Structural Difference in Sites on the Surface Membrane of Normal and Transformed Cells

250

An electron microscopic stain for specific saccharides was prepared by the conjugation of ferritin to concanavalin A, a plant agglutinin that specifically binds to oligosaccharides containing terminal d-glucose, dmannose, or sterically related sugar residues. A technique was developed to allow topological visualization of erythrocyte and other membranes by means of transmission electron microscopy, and the distribution of the binding sites for ferritin-concanavalin A on such membrane preparations was determined. The conjugate was found to bind specifically to the outer, but not the inner, surface of erythrocyte membranes. The number of conjugate molecules bound per unit area of the membrane was larger for rabbit than for human erythrocytes.Complex oligo-and polysaccharides are known to be associated with the plasma membranes of a wide variety of cell types (1, 2). Electron microscopic studies of membrane-bound polysaccharides have utilized staining techniques adapted from light microscopy, such as colloidal-iron and colloidalthorium staining (3-7) for acid mucopolysaccharides, or the periodic acid-silver methenamine procedure (8-13) for 1,2-dihydroxy alcohols and a-amino alcohols. These procedures suffer from limitations of specificity and of inadequate resolution.Plant agglutinins are commonly occurring proteins (14) that bind to specific sugar ligands; several of these proteins have been isolated and characterized. One of these is concanavalin A, an agglutinin isolated from Jack beans (15), which binds reversibly to oligosaccharides containing terminal a-D-glucopyranosyl, a-D-mannopyranosyl, and sterically related sugar residues (16, 17), but not to other oligosaccharides. Concanavalin A has recently been used for gross studies of the surface architecture of cell membranes (18)(19)(20). We report the preparation of a specific electron microscopic stain for such terminal sugar residues by the conjugation of concanavalin A to ferritin (21), and the use of this conjugate to determine the distribution of concanavalin A binding sites on erythrocyte membranes. We have also developed a method for preparing membrane specimens to allow observations of the topological distribution of ferritin conjugates on membrane surfaces. MATERIALS AND METHODSHorse-spleen ferritin (6>X recrystallized, Miles-Pentex) was further purified by crystallization and ultracentrifugation (22). Concanavalin A (Miles-Yeda) was obtained as a pure protein by elution from an adsorbent column (16). Glutaraldehyde was freshly prepared by distillation of 50% glutaraldehyde obtained from Union Carbide.Ferritin-conjugated concanavalin A (Fer-Con A) was prepared by the glutaraldehyde-coupling method of Avrameas (23), since alkaline pH values had to be avoided to prevent aggregation of concanavalin A (24). To a solution containing 9% ferritin and 2.5% concanavalin A in a buffer of 0.5 M NaClI-0.05 M sodium phosphate (pH 6.8) was carefully added, with stirring, freshly distilled 0.5% glutaraldehyde, to a final concentration of 0.05%. After 45-60 mi...

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Ferritin-Conjugated Plant Agglutinins as Specific Saccharide Stains for Electron Microscopy: Application to Saccharides Bound to Cell Membranes

Nicolson

Singer

1971

250

“…Measurements of the binding of radioactively labeled Con A to cells cultured under different conditions have shown that the change in structure of the surface membrane in cell transformation can be explained by three types of changes in binding sites. There can be an exposure of cryptic sites, a concentration of exposed sites by a decrease in cell size, and a rearrangement of exposed sites without a decrease in cell size resulting in a clustering of sites (4)(5)(6)(7). The use of ferritin-conjugated Con A (8) has provided electron microscopic evidence that binding sites for Con A can be clustered (8,9).…”

mentioning

confidence: 99%

Differences in the Binding of Fluorescent Concanavalin A to the Surface Membrane of Normal and Transformed Cells

