scholarly journals DETECTION OF PLASMA MEMBRANE CARBOHYDRATES WITH LECTIN PEROXIDASE CONJUGATES

1973 ◽  
Vol 59 (2) ◽  
pp. 436-443 ◽  
Author(s):  
Nicholas K. Gonatas ◽  
Stratis Avrameas
Keyword(s):  
Plasma Membrane ◽  
Horseradish Peroxidase ◽  
Cell Surface ◽  
Lymphoid Cell ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Cell Interaction ◽  
Lymphoid Cells

With the use of the cytochemical stain for horseradish peroxidase of Graham and Karnovsky (1966. J. Histochem. Cytochem. 14:291), conjugates of horseradish peroxidase with ricin, wheat germ agglutinin, and phytohemagglutinin were employed for the morphologic demonstration of d-galactose (ricin), N-acetylglucosamine (wheat germ), and N-acetylgalactosamine (phytohemagglutinin) containing moieties on the surface of unfixed, or paraformaldehyde-fixed rat lymphoid cells. D-Galactose, or d-galactose containing disaccharides inhibited the interaction between ricin peroxidase and lymphoid cell surface; also, N-acetylglucosamine inhibited the wheat germ peroxidase-lymphoid cell interaction, but N-acetylgalactosamine failed to inhibit the reaction between phytohemagglutinin peroxidase and the surface of lymphoid cells.

scholarly journals The use of horseradish peroxidase–lectin conjugates to monitor the purity of plasma-membrane preparations obtained from isolated cells

1981 ◽  
Vol 198 (2) ◽  
pp. 421-423 ◽  
Author(s):  
J. Paul Banga ◽  
Philip Penfold ◽  
Ivan M. Roitt
Keyword(s):  
Plasma Membrane ◽  
Horseradish Peroxidase ◽  
Positive Reaction ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Ultrastructural Examination ◽  
Isolated Cells ◽  
Intact Cells

The plasma membranes of viable murine EL4 tumour cells were labelled with horseradish peroxidase-conjugated wheat-germ agglutinin. After disruption of the labelled intact cells, plasma-membrane purification could be monitored by ultrastructural examination of the various fractions for positive reaction product on the membrane vesicles.

An ultrastructural study of the effects of wheat germ agglutinin (WGA) on cell cortex organization during the first cleavage of Xenopus laevis eggs. II. Cortical wound healing

1979 ◽  
Vol 37 (1) ◽  
pp. 59-67
Author(s):  
M. Geuskens ◽  
R. Tencer
Keyword(s):  
Wound Healing ◽  
Plasma Membrane ◽  
Electron Microscope ◽  
Xenopus Laevis ◽  
Ultrastructural Study ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Cell Cortex ◽  
Animal Pole ◽  
Annular Zone

Uncleaved fertilized eggs of Xenopus laevis treated with wheat germ agglutinin (WGA) have been pricked at the animal pole both inside and outside the regressed furrow region. The wounded cortex of both regions has been studied with the electron microscope and compared with the same region of wounded, untreated eggs. In all 3 cases, filaments are organized in an annular zone in the damaged cortex. When the surface is pricked outside the regressed furrow of WGA-treated embryos, bundles of microfilaments radiate from the ring and extend in deep folds which form a ‘star’ around the wound at the surface of the embryo. However, when the surface is pricked in the new membrane of the regressed furrow, filaments are intermingled with internalized portions of the plasma membrane. It is suggested that, when the surface is pricked outside the furrow region, more filaments are mobilized to counteract the tangential retraction of the membrane which has acquired more rigidity after WGA binding.

scholarly journals Isolation of the full-length murine erythropoietin receptor using a baculovirus expression system

Blood ◽  
1996 ◽  
Vol 87 (3) ◽  
pp. 926-937 ◽  
Author(s):  
JL Spivak ◽  
LS Avedissian ◽  
JH Pierce ◽  
D Williams ◽  
WD Hankins ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Affinity Chromatography ◽  
Molecular Mass ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Erythropoietin Receptor ◽  
Receptor Protein ◽  
Sf9 Cells ◽  
Con A ◽  
Whole Cell

The full-length murine erythropoietin receptor was expressed in Spodoptera frugiperda (Sf9) cells using a recombinant baculovirus vector. Erythropoietin receptor protein production was maximal 48 hours after infection, as determined by metabolic labeling and immunoblotting; receptor protein varied in molecular mass from 62 to 76 kD. Erythropoietin receptors produced in Sf9 cells could be solubilized using CHAPS in a form capable of binding erythropoietin, and the solubilized receptor bound to immobilized Concanavalin A (Con A) and wheat germ agglutinin, as well as to immobilized recombinant human erythropoietin. Analysis of the distribution of erythropoietin receptors in Sf9 plasma membrane and cytosol fractions using lectin affinity chromatography revealed that membrane-bound receptor had a higher apparent molecular mass and contained the bulk of receptors that bound to wheat germ agglutinin. The receptor was purified by sequential affinity chromatography on Con A-Sepharose and immobilized erythropoietin. Erythropoietin receptors expressed in Sf9 cells were inserted into the plasma membrane in the correct orientation, bound 125I-erythropoietin with a single affinity (kD, 330 pmol/L), and were internalized after ligand binding. However, kD varied inversely with the number of cell surface receptors. Solubilized erythropoietin receptors in whole-cell lysates and isolated plasma membranes exhibited high-affinity binding, with kD values of 92 and 57 pmol/L, respectively. Erythropoietin bound to the surface of infected Sf9 cells could be cross-linked to two proteins with molecular masses of 90 and 65 kD using the homobifunctional cross-linker, disuccinimidyl suberate (DSS). Similar results were obtained with solubilized receptors in whole-cell lysates, and both proteins could be immunoprecipitated by an antiserum to the erythropoietin receptor carboxyl-terminal domain.

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