding site distribution of concanavalin agglutinin (Con A) and wheat germ agglutinin (WGA) on embryo sacs at various developmental stages of Torenia fournieri L was studied by using a cooled Charge Coupled Device (CCD) and fluorescent Con A and WGA probes. The distribution patterns of Con A and WGA binding sites on embryo sacs changed during the fertilization process. The fluorescent signal indicating Con A binding sites was distributed evenly on the surface of the embryo sac wall before anthesis, was much denser on the micropylar end of the embryo sac wall and looked like a corona on the day of anthesis. After pollination, stronger fluorescence was present on the micropylar end of the embryo sac wall and the filiform apparatus (FA), showing an obvious polar distribution. When the pollen tube entered the embryo sac and reached a synergid, the fluorescence was still concentrated on the micropylar end and FA, and started to appear on the synergid. After fertilization, the polar distribution of the fluorescence gradually disappeared and an even distribution pattern was observed again on the embryo sac wall. These results revealed that the dynamic distribution of Con A binding sites was temporally coupled with the process of fertilization. WGA binding site distribution on the embryo sac was also investigated and showed a simple pattern but also regularly changed during the process of fertilization. The variation of these lectin binding sites during the fertilization process suggests that lectin binding site interactions may play a role in the process.  相似文献   

7.
Summary Five Fluorescein-isothiocyanate (FITC)-labelled lectins were used to study the postnatal development of carbohydrate constituents in the rat ventral prostate: Concanavalin A (Con A), wheat germ agglutinin (WGA), peanut agglutinin (PNA),Dolichos biflorus agglutinin (DBA) andRicinus communis agglutinin I (RCA-I) With all the lectins, tested, except RCA-I, specific binding sites could be shown for every stage of differentiation in the glandular epithelium. Binding sites for Con A, WGA, PNA and DBA were found from day 10 to 13 post partum onwards. Each lectin showed a characteristic localization. Binding sites for the lectins used changed to different extents during the following two weeks. After the 24th day post partum no further changes in the lectin binding pattern could be found. The development of the lectin binding properties showed that the changes in carbohydrate-containing constituents of the prostate correlate with the beginning of prostatic secretion and to prostatic epithelial differentiation. In the periacinar stroma the development of the lectin binding pattern was similar to that in the glandular epithelium. The changes of stromal binding sites for Con A and WGA during epithelial differentiation may reflect the changes of epithelial-stromal interactions in the prostate.  相似文献   

8.
Search for the presence of lectin-binding sites on Toxoplasma gondii   总被引:4,自引:0,他引:4  
Evidence for the presence of carbohydrate on the surface membrane of Toxoplasma gondii trophozoites and on the cell wall of toxoplasma brain cysts was sought by fluorescent lectin staining. Using FITC-conjugated preparations of Concanavalin A (Con A), wheat germ agglutinin (WGA), or soy bean agglutinin (SBA), we have failed to obtain evidence for the binding of these lectins on the surface of T. gondii trophozoites. In contrast, the three test lectins bound effectively and specifically to the wall of toxoplasma brain cysts. Prefixation of cysts with glutaraldehyde or brief trypsinization of cysts did not affect the intensity of cyst wall fluorescence when stained with FITC-conjugated Con A, SBA, or WGA. The results are interpreted to indicate that whereas exposed Con A, SBA, and WGA binding sites are associated with the wall of toxoplasma brain cysts, such lectin-binding saccharide residues are not present on the surface of trophozoites in exposed or reactive form.  相似文献   

9.
Organelles isolated from leaves of spinach (Spinacia oleracea L.) were prefixed in glutaraldehyde and then incubated with ferritin conjugates of four lectins — Concanavalin A (Con A), Ricinus communis L. agglutinin, MW 120,000 (RCA), soybean agglutinin (SBA), and wheat germ agglutinin (WGA) — in order to probe their cytoplasmic surfaces for saccharide residues. In each case the major leaf organelles, including microbodies, mitochondria and chloroplast derivatives, failed to exhibit labeling when examined with the electron microscope. Tobacco (Nicotiana tabacum L.) leaf protoplasts, incubated simultaneously with and under identical conditions to the spinach organelles, showed specific labeling of their plasma membranes with all four lectin conjugates, thus establishing the efficacy of the procedure for demonstrating the presence of binding sites when they exist. Further attempts to show binding of one of the lectins, Con A, by labeling with fluorescein-Con A and by organelle agglutination, yielded results consistent with the absence of ultrastructural labeling. It is concluded that no saccharide residues recognized by the four lectins are present on the cytoplasmic surfaces of organelles and that those residues reported to be constituents of intracellular membranes, therefore, are most likely exposed on the luminal (extracytoplasmic) surfaces.Abbreviations Con A Concanavalin A - RCA Ricinus communis agglutinin, MW 120,000 - SBA soybean agglutinin - WGA wheat germ agglutinin  相似文献   

