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1.
2.
Summary Protoplasts isolated from cell suspension cultures of carrot (Daucus carota L.) and leaves of tobacco (Nicotiana tabacum L.) were treated with three lectins specific for galactosyl residues. After incubation with RCA I (Ricinus communis agglutinin, molecular weight 120,000) conjugated to ferritin or fluorescein, freshly isolated protoplasts displayed heavy labeling of their surfaces. Moreover, they agglutinated rapidly when exposed to low concentrations of RCA I. In parallel studies, PNA (peanut agglutinin) also bound extensively to the protoplast plasma membranes whileBandeiraea simplicifolia lectin I attached relatively weakly. When protoplasts were cultured for two days and then incubated with conjugates of RCA I and PNA, additional binding sites were revealed on the regenerating walls.The results indicate that galactosyl residues are distributed densely over the surface of plant protoplasts. They also allow inferences to be made regarding the positions and linkages of the galactose groups being recognized by the lectins. Moreover, they open up the question whether the galactosyl moieties detected in the wall derive from those labeled on the plasma membrane. To conclude, we make comparisons with binding by concanavalin A, and predict that galactose-recognizing lectins will join and in certain respects prove superior to concanavalin A as probes of the plant cell surface.  相似文献   

3.
Summary The composition of the surface coat in embryonic cells ofXenopus laevis was examined by agglutination and fluorescent staining with lectins.Cells of early and mid gastrula stages were agglutinated by lectins specific for D-mannose, D-galactose, L-fucose, N-acetyl-D-glucosamine and N-acetyl-D-galactosamine. No differences in agglutinability among ectoderm, mesoderm and endoderm cells were observed with lectins specific for D-mannose, D-galactose and N-acetyl-D-galactosamine, though agglutination of gastrula cells with fluorescent lectins revealed considerable differences in the intensity of lectin binding among cells within an aggregate. These differences in amount of lectin bound were not related to cell size or morphology. Patches of fluorescent material formed on the cells, suggesting that lectin receptors are mobile in the plane of the plasma membrane.In the early cleavage stages intensive lectin binding occurs only at the boundary between preexisting and nascent plasma membranes. The external surface of the embryo has few lectin receptors up to the late gastrula stage. The unpigmented nascent plasma membranes, when exposed to fluorescent lectins, do not assume any fluorescence distinguishable from the background autofluorescence of yolk, in stages up to the mid-blastula. From this stage onwards lectin binding was observed on the membranes of the reverse side of surface layer cells and on the membranes of deep layer cells. During gastrulation there is an accumulation of lectin-binding material on surfaces involved in intercellular contacts.The significance of lectin binding material for morphogenesis is discussed.  相似文献   

4.
Summary The claim that osmium-containing deposits which lack lead are frequently and incorrectly interpreted as enzymatic reaction products in lead precipitation techniques for ATPase localization in plants is without foundation. Proper controls clearly demonstrate the enzymatic origin of membrane-located deposits and the presence of lead is confirmed by analytical electron microscopy.  相似文献   

5.
The effects of ribosome-inactivating proteins (RIPs) from Ricinus communis and from Viscum album on the water permeability, Pf, and the surface dielectric constant, epsilon, of model membranes were studied. Pf was calculated from microelectrode measurements of the ion concentration distribution in the immediate vicinity of a planar membrane, and epsilon was obtained from the fluorescence of dansyl phosphatidylethanolamine incorporated into unilamellar vesicles. Pf and epsilon of fully saturated phosphatidylcholine membranes were affected only in the presence of a lectin receptor (monosialoganglioside, GM1) in the bilayer. It is suggested that the membrane area occupied by clustered lectin-receptor complexes is markedly less permeable to water. Protein binding to the receptor was not a prelude for hydrophobic lipid-protein interactions when the membranes were formed from a mixture of natural phospholipids with a high content of unsaturated fatty acids. These membranes, characterized by a high initial water permeability, were found to interact with the RIPs unspecifically. From a decrease of both Pf and epsilon it was concluded that not only water partitioning but also protein adsorption correlates with looser packing of polyunsaturated lipids at the lipid-water interface.  相似文献   

