scholarly journals Wheat Germ Agglutinin Conjugated Fluorescent pH Sensors for Visualizing Proton Fluxes

2019 ◽  
Author(s):  
Lejie Zhang ◽  
Mei Zhang ◽  
Karl Bellve ◽  
Kevin Fogarty ◽  
Maite A. Castro ◽  
...  
Keyword(s):  
Small Molecule ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Plasma Membranes ◽  
Proton Channel ◽  
Primary Cells ◽  
Channel Activity ◽  
Ph Sensors ◽  
Single Plane ◽  
Extracellular Side

AbstractSmall molecule fluorescent wheat germ agglutinin (WGA) conjugates are routinely used to demarcate mammalian plasma membranes because they bind to the cell’s glycocalyx. Here we describe the derivatization of WGA with a pH sensitive rhodamine fluorophore (pHRho: pKa = 7) to detect proton channel fluxes and extracellular proton accumulation and depletion from primary cells. We found that WGA-pHRho labeling was uniform, did not appreciably alter the voltage-gating of glycosylated ion channels, and the extracellular changes in pH directly correlated with proton channel activity. Using single plane illumination techniques, WGA-pHRho was used to detect spatiotemporal differences in proton accumulation and depletion over the extracellular surface of cardiomyocytes, astrocytes, and neurons. Because WGA can be derivatized with any small molecule fluorescent ion sensor, WGA conjugates should prove useful to visualize most electrogenic and non-electrogenic events on the extracellular side of the plasma membrane.

scholarly journals Wheat germ agglutinin–conjugated fluorescent pH sensors for visualizing proton fluxes

2020 ◽  
Vol 152 (6) ◽  
Author(s):  
Lejie Zhang ◽  
Mei Zhang ◽  
Karl Bellve ◽  
Kevin E. Fogarty ◽  
Maite A. Castro ◽  
...  
Keyword(s):  
Small Molecule ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Plasma Membranes ◽  
Proton Channel ◽  
Primary Cells ◽  
Channel Activity ◽  
Ph Sensors ◽  
Single Plane ◽  
Extracellular Side

Small-molecule fluorescent wheat germ agglutinin (WGA) conjugates are routinely used to demarcate mammalian plasma membranes, because they bind to the cell’s glycocalyx. Here, we describe the derivatization of WGA with a pH-sensitive rhodamine fluorophore (pHRho; pKa = 7) to detect proton channel fluxes and extracellular proton accumulation and depletion from primary cells. We found that WGA-pHRho labeling was uniform and did not appreciably alter the voltage gating of glycosylated ion channels, and the extracellular changes in pH correlated with proton channel activity. Using single-plane illumination techniques, WGA-pHRho was used to detect spatiotemporal differences in proton accumulation and depletion over the extracellular surface of cardiomyocytes, astrocytes, and neurons. Because WGA can be derivatized with any small-molecule fluorescent ion sensor, WGA conjugates should prove useful to visualize most electrogenic and nonelectrogenic events on the extracellular side of the plasma membrane.

scholarly journals Partial purification and characterization of oestrogen receptors in subfractions of hepatocyte plasma membranes

1980 ◽  
Vol 191 (3) ◽  
pp. 743-760 ◽  
Author(s):  
Richard J. Pietras ◽  
Clara M. Szego
Keyword(s):  
Plasma Membrane ◽  
Binding Sites ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Low Density ◽  
Marker Enzymes ◽  
Low Density Plasma ◽  
Density Plasma