10.
1. Oxytocinase enzymes were purified from maternal serum and human placenta, run by SDS-PAGE and transferred onto nitrocellulose. Both enzymes were homogeneous in protein staining with Mr of 145,000. 2. Both serum and placental oxytocinases bound concanavalin A (Con A), limax flavus agglutinin (LFA) and wheat germ agglutinin (WGA). The WGA-binding of the placental enzyme was more strongly inhibited by 0.2 M N-acetylglucosamine than that of the serum enzyme which may indicate a higher sialic acid content in the serum enzyme. 3. Neuraminidase treatment did not affect the binding of Con A but decreased the binding of WGA to serum and placental enzymes. Serum enzyme showed a pl 4.7 on isoelectric focusing.  相似文献   

11.
The acrosome is a large secretory vesicle of the sperm head that carries enzymes responsible for the digestion of the oocyte's investments. The event leads to sperm penetration and allows fertilization to occur. Release of the acrosomal enzymes is mediated by the interaction between sperm acrosomal and plasma membranes (acrosome reaction). Biochemical characterization of the acrosomal membrane has been restrained by a lack of methods to isolate uncontaminated fractions of the membrane. Here, we use new methods to expose the membrane to in situ cytochemical labeling by lectin-gold complexes. We study the topology and relative density of glycoconjugates both across and along the plane of the acrosomal membrane of boar sperm. Detachment of the plasma membrane from glutaraldehyde-fixed cells exposed the cytoplasmic surface of the acrosome to the lectin markers; freeze-fractured halves of the acrosomal membrane were marked by "fracture-label" (Aguas, A. P., and P. Pinto da Silva, 1983, J. Cell Biol. 97:1356-1364). We show that the cytoplasmic surface of the intact acrosome is devoid of binding sites for both concanavalin A (Con A) and wheat germ agglutinin (WGA). By contrast, it contains a high density of neuraminidase-resistant anionic sites detected by cationic ferritin. On freeze-fractured sperm, the receptors for Con A partitioned with the exoplasmic membrane half of the acrosomal membrane. The Con A-binding glycoconjugates were accumulated on the equatorial segment of the membrane. A low density of WGA receptors, as well as of intramembrane particles, was found on the freeze-fracture halves of the acrosomal membrane. The plasma membrane displayed, in the same preparations, a high density of receptors for both Con A and WGA. We conclude that the acrosome is limited by a membrane poor in glycoconjugates, which are exclusively exposed on the exoplasmic side of the bilayer. Regionalization of Con A receptors on the acrosome shows that sperm intracellular membranes, like the sperm surface, express domain distribution of glycocomponents.  相似文献   

12.
The ability of seven lectins to bind to newt epidermal cells and influence their motility was examined. Of the seven fluoresceinated lectins applied to frozen sections containing intact newt skin and migrating epidermis (wound epithelium), only Con A (concanavalin A), WGA (wheat germ agglutinin), and PNA (peanut agglutinin) produced detectable epidermal fluorescence. Con A and WGA each heavily labeled all layers of intact epidermis, but PNA bound only to the more superficial layers. In contrast to a single population of labeled cells in migrating epidermal sheets after treatment with Con A, there were both labeled and unlabeled cells after exposure to either WGA or PNA. The wound bed was labeled by both Con A and WGA, but not by PNA. DBA (Dolichos bifloris agglutinin), RCA I (Ricinus communis agglutinin), and UEA (Ulex europaeus agglutinin), did not produce significant fluorescence with either migrating or intact epidermis. In general, inhibitory effects on epidermal motility correlated with the binding studies. Thus, Con A, WGA, and PNA, the lectins which clearly bound to the epidermis, all produced a concentration-dependent depression in the rate of epidermal wound closure. RCA was somewhat paradoxical in that it was moderately inhibitory despite showing essentially no binding. The effects of SBA and UEA were equivocal. DBA had no effect. These results indicate that the inhibition of motility produced by Con A that we have described previously is not peculiar to this mannose-binding lectin, but is shared by at least one lectin with an affinity for D-GlcNAc (WGA), and one with an affinity for B-D-Gal(1-3)-D-GalNAc (PNA).(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

