scholarly journals Subfractionation of cardiac sarcolemma with wheat-germ agglutinin

1989 ◽  
Vol 264 (3) ◽  
pp. 885-892 ◽  
Author(s):  
J H M Charuk ◽  
S Howlett ◽  
M Michalak
Keyword(s):  
Plasma Membrane ◽  
Binding Sites ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Freeze Fracture ◽  
Protein Composition ◽  
Tubular Structures ◽  
Electron Microscopic ◽  
Cardiac Sarcolemma ◽  
Inside Out

The properties of highly purified bovine cardiac sarcolemma subfractionated with the lectin, wheat-germ agglutinin (WGA) were studied. Two different membrane subfractions were isolated, one which was agglutinated in the presence of 1.0 mg of WGA/mg of protein (WGA+ vesicles) and a second fraction which failed to agglutinate (WGA- vesicles). These two membrane fractions had quantitatively different rates of Na+/K+-dependent, ouabain-sensitive ATPase and Na+/Ca2+ exchange activities, yet a similar protein composition, which suggests that they were both derived from the plasma membrane. WGA- vesicles had a decreased number of [3H]quinuclidinyl benzilate-binding sites and no detectable [3H]nitrendipine-binding sites. Electron-microscopic and freeze-fracture analysis showed that the WGA+ fraction was composed of typical spherical sarcolemmal vesicles, whereas the WGA- fraction primarily contained elongated tubular structures suggestive of the T-tubule vesicles which were previously isolated from skeletal muscle. Assays of marker enzymes revealed that these fractions were neither sarcoplasmic reticulum nor plasma membrane from endothelial cells. Moreover, WGA agglutination did not result in the separation of right-side-out and inside-out vesicles. On the basis of these findings we propose that the WGA+ fraction corresponds to highly purified sarcolemma, whereas the WGA- fraction may be derived from T-tubule membranes.

scholarly journals Freeze-fracture cytochemistry: localization of wheat-germ agglutinin and concanavalin A binding sites on freeze-fractured pancreatic cells.

1981 ◽  
Vol 91 (2) ◽  
pp. 361-372 ◽  
Author(s):  
P Pinto da Silva ◽  
M R Torrisi ◽  
B Kachar
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Concanavalin A ◽  
Binding Sites ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Freeze Fracture ◽  
Combined Application ◽  
Pancreatic Cells ◽  
Critical Point Drying

The combined application of thin-section and critical-point-drying "fracture-label" is used to determine the pattern of distribution and partition of wheat-germ agglutinin and concanavalin A binding sites on the membrane faces of freeze-fractured exocrine and endocrine rat pancreatic cells. Whereas the exoplasmic face of plasma membrane is preferentially labeled by both lectins, the endoplasmic reticulum and nuclear envelope are strongly and uniformly labeled by concanavalin A but not by wheat-germ agglutinin. The results support current views in the glycosylation of membrane proteins and do not support the backflow of sialidated glycoproteins to the endoplasmic reticulum.

scholarly journals Compartmentalization of intracellular membrane glycocomponents is revealed by fracture-label.

1984 ◽  
Vol 98 (1) ◽  
pp. 29-34 ◽  
Author(s):  
M R Torrisi ◽  
P Pinto da Silva
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Binding Sites ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Secretory Granules ◽  
The Other ◽  
Rat Tissues ◽  
Intracellular Membrane ◽  
Definition Of

We used thin-section fracture-label to determine the distribution of wheat-germ agglutinin binding sites in intracellular membranes of secretory and nonsecretory rat tissues as well as in human leukocytes. In all cases, analysis of the distribution of wheat germ agglutinin led to the definition of two endomembrane compartments: one, characterized by absence of the label, includes the membranes of mitochondria and peroxisomes as well as those of the endoplasmic reticulum and nuclear envelope; the other, strongly labeled, comprises the membrane of lysosomes, phagocytic vacuoles, and secretory granules, as well as the plasma membrane. The Golgi apparatus was weakly labeled in all studied tissues.

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 Freeze-fracture cytochemistry: partition of glycophorin in freeze-fractured human erythrocyte membranes.

1982 ◽  
Vol 93 (2) ◽  
pp. 463-469 ◽  
Author(s):  
P P da Silva ◽  
M R Torrisi
Keyword(s):  
Human Erythrocyte ◽  
Binding Sites ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Transmembrane Protein ◽  
Freeze Fracture ◽  
Membrane Skeleton ◽  
Erythrocyte Membranes ◽  
Con A ◽  
Human Erythrocyte Membranes

Thin-section and critical-point-dried fracture-labeled preparations are used to determine the distribution and partition of glycophorin-associated wheat germ agglutinin (WGA) binding sites over protoplasmic and exoplasmic faces of freeze-fractured human erythrocyte membranes. Most wheat germ agglutinin binding sites are found over exoplasmic faces. Label is sparse over the protoplasmic faces. These results contrast with previous observations of the partition of band 3 component where biochemical analysis and fracture-label of concanavalin A (Con A) binding sites show preferential partition of this transmembrane protein with the protoplasmic face. Presence of characteristic proportions of WGA and Con A binding sites over each fracture face is interpreted to indicate the operation of a stochastic process during freeze-fracture. This process appears modulated by the relative expression of each transmembrane protein at either surface as well as by their association to components of the erythrocyte membrane skeleton.

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.

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