To assess the subcellular distribution of oestrogen-binding components in their native state, plasma membrane and other cell fractions were prepared from hepatocytes in the absence of [3H]oestradiol-17β. Cells from livers of ovariectomized rats were disrupted, with submaximal homogenization in buffered isotonic sucrose with CaCl2 and proteinase inhibitor, and fractionated by using isotonic media. Fractions were characterized by determinations of enzyme activities, biochemical constituents and ligand binding. Specific binding of 2nm-[3H]oestradiol-17β to intact cells and their fractions was detemined after equilibration for 1.5h at 4°C. More than 92% of the radioactivity from representative preparations was verified as authentic oestradiol by thin-layer chromatography. Activities of plasma-membrane marker enzymes as well as binding sites for oestrogen and for wheat germ agglutinin were present principally in particulate fractions, rather than in 105000g-supernatant fractions. However, by using alternative homogenization procedures (i.e. hypotonic media), known to fragment and strip structural components, oestradiol-binding sites and activities of plasma-membrane marker enzymes were distributed predominantly into cytosol. By using the more conservative procedures, plasma membranes of low (ρ=1.13–1.16) and high (ρ=1.16–1.18) density were purified from crude nuclear fractions. A second low-density subfraction of plasma membrane was prepared from microsome-rich fractions. Activities of plasma-membrane marker enzymes were enriched to about 28 and four times that of the homogenate in plasma membranes of low and high density respectively. Binding sites for wheat germ agglutinin and oestradiol were concentrated in low-density plasma membranes to 46–63 times that of the homogenate. Specific binding of oestrogen in low-density plasma membranes purified from crude nuclei was saturable, with an apparent association constant of 3.5nm. At saturation, such oestradiol receptors corresponded to 526fmol/mg of membrane protein. A Hill plot showed a moderate degree of positive co-operativity in the interaction of hormone with plasma membranes. Specific binding of [3H]oestradiol-17β was reduced by a 200-fold molar excess of unlabelled oestradiol-17β, oestriol or diethylstilbestrol, but not by oestradiol-17α, cortisol, testosterone or progesterone. Binding was also blocked by prior exposure of membranes to trypsin or to 60°C, but remained essentially undiminished by extraction of membranes with either hypotonic or high-salt buffers. Extraction with 0.1% (v/v) Triton X-100 partially solubilized the oestrogen-binding component(s) of plasma membranes. Particle-free extracts were resolved on 5–20% (w/v) sucrose density gradients with either 0.01m- or 0.4m-KCl, and the fractions were analysed by adsorption to hydroxyapatite. In low-salt gradients macromolecule-bound oestrogen sedimented at predominantly 7.4S and binding was 1560 times that of the homogenate. Under high-salt conditions oestradiol-binding activity occurred at both 3.6S and 4.9S.

scholarly journals Analysis of cholecystokinin-binding proteins using endo-beta-N-acetylglucosaminidase F.

1984 ◽  
Vol 99 (3) ◽  
pp. 1110-1116 ◽  
Author(s):  
S A Rosenzweig ◽  
L D Madison ◽  
J D Jamieson
Keyword(s):  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Plasma Membranes ◽  
Binding Proteins ◽  
Neuraminidase Treatment ◽  
Similar Treatment ◽  
Definitive Evidence ◽  
Electrophoretic Mobilities ◽  
Significant Enrichment ◽  
Polypeptide Backbone

We have previously shown that the cholecystokinin (CCK)-binding proteins in rat pancreatic plasma membranes consist of a major Mr 85,000 and minor Mr 55,000 and Mr 130,000 species as revealed by affinity labeling with 125I-CCK-33 using the cross-linker, disuccinimidyl suberate. The glycoprotein nature of these species was investigated using endoglycosidase F (endo F) and neuraminidase treatment and wheat germ agglutinin-agarose chromatography. Treatment of affinity-labeled membranes with endo F resulted in increased electrophoretic mobilities of all three binding proteins, indicating removal of N-linked oligosaccharide side chains. Endo F treatment of each protein in gel slices indicated the following cleavage relationships: Mr 85,000----65,000; Mr 55,000----45,000; Mr 130,000----110,000. Using limiting enzyme conditions to digest each protein contained in excised SDS gel slices, three and four products, respectively, were identified for the Mr 85,000 and 55,000 proteins. Similar treatment of the Mr 130,000 protein revealed only the Mr 110,000 product. These results indicated that the Mr 85,000 protein has at least three, the Mr 55,000 protein has at least four, and the Mr 130,000 protein has at least one, N-linked oligosaccharide side chain(s) on their polypeptide backbone. Neuraminidase treatment of affinity-labeled membranes caused slight increases in the electrophoretic mobilities of all three proteins, indicating the presence of sialic acid residues. Solubilization of affinity-labeled membranes in Nonidet P-40 followed by affinity chromatography on wheat germ agglutinin-agarose revealed that all three CCK-binding proteins specifically interact with this lectin and can be eluted with N-acetyl-D-glucosamine. Analysis of the proteins present in the eluted fractions by silver staining indicated a significant enrichment for proteins having molecular weights corresponding to the major CCK-binding proteins in comparison to the pattern of native membranes. Taken together, these studies provide definitive evidence that the CCK-binding proteins in rat pancreas are (sialo)glycoproteins.

scholarly journals Localization of axonally transported 125I-wheat germ agglutinin beneath the plasma membrane of chick retinal ganglion cells.