6.
Lymphocytes from murine lymph node, cultured in the presence of an optimally mitogenic dose of phytohaemagglutinin, were stained with fluoresceinated lectins and analysed by flow cytometry. A marked increase in the ability of lymphocytes to bind wheat-germ agglutinin was observed that is particularly pronounced for the blast cells, reaching a maximum at about 40 h, when they are 5.5-times brighter than cells at zero time. The corresponding intensification of the small cells is 2-fold. Much smaller increases in binding accompanying blast transformation were observed when fluoresceinated concanavalin A or Lens culinaris haemagglutinin were used. Polyacrylamide gel electrophoresis of plasma membranes followed by treatment of the gels with radioactively labelled lectins and autoradiography also showed a very distinct increase in the binding of wheat-germ agglutinin to membranes from mitogen-stimulated porcine lymphocytes. Less marked changes in the binding of concanavalin A Lens culinaris heamagglutin and Ricinus communis agglutinin 120 were also noted. The apparent multiplicity of glycoproteins that bind each lectin, suggests that in each case the sites are heterogeneous. We conclude that lymphocytes stimulated by the T-cell mitogen phytohaemagglutinin expose new glycoprotein receptors for wheat-germ agglutinin that are most abundant on blast cells at 40 h. Attempts to characterize the receptor biochemically suggest that the carbohydrate moiety recognised by wheat-germ agglutinin is present on a glycoprotein of approx. 120 kDa molecular mass and also possibly on glycoproteins of 170–190 kDa.  相似文献   

7.
T. Hogetsu 《Protoplasma》1990,156(1-2):67-73
Summary Binding of fluorescein-conjugated wheat-germ agglutinin (F-WGA) and some other lectins to tissues from various plants were examined by epifluorescence microscopy. F-WGA bound specifically to the walls of tracheary elements (TEs) and phloem cells of pea roots. The binding sites in TEs were localized only in the secondary thickening and became evident at very early stages of differentiation. Fluorescein-conjugated derivatives ofSolanum tuberosum lectin,Lycopersicon esculentum lectin, andDatura stramonium lectin, which bind N-acetylglucosamine residues as WGA, also bound to the secondary thickening of TEs of pea roots. The binding sites for F-WGA were not removed by extraction with hot EDTA and proteinase K, but removed by extraction with an alkali solution. The alkali-extracted binding sites from the roots were precipitated together with hemicelluloses by 80% ethanol. These results indicate that the binding sites are not present on pectins, proteins, or cellulose, but hemicelluloses. Localized distribution of the binding sites for F-WGA in TEs was found also in a variety of angiosperm plants.Abbreviations BSL-II Bandeiraea simplicifolia lectin II - DSL Datura stramonium lectin - F fluorescein-conjugated - LEL Lycopersicon esculentum lectin - MT microtubule - STL Solanum tuberosum lectin - TE tracheary element - WGA wheat-germ agglutinin  相似文献   

8.
Summary Buoyant density differences between different types of protoplasts were used in an iso-osmotic density gradient system to enrich protoplast fusion mixtures for heterokaryocytes. Protoplasts of maize stem and wheat mesophyll, as well as epidermis, stem parenchyma and mesophyll protoplasts of two amphihaploid, light sensitive tobacco mutants were fused with polyethylene glycol using conventional methods and a new rolling tube technique. The protoplast combinations used for fusion involved protoplast types with considerably different buoyant densities. Enriched fractions of maize-wheat heterokaryocytes of intermediate density were recovered which contained up to 31% mostly binucleate heterokaryocytes (a 2–7 fold relative enrichment). Tobacco heterokaryocytes recovered analogously from enriched intermediate fractions readily divided and gave rise to an increased number of light resistant calluses when compared with cultures from non-fractionated fusion mixtures. Maize-wheat fusion products, however, failed to divide.  相似文献   

