Mechanism of human lymphocyte stimulation by concanavalin A: role of valence and surface binding sites.

Abstract

A monovalent form of concanavalin A (m-Con A) has been prepared to determine the importance of valence for human lymphocyte surface binding and subsequent lymphocyte stimulation as measured by blast transformation and cytotoxicity. Concanavalin A (Con A) was fragmented by a proteolytic process and the m-Con A derivative was isolated by elution with an ascending D-glucose gradient on a Sephadex CG200 column. The molecular weight of m-Con A was 18,000 by sodium dodecyl sulfate-polyacrylamide electrophoresis. Equilibrium dialysis with a-methyl Dglucoside and subsequent METHODSPreparation of m-Con A. Con A was fragmented by a modification of the method of Evans and Jones (14). For this procedure, Con A (150 mg) was dissolved in 5.0 ml 0.1 M HCI to which 2 mg of porcine pepsin (California Biomedical Research Co.) was added. The mixture was incubated for 120 min at 400, after which the reaction was stopped by adjusting the pH to neutrality with 0.1 M NaOH. Subsequently, 20.0 ml of phosphate-buffered saline containing 0.9 mM CaC12 and 1.05 mM MgCl2 was added, and the precipitate was removed by centrifugation at 20,000 X g for 20 min. Trypsin treated with L-1-tosylamide-2-phenylethyl-chloromethyl ketone (TPCK) (120 mg) and protease type 6 (40 mg; Sigma Chemical Co.) were then added and the mixture was incubated at 400 for 120 min. Finally, the reaction was stopped by cooling on ice and again the precipitate was removed by centrifugation.The cooled reaction mixture was introduced at a flow rate of 6.0 ml/hr to a column (35.0 cm X 1.5 cm) of Sephadex G-200 equilibrated with phosphate-buffered saline. Subsequently, the column was washed with buffer by means of a peristaltic pump at a flow rate of 5.0 ml/hr and 5.0 ml fractions were collected. Protein concentration in the eluant was determined by continuous monitoring at a wavelength of 280 nm. After 24 hr D.glucose was added to the elution buffer in an ascending linear gradient from 0 to 35 mM using a Pharmacia Ultragrad 2000 gradient maker. A flow rate of 6.0 ml/hr was maintained and 3.0 ml fractions were collected. Each fraction was subsequently dialyzed extensively against phosphate-buffered saline and the total protein concentration of the pooled fraction was measured by the method of Lowry et al. (15).After dialysis, each fraction eluted during the application of the linear glucose gradient was assayed for agglutinating ability. Fresh washed rabbit erythrocytes were prepared and agglutination studies were performed as previously described (16). Rabbit erythrocytes were regularly agglutinated at concentrations of 1 ug/ml of untreated Con A. Those fractions obtained after elution with D-glucose (and subsequent dialysis against phosphate-buffered saline) that failed to cause agglutination after 40 min at protein concentrations greater than 10 ,gg/ml were pooled and subjected to further characterization. This pooled fraction was also evaluated for its ability to agglutinate human lymphocytes in a similar manner to the assay for rabbit erythrocyte agglutination. ...

Recent Advancement on Isolation, Activation and Cryopreservation of Lymph Node Cells in Mice.

“…These include increased phospholipid incorporation into the membrane, increased flux of ionic potassium, calcium and increased uptake of nucleotides, sugars, and amino acids. 20 In another study, Con A covalently bound to acrylic polymer particles and stimulated rat lymphocytes into DNA synthesis. The average maximum number of Con A that bound was about 5x10 6 per cell.…”

Role of the valence of concanavalin A in the activation of guinea pig polymorphonuclear leukocytes.

“…By virtue of its tetravalence, ConA may function to crosslink carbohydrate-containing receptors into clusters which, in turn, may lead to metabolic alterations (3). In order to evaluate this hypothesis, monovalent derivatives have been prepared by proteolytic treatment (18) or by a combination of succinylation and photoaffinity labeling (2, 19). Their actions have been most extensively studied in lymphocytes.…”