1983 ◽  
Vol 96 (2) ◽  
pp. 373-381 ◽  
Author(s):  
J H LaVail ◽  
I K Sugino ◽  
D M McDonald
Keyword(s):  
Plasma Membrane ◽  
Retinal Ganglion Cells ◽  
Optic Tectum ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Plasma Membranes ◽  
Ganglion Cells ◽  
Retinal Ganglion ◽  
Electron Microscopic Autoradiography ◽  
Neuron Cell Body

The distribution of 125I-wheat germ agglutinin (WGA) transported by axons of chick retinal ganglion cells to layer d of the optic tectum was studied by electron microscopic autoradiography. We found that 52% of the radioactivity was located in axons and axon terminals in the contralateral optic tectum 22 h after intravitreal injection of affinity-purified 125I-WGA. Axons comprised 43% of the volume of layer d. Dendrites, glial cells, and neuron cell bodies contained 20%, 17%, and 3% of the label, whereas these structures comprised 24%, 21%, and 2% of the tissue volume, respectively. We also measured the distances between the autoradiographic silver grains and the plasma membranes of these profiles, and compared observed distributions of grains to theoretical distributions computed for band-shaped sources at various distances from the plasma membranes. This analysis revealed that the radioactive source within axons was distributed in a band of cytoplasm extending in from the plasma membrane a distance of 63 nm. Because WGA is known to bind to specific membrane glycoconjugates, we infer that at least some glycoconjugates may be concentrated within an annular region of cytoplasm just beneath the axonal plasma membrane after axoplasmic transport from the neuron cell body.

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.

Effect of in vivo γ-irradiation on the binding of wheat germ agglutinin on lymphocyte plasma membranes

1986 ◽  
Vol 883 (3) ◽  
pp. 407-412 ◽  
Author(s):  
Philippe Moullier ◽  
Denis Daveloose ◽  
Michel Dubos ◽  
Francois Leterrier ◽  
Johan Hoebeke
Keyword(s):  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Plasma Membranes ◽  
Γ Irradiation

scholarly journals Purification of rat liver plasma membranes by wheat-germ-agglutinin affinity partitioning

1991 ◽  
Vol 273 (1) ◽  
pp. 173-177 ◽  
Author(s):  
A Persson ◽  
B Johansson ◽  
H Olsson ◽  
B Jergil
Keyword(s):  
Rat Liver ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Plasma Membranes ◽  
Bottom Phase ◽  
Phase System ◽  
Marker Enzyme ◽  
Liver Plasma Membranes ◽  
Rat Liver Plasma Membranes ◽  
Affinity Partitioning

Rat liver plasma membranes were separated from other cellular membranes by affinity partitioning in an aqueous polymer two-phase system by using the lectin wheat-germ agglutinin covalently bound to dextran as the affinity ligand. In borate buffer the bulk of membranes partitioned in the poly(ethylene glycol)-rich top phase, whereas plasma membranes were pulled selectively into the dextran-rich bottom phase in the presence of ligand. The purity and yield of plasma membranes prepared by lectin affinity partitioning and by conventional sucrose-density-gradient centrifugation was similar, as judged from marker-enzyme activities. The affinity procedure, not dependent on lengthy centrifugations, is fast and gentle and will be advantageous when studying labile components.

Failure of 6D1 or Exposure of Platelets to ADP to Affect Agglutination Induced by Wheat Germ Agglutinin

1989 ◽  
Vol 62 (02) ◽  
pp. 815 ◽  
Author(s):  
Marjorie B Zucker ◽  
Robert A Grant ◽  
Evelyn A Mauss
Keyword(s):  
Wheat Germ Agglutinin ◽  
Wheat Germ
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