High and low affinity binding sites for Concanavalin A on normal human fibroblasts in vitro

Feller, Martha; Richardson, Christopher A.; Behnke, W. David; Gruenstein, Eric

doi:10.1016/0006-291x(77)90959-7

Cited by 18 publications

(3 citation statements)

References 24 publications

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“…ConcanavalinAbinds to highand low-affinity sites on the surface of fibroblasts (Feller et al, 1977). Approximately 1 % of the sites are of high affinity, with an affinity constant 1000 times greater than the remaining 99%.…”

Section: Introductionmentioning

confidence: 99%

Quantitation of membrane receptor distributions by image correlation spectroscopy: concept and application

Petersen

Höddelius

Wiseman

et al. 1993

Biophysical Journal

359

333

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Measurement of receptor distributions on cell surfaces is one important aspect of understanding the mechanism whereby receptors function. In recent years, scanning fluorescence correlation spectroscopy has emerged as an excellent tool for making quantitative measurements of cluster sizes and densities. However, the measurements are slow and usually require fixed preparations. Moreover, while the precision is good, the accuracy is limited by the relatively small amount of information in each measurement, such that many are required. Here we present a novel extension of the scanning correlation spectroscopy that solves a number of the present problems. The new technique, which we call image correlation spectroscopy, is based on quantitative analysis of confocal scanning laser microscopy images. Since these can be generated in a matter of a second or so, the measurements become more rapid. The image is collected over a large cell area so that more sampling is done, improving the accuracy. The sacrifice is a lower resolution in the sampling, which leads to a lower precision. This compromise of precision in favor of speed and accuracy still provides an enormous advantage for image correlation spectroscopy over scanning correlation spectroscopy. The present work demonstrates the underlying theory, showing how the principles can be applied to measurements on standard fluorescent beads and changes in distribution of receptors for platelet-derived growth factor on human foreskin fibroblasts.

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Section: Introductionmentioning

confidence: 99%

Quantitation of membrane receptor distributions by image correlation spectroscopy: concept and application

Petersen

Höddelius

Wiseman

et al. 1993

Biophysical Journal

359

333

View full text Add to dashboard Cite

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“…The amount of WGA bound on intact Raji cells was almost equal at the high-affinity and low-affinity binding sites (0.276 X 10-4 and 0.262 X 10-4 nmol^g of protein, respectively), corresponding to 1.73 X 106 high-affinity and 1.65 X 106 low-affinity sites. In contrast, the numbers of high-and low-affinity binding sites for Con A on intact fibroblasts differ by more than 2 orders of magnitude, being 8 X 105 and 3 X 10s sites per cell, respectively (Feller et al, 1977). In the present work, nearly equal amounts of WGA were bound at high-and low-affinity sites in the case of isolated nuclei (0.75 X ÍO'4 and 0.70 X 4 nmol/^g of protein, respectively), but the values differed somewhat for plasma membranes (9.82 X 10"4 and 6.5 X 10"4 nmol/Vg of protein, respectively).…”

Section: Discussionmentioning

confidence: 84%

Comparison of binding sites for wheat germ agglutinin on Raji lymphoblastoid cells and their isolated nuclei and plasma membranes

Jett¹,

Jamieson²

1981

Biochemistry

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Raji lymphoblastoid cells and the cell nuclei and plasma membranes isolated by the glycerol-lysis technique [Jett, M., Seed, T., & Jamieson, G. A. (1977) J. Biol. Chem. 252, 2134-2142] have been examined for their ability to bind wheat germ agglutinin. Intact cells and isolated nuclei showed similarities (i) in the total number of binding sites (3.38 X 10(6) and 4.06 X 10(8), respectively), indicating at least a 2-fold higher receptor density on the nuclei, (ii) in the ratios of the number of high-affinity sites and low-affinity sites (1.05 and 1.07), and (iii) in the apparent association constants at the high-affinity sites (28 nM and 48 nM) and at the low-affinity site (116 nM and 370 nM). Isolated plasma membranes had a similar number of total binding sites calculated on an equivalent cell basis (2.01 X 10(6)) but showed differences in the ratio of high- to low-affinity sites (1.5) and in their apparent association constants (3 nM and 22 nM). These results suggest similarities in the lectin receptors on the outer surface of lymphoblastoid cells and the cell nuclei. The differences obtained with isolated membranes may be due to inversion of the membrane vesicles or to their decreased rigidity as compared with the intact cell.

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“…To test whether S-FCS can discriminate between the high and low affinity components of certain receptor systems, we performed S-FCS experiments of SconA as a function of concentration on 3T3 Swiss mouse fibroblasts. The motivation for using the concanavalin A receptor system is that the concanavalin A receptors are known to possess both high and low affinity sites on human fibroblasts (21). Binding studies with radiolabeled concanavalin A reported Scatchard plots with two distinct lines corresponding to binding constants on the order of 10+9 and 10+6 M-for the high and low binding sites.…”

Section: Resultsmentioning

confidence: 99%

Relative ligand binding to small or large aggregates measured by scanning correlation spectroscopy

St-Pierre¹,

Petersen²

1990

Biophysical Journal

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Cell surface receptors transduce signals, required to produce cellular activity, that may be mediated by ligand-induced receptor aggregation. Several receptor systems exhibit both low and high ligand affinities and some models of receptor activation associate receptor clusters with high or low ligand binding affinity. In the present work succinyl concanavalin A, which binds with both high and low affinity to receptors, was studied on 3T3 Swiss mouse fibroblasts, where preaggregation of receptors has been postulated. Scanning fluorescence correlation spectroscopy measurements were used to determine the relationship between the degree of ligand binding and the state of receptor aggregation. Correlation analysis of fluorescence fluctuations across the cell surface reveal that the variance of the fluctuations (quantitated by g[0]) increased when the ligand concentration was varied from 0.33 to 67 mg/L. The g(0) values reached a plateau at concentrations greater than approximately 10 mg/L. These data are incompatible with homogeneous receptor distributions or equal affinity receptor binding but are compatible with a partly aggregated receptor system with high affinity binding to small aggregates, and low affinity binding to large aggregates. Computer simulated scanning fluorescence correlation spectroscopy experiments confirm that background fluorescence from the cell does not account for the experimentally observed effects.

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High and low affinity binding sites for Concanavalin A on normal human fibroblasts in vitro

Cited by 18 publications

References 24 publications

Quantitation of membrane receptor distributions by image correlation spectroscopy: concept and application

Quantitation of membrane receptor distributions by image correlation spectroscopy: concept and application

Comparison of binding sites for wheat germ agglutinin on Raji lymphoblastoid cells and their isolated nuclei and plasma membranes

Relative ligand binding to small or large aggregates measured by scanning correlation spectroscopy

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