Shoham

Sachs

1972

Self Cite

The binding offluorescein-conjugated Concanavalin A to the cell surface has been studied in normal and transformed cells in interphase and mitosis. Binding to the cell surface was in the form of an incomplete ring of fluorescence, and the binding was inhibited by a-methyl-D-mannopyranoside. All the cells were fluorescent when treated with 25-250 jug/ml of fluorescent Concanavalin A. With 10 Mg, the cells were all fluorescent after 30 min of binding, but after 0. As a result of changes in the surface membrane, Concanavalin A (Con A) (1) agglutinates transformed interphase cells, but agglutinates normal interphase cells only after they have been treated with trypsin (2). This agglutination is temperature sensitive (3). Measurements of the binding of radioactively labeled Con A to cells cultured under different conditions have shown that the change in structure of the surface membrane in cell transformation can be explained by three types of changes in binding sites. There can be an exposure of cryptic sites, a concentration of exposed sites by a decrease in cell size, and a rearrangement of exposed sites without a decrease in cell size resulting in a clustering of sites (4-7). The use of ferritin-conjugated Con A (8) has provided electron microscopic evidence that binding sites for Con A can be clustered (8, 9).Here, we determine whether the binding of fluoresceinconjugated Con A (Fl-Con A) in nonsaturation conditions, as measured by microscopically visible surface fluorescence, can be used to detect differences between normal and transformed interphase and mitotic cells. Cells were cultured under conditions (5) in which a similar number of radioactivelylabeled Con A molecules were bound to normal and transformed cells at saturation. Fluorescein-Conjugated Con A. Con A was prepared by Miles-Yeda from Jack bean meal (Sigma Chemical Co.) by two crystallizations (1), and was stored lyophilized at -20°. Conjugation with fluorescein isothiocyanate (Sylvana Co.) was done according to Nairn (12), except that the reaction mixture was kept for 120 min at room temperature. The fluorescein to protein ratio was 1.4-1.7. Fluorescent Con A (Fl-Con A) agglutinated transformed cells to the same extent as native Con A at 5-500 .ug/ml. The agglutination was inhibited by 0.1 M a-methyl-D-mannopyranoside (Pfanstiehl Labs.). Treatment of cells for 30 min with 500 ug/ml of nonfluorescent Con A before incubation with 50 jug/ml of FlCon A, inhibited the cell surface fluorescence.Assay of Fl-Con A Binding. Binding of Fl-Con A was tested on cells without any fixation. For the studies with interphase cells, 106 cells were seeded per 100-mm petri dish; 3-4 days later, the cultures were washed with phosphate-buffered saline (P04-NaCl) (pH 7.2) and removed from the petri dishes with 0.02% EDTA solution (2). These cultures contained not more than 2% mitotic cells. Trypsinized cells were treated with 10,jg/ml of lyophilized and crystallized trypsin (Worthington Corp.) for 10 min at 370 after dissociation with EDTA. The trypsinization wa...

“…After Other Determinations. Cell-surface area was determined by centrifuging a known number of cells in centrifuge tubes containing a graduated capillary tube of 1-mm diameter (16 (Fig. 3).…”

mentioning

confidence: 99%

Regulation of Acetylcholine Receptors in Relation to Acetylcholinesterase in Neuroblastoma Cells

Simantov

Sachs

1973

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Purified α-toxin from Naja nigricollis snake venom labeled by [ 3 H]acetylation binds specifically to the acetylcholine receptors of mouse neuroblastoma cells. Toxin binding was inhibited by inhibitors for nicotinic and muscarinic acetylcholine receptors. Clones of neuroblastoma cells were selected for low acetylcholinesterase (EC 3.1.1.7) activity with antibodies against this enzyme. Selection for an 80-fold decrease in acetylcholinesterase activity was not associated with any decrease in the number of acetylcholine receptors (3.4 × 10 7 per cell). Removal or inactivation of 80% of the acetylcholine receptors by proteolytic enzymes or by compounds that block sulfhydryl groups did not change the activity of acetylcholinesterase on the cell surface. In addition to these results on the separation between acetylcholine receptors and acetylcholinesterase, a common regulation was found in that both the number of acetylcholine receptors and the activity of acetylcholinesterase were increased 5- to 10-fold when the cells stopped to multiply or were induced to differentiate by dibutyryl-cyclic AMP. It is suggested that there are different genes for the acetylcholine receptor and acetylcholinesterase, and that both are regulated during growth and differentiation by a common regulatory gene.