13.
Bloodstream trypomastigote and culture procyclic (insect midgut) forms of a cloned T. rhodesiense variant (WRAT at 1) were tested for agglutination with the lectins concanavalin A (Con A), phytohemagglutinin P (PP), soybean agglutinin (SBA), fucose binding protein (FBP), wheat germ agglutinin (WGA), and castor bean lectin (RCA). Fluorescence-microscopic localization of lectin binding to both formalin-fixed trypomastigotes and red cells was determined with fluorescein isothiocyanate (FITC)-conjugated Con A, SBA, FBP, WGA, RCA, PNA (peanut agglutinin), DBA (Dolichos bifloris), and UEA (Ulex europaeus) lectins. Electron microscopic localization of lectin binding sites on bloodstream trypomastigotes was accomplished by the Con A-horseradish peroxidase-diamino-benzidine (HRP-DAB) technique, and by a Con A-biotin/avidin-ferritin method. Trypomastigotes, isolated by centrifugation or filtration through DEAE-cellulose or thawed after cryopreservation, were agglutinated by the lectins Con A and PP with agglutination strength scored as Con A greater than PP. No agglutination was observed in control preparations or with the lectins WGA, FBA or SBA. Red cells were agglutinated by all the lectins tested. Formalin-fixed bloodstream trypomastigotes bound FITC-Con A and FITC-RCA but not FITC-WAG, -SBA, -PNA, -UEA or -DBA lectins. All FITC-labeled lectins bound to red cells. Con A receptors, visualized by Con A-HRP-DAB and Con A-biotin/avidin-ferritin techniques, were distributed uniformly on T. rhodesiense bloodstream forms. No lectin receptors were visualized on control preparations. Culture procyclics lacked a cell surface coat and were agglutinated by Con A and WGA but not RCA, SBA, PP and FBP. Procyclics were not agglutinated by lectins in the presence of competing sugar at 0.25 M.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

14.
Summary The affinity of rat peripheral nerve fibres for concanavalin A (Con A) and wheat germ agglutinin (WGA) was tested in semithin sections of Epon-embedded material. A two-step post-embedding technique was used. As a first step, Con A and WGA were used in pure form. As a second step, peroxidase-gold (for Con A) and ovomucoid-gold (for WGA) complexes were applied. The lectin-binding sites, visualized by means of signal amplification with the photochemical silver reaction, were associated mainly with the myelin sheaths and the surfaces of Schwann cells.  相似文献   

15.
Surface changes of hemocytes from the migratory grasshopper,Melanoplus sanguinipes (Fab.), following wounding and/or infection withBeauveria bassiana (Bals.) Vuillemin were assessed using fluorescein labelled wheat germ agglutinin (WGA) and concanavalin A (Con A). Binding was observed on outer wall of hemocytes when either Con A or WGA conjugates were tested. After infection there was an increase in the percentage of hemocytes which bound to WGA and which remained so for 24 h. However, after wounding this increase in binding subsided after only 1 h. Furthermore, it was found that the increase in WGA-binding occurred within the first 15 min of wounding or infection. In addition, ConA binding in males only increased 3-fold in fungus-injected insects within 1 h of treatment and remained high for 24 h. These results indicated the presence of mannose (and/or glucose) residues (Con A specificity) and N-acetyl-D-glucosamine residues (wheat germ agglutinin; WGA specificity) on outer wall surfaces of the hemocytes. The implications of these results in the immune response ofM. sanguinipes to infection withB. bassiana are discussed.  相似文献   

16.
The distribution of structural and secretory glycoconjugates in the gastric region of metamorphosing Xenopus laevis was studied by the avidin-biotin-peroxidase (ABC) histochemical staining method using seven lectins (concanavalin A, Con A; Dolichos biflorus agglutinin, DBA; peanut agglutinin, PNA; Ricinus communis agglutinin I, RCA-I; soybean agglutinin, SBA; Ulex europeus agglutinin I, UEA-I; and wheat germ agglutinin, WGA). Throughout the larval period to stage 60, the epithelium consisting of surface cells and gland cells was stained in various patterns with all lectins examined, whereas the thin layer of connective tissue was positive only for RCA-I. At the beginning of metamorphic climax, the connective tissue became stained with Con A, SBA, and WGA, and its staining pattern varied with different lectins. The region just beneath the surface cells was strongly stained only with RCA-I. With the progression of development, both the epithelium and the connective tissue gradually changed their staining patterns. The surface cells, the gland cells, and the connective tissue conspicuously changed their staining patterns, respectively, for Con A and WGA; for Con A, PNA, RCA-I, SBA, and WGA; and for Con A, RCA-I, and WGA. At the completion of metamorphosis (stage 66), mucous neck cells became clearly identifiable in the epithelium, and their cytoplasm was strongly stained with DBA, PNA, RCA-I, and SBA. These results indicate that lectin histochemistry can provide good criteria for distinguishing among three epithelial cell types, namely, surface cells, gland cells, and mucous neck cells, and between adult and larval cells of each type.  相似文献   