9.
Summary A simple effective technique for the fractionation of protoplast populations is described. Protoplasts are separated by low-speed centrifugation in an iso-osmotic, discontinuous density gradient system on the basis of differences in their buoyant densities. At a constant osmolality of 660±20 mOs/kg H2O, the gradients provide a density range from 1.017 to 1.069 g/cm3 at 20 °C which corresponds to the buoyant densities of most protoplast types studied. Characteristics of the KMC/S-density gradient system and factors affecting the fractionation were investigated. Protoplasts were isolated from various tissues and cultivars of tobacco, barley, wheat, rye, oat and maize. Their density-dependent distribution profiles in KMC/S-gradients and their average buoyant densities were determined under standardized conditions. Great differences in the buoyant densities were found between protoplasts of different tissues. Mixed populations of two types of protoplasts, differing in buoyant density by about 15–20 mg/cm3, were separated to give highly purified fractions. Factors affecting the buoyant densities of protoplasts have been investigated. Ploidy level and species differences did not significantly affect the fractionation profiles. However, an age-dependent variation in the average buoyant density of tobacco mesophyll protoplasts was observed. Fractionation of tobacco mesophyll protoplasts and their subsequent regeneration to plants demonstrates the practicability and physiological compatibility of the KMC/S-density gradient system under sterile conditions. The morphogenetic potential of protoplasts was not affected by the separation procedure or the gradient components.  相似文献   

10.
Upon subfractionation of certain plant seed homogenates on sucrose density gradients, we encountered problems in defining the location and amount of mitochondria using marker enzymes. In order to overcome the inherent limitations of enzyme assays, we utilized a heterologous DNA probe specific foratp6 in maize orBrassica tournefortii to detect mitochondria. The samples were treated with SDS, proteinase K, and RNase A followed by agarose gel electrophoresis, and blotting. The immobilized DNA was detected with [32P]-labelled probes, and quantified using a phosphor imager. The assay is specific, sensitive, and independent of species, cell type, and developmental stage, thus circumventing the need for expressed protein to assay enzyme activity.  相似文献   

11.
Summary Microorganism useful for the induction of enzymes lytic towards walls of filamentous fungusCochliobolus lunatus were studies. Production of specificTrichoderma viride mycolytic enzymes was studied in a laboratory fermentor. The product with high chitinase and relatively low protease activity gave better yields ofC. lunatus protoplasts than commercial Novozym 234.  相似文献   

12.
Ribosome-inactivating proteins (RIPs) are enzymes that inhibit protein synthesis after depurination of a specific adenine in rRNA. The RIP family members are classified as type I RIPs that contain an RNA-N-glycosidase domain and type II RIPs that contain a lectin domain (B chain) in addition to the glycosidase domain (A chain). In this work, we identified 30 new plant RIPs and characterized 18 Ricinus communis RIPs. Phylogenetic and functional divergence analyses indicated that the emergence of type I and II RIPs probably occurred before the monocot/eudicot split. We also report the expression profiles of 18 castor bean genes, including those for ricin and agglutinin, in five seed stages as assessed by quantitative PCR. Ricin and agglutinin were the most expressed RIPs in developing seeds although eight other RIPs were also expressed. All of the RIP genes were most highly expressed in the stages in which the endosperm was fully expanded. Although the reason for the large expansion of RIP genes in castor beans remains to be established, the differential expression patterns of the type I and type II members reinforce the existence of biological functions other than defense against predators and herbivory.  相似文献   

13.
Lectins localized in the plasma membranes seem to be of special importance for the intercellular interaction mechanisms. We describe the isolation of mannose-binding proteins by Triton X-100 extraction and affinity chromatography on agarose-bound mannose. The isolation procedure was performed with whole GH3 cells as well as with isolated plasma membranes. For the isolation of plasma membranes of GH3 cells a mechanical pump was used for the disruption. After differential centrifugation an enriched plasma membrane fraction was achieved by discontinuous sucrose gradient centrifugation. The whole fractionation procedure was controlled by total balance sheets for the marker enzymes of the different cell organelles. The plasma membrane fraction was further characterized by gel electrophoresis and electron microscopy. The SDS gel electrophoresis patterns of the proteins, resulting from the Triton X-100 extraction and the affinity chromatography, are nearly identical for whole cells as well as for the enriched plasma membrane fraction. Therefore we presume these mannose-specific proteins to be plasma membrane bound, showing the molecular properties of integral proteins and having a molecular weight of Mr 67 000, 57 000, 47 000.  相似文献   

