Mechanism of lymphocyte activation: the binding of phytohemagglutinin to the lymphocyte surface

Abstract

The dynamics of phytohemagglutinin (PHA)-lymphocyte interaction was studied using ¹²⁵I-labeled PHA (leucoagglutinin) and pig mesenteric lymph node lymphocytes that had been depleted of erythrocytes, dead cells, adherent cells and immunoglobulin-bearing cells. Evidence was obtained that PHA stimulated the majority of the lymphocytes to transform. Binding of PHA at 37°