17.
Using fibroblastic CHO cells, we have examined (1) the internalization and redistribution of surface binding sites for the lectins Concanavalin A and wheat germ agglutinin and (2) the sensitivity of these processes to putative inhibitors of cytoskeletal activity. Following initial exposure to fluorescein conjugated Con A (CAF) or WGA (WGAF) at 4° C, kinetic analysis of internalization and intracellular aggregation of lectin at 37°C indicated more rapid aggregate formation in the case of WGA than in the case of Con A. Treatment with tertiary amine local anesthetics (tetracaine, dibucaine, and xylocaine) or with a lysosomatrophic amine, m-dansyl cadaverine, blocked internalization of Con A but not of WGA. Similar differential effects on redistribution of Con A and WGA were not however observed with the antimicrotubule agents colchicine and nocodazole. Simultaneous treatment of cells with WGAF and with rhodamine labeled Con A (CAR) resulted in the accumulation of both labels in a central perinuclear aggregate; a similar experiment in the presence of local anesthetic resulted in a diffuse peripheral distribution of CAR and a central aggregate of WGAF. These results suggest (1) CHO cells possess at least two distinct pathways for lectin internalization and redistribution, which can be discriminated in terms of drug sensitivity; (2) CHO cells can sort out and independently internalize different populations and lectin binding sites; and (3) different pathways may be a manifestation of biochemically distinct linkages between cytoskeletal elements and various groups of surface glycoproteins. Present findings concur with our previous results concerning the mutual independence of the surface binding sites for Con A and WGA (Emerson and Juliano, 1982).  相似文献   

18.
Histotopography of lectin receptor sites in adult mice ovary, oviduct, uterus, testis and epididymis has been investigated on light-optic level by means of lectin-peroxidase technique. Paraffin sections are treated with peanut agglutinin (PNA), soybean agglutinin (SBA), wheat germ agglutinin (WGA) and Laburnum anagyroides lectin (LAL), conjugated with horseradish peroxidase. Concanavalin A (Con A) receptor sites are visualized by indirect method. The usefulness of lectins for selective histochemic evaluation of definite organ structures is demonstrated. Zona pellucida, luteocytes, oviductal and uterine epitheliocytes are rich in receptor sites for all lectin used in the investigation. The most intense binding to zonae pellucidae glycoconjugates possess WGA and LAL, to luteocytes--PNA, SBA and LAL, to oviductal and uterine epitheliocytes--Con A and LAL. The preferential SBA binding to the acrosomal system and plasma membranes of spermatogenic cells is demonstrated. Changes in lectin-binding patterns during the maturation of intraovarian oocytes and spermatogenic cells are also studied. The perspectives of practical application of the results obtained, as well as trends in further lectin histochemistry investigations of the reproductive system are discussed.  相似文献   

19.
To add an additional aspect to the process of recognition and removal of senescent human erythrocytes from the circulation, the binding of wheat germ agglutinin (WGA) to separated young, old and sialidase-treated human erythrocytes is evaluated with the immune-electron microscopical method. WGA/gold conjugate binding to old erythrocytes was lower (27%) than to young erythrocytes and even lower following treatment with sialidase (82%), exhibiting a clustered, non-continuous labeling pattern in all three erythrocyte populations, thus showing a possible redistribution of WGA binding sites. The decrease in bound WGA/gold particles correlates well with the previously reported decrease in surface sialic acid on old erythrocytes. The binding of WGA/gold are indicative of the changes occurring on erythrocyte membrane surfaces when interacting with different agglutinins.  相似文献   

20.
The binding of seven lectins (concanavalin A, Con A; Dolichos biflorus agglutinin, DBA; peanut agglutinin, PNA; Ricinus communis agglutinin I, RCA-I; soybean agglutinin, SBA; Ulex europeus agglutinin, UEA-I; and wheat germ agglutinin, WGA) to the small intestine in metamorphosing Xenopus laevis was studied by the avidin-biotin-peroxidase (ABC) method. The staining pattern of the epithelium with all lectins except for UEA-I and Con A changed gradually during metamorphic climax; the main component of the epithelium, absorptive cells, gradually became positive for DBA, PNA, and SBA and the scattered goblet cells for RCA-I and WGA. On the other hand, the change of the staining pattern in the connective tissue occurred only for Con A, RCA-I, and WGA, and this change took place rapidly at the beginning of climax (stage 60). Increased staining for Con A and WGA at stage 60 was observed only in a group of connective tissue cells close to the epithelium and in the basement membrane. As metamorphosis progressed, this localization of the staining intensity became less clear. At the completion of metamorphosis (stage 66), the absorptive cells were stained with all lectins except for UEA-I, whereas the goblet cells stained only with RCA-I and WGA. These results indicate that lectin histochemistry can distinguish between larval and adult cells of both two epithelial types (absorptive and goblet cells). The technique may also identify a group of connective tissue cells, close to the epithelium, that possibly induce the metamorphic epithelial changes.  相似文献   

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