14.
Insect feeding trials were carried out to determine the effects of a range of mannose-specific lectins on third instar nymphs of the rice brown planthopper (BPH), Nilaparvata lugens. Stål. Dose response curves show that Galanthus nivalis agglutinin (GNA) has the strongest toxic effect of the lectins tested, and is effective at concentrations considerably lower than those previously reported. Narcissus pseudonarcissus agglutinin (NPA) and Allium sativum agglutinin (ASA) exhibit a significant antimetabolic effect towards the insect but were less effective (on a molar basis) than GNA. LC50 values for GNA, NPA and ASA are approximately 4 μM, 11 μM and >40 μM respectively. These mannose-specific lectins are serologically identical, but differ in the number of subunits per protein molecule; ASA is a dimer, NPA is a trimer and GNA is a tetramer. The results obtained support the hypothesis, that the effectiveness of the mannose-binding lectins as antimetabolites is determined by the number of subunits per molecule. Two N-acetylglucosamine binding lectins, the dimeric Oryza sativa agglutinin (OSA) and the monomeric Urtica dioica agglutinin (UDA), were also tested but at a concentration of 0.1% w/v exhibited no significant antimetabolic effect towards BPH, although the related lectin wheatgerm agglutinin (WGA) has previously been demonstrated to be toxic towards the insect.  相似文献   

15.
Taro Colocasia esculenta (L. Schott) and rice (Oryza sativa L.) form a major part of the staple diet of pacific islanders. Pest constraints hamper the sustainability of taro and rice production in the Pacific region. Insect feeding trials were conducted in vitro to determine the effects of plant lectins against planthopper pests of taro and rice. Lectins were incorporated into artificial diet at 0.1% (w/v) level. The lectins Galanthus nivalis agglutinin (GNA) and concanavalin A (Con A) showed significant antimetabolic effects towards third instar nymphs of taro planthopper (Tarophagous proserpina Kirkaldy) whilst Pisum sativum agglutinin (PSA) showed no significant effects toward the insect. Psophocarpus tetragonolobus agglutinin (PTA) showed significant antimetabolic effects towards third instar nymphs of rice brown planthopper (Nilaparvata lugens Stål). PTA also reduced honeydew excretion levels of rice brown planthopper, over a 24-hour period, demonstrating antifeedant properties of the protein.  相似文献   

16.
Murine monoclonal antibodies to protoplast membrne antigens were generated using mouse myelomas and spleen cells from mice immunized with Nicotiana tabacum L. leaf protoplasts. For selecting antibody-secreting clones, a sensitive and rapid enzyme-linked immunosorbent assay (ELISA) for monoclonal antibody binding to immobilized cellular membrane preparations or immobilized protoplasts was developed. With intact protoplasts as immobilized antigen, the ELISA is selective for antibodies that bind to plasma-membrane epitopes present on the external surface of protoplasts. Using the membrane ELISA, a total of 24 hybridoma lines were identified that secreted antibodies to plant membrane epitopes. The protoplast ELISA and subsequent immunofluorescence studies identified four hybridoma lines as secreting antibodies which bound to the external surface of protoplasts and cells. The corresponding antigens were not species- or tissue-specific, were periodatesensitive, and were located in membranes which equilibrated broadly throughout a linear sucrose gradient. When protein blots of electrophoretically separated membrane proteins were probed with these antibodies, a band of Mr 14 kilodaltons (kDa) and a smear of bands of Mr 45–120 kDa were labeled. An additional set of three antibodies appeared by immunofluorescence to bind to the plasma membrane of broken but not intact protoplasts and labeled membranes equilibrating at a density of approx. 1.12 kg·l-1 in a linear sucrose density gradient. These classes of monoclonal antibodies enlarge the library of monoclonal antibodies (Norman et al. 1986, Planta 167, 452–459) available for the study of plant plasma-membrane structure and function.Abbreviations ELISA Enzyme-linked immunosorbent assay - Ig immunoglobulin - kDa kilodalton - Mr relative molecular mass - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis  相似文献   

17.
Insect feeding trials were carried out to determine the effects of incorporating a range of plant derived proteins into artificial diets fed to leafhopper and planthopper pests of rice. The lectins Galanthus nivalis agglutinin (GNA) and wheat germ agglutinin (WGA), and the enzyme soy bean lipoxygenase (LPO) were shown to exhibit significant antimetabolic effects towards first and third instar nymphs of rice brown planthopper (Nilaparvata lugens Stål) when incorporated into artificial diet at 0.1% (w/v), 0.1% (w/v) and 0.08% (w/v) levels respectively. The lectin GNA was also shown to exhibit a significant antimetabolic effect towards third instar nymphs of the rice green leafhopper (Nephotettix cinciteps Uhler). A number of inert proteins, lectins, protein inhibitors and enzymes also tested showed relatively little or no effect towards both insects.  相似文献   

18.
Summary Immunofluorescence methods were developed for examining the distribution of microtubules in freshly isolated and cultured protoplasts and regenerated somatic embryos of white spruce (Picea glauca). Freshly isolated protoplasts consisted of both uniand multinucleate types. Uninucleate protoplasts established parallel cortical microtubules during cell wall formation and cell shaping, divided within 24 h and developed into somatic embryos in culture. Dividing cells were characterized by preprophase bands (PPBs) of microtubules, atypical spindle microtubules focused at the poles and a typical phragmoplast at telophase. Multinucleate protoplasts also established parallel arrays of cortical microtubules during cell wall formation. In addition their nuclei divided synchronously within 4 days, then cell walls formed between the daughter nuclei. Individual multinucleate protoplast-derived colonies subsequently gave rise to elongate suspensor cells thereby forming embryo-like structures by 7 days.  相似文献   

19.
Two lectin proteins were purified from the corms of Pinellia ternata and Lycoris radiata. Both P. ternata agglutinin (PTA) protein and L. radiata agglutinin (LRA) protein formed polymers and coagulated both rabbit red blood cells and yeast cells. The two proteins were each diluted to different concentration and then mixed with pinewood nematodes, and nematode survival was measured. Results showed that the two lectin proteins showed significant levels of resistance against nematodes and the nematode population was significantly reduced, compared to PBS buffer without protein control group. The mean number of nematodes of two lectin proteins group was significantly lower than that of control group constantly throughout the assay period with differences being very significant at P < 0.01 after 24 h. After 96 h, when 500 μg/ml proteins were used, nematode number significantly declined to an average of 26 (approximately 43% of the controls) and 32.2 (approximately 53.3% of the controls) nematodes at LRA and PTA protein, respectively, compared to the control group. Results also indicated that higher concentrations of protein were more toxic to the pinewood nematode. Even when the concentration was as low as 30 μg/ml, the toxic proteins retained their anti-nematode activity. Furthermore, pinewood nematode was exposed to the proteins for longer, more pinewood nematodes were killed. Our results indicated the two lectin proteins both apparently have a toxic effect on the pinewood nematode that affects its survival in vitro.  相似文献   

20.
Protoplasts of Azotobacter vinelandii were formed by incubating whole cells in lysozyme and EDTA in Tris-HCl buffer (0.05 M, pH 8.0) supplemented with sucrose (15% w/v). This appeared to be related to the special chelating ability of EDTA and Tris-HCl since substitution of the former by nitrilotriacetic acid or by trisodium citrate and the latter by veronal-acetate buffer or tris-maleate buffer over a pH range of 5.2 to 8.6 yielded only spheroplasts. Of nine strains of Azotobacter studied, only A. vinelandii strain 12837 and strain 0 formed protoplasts.  相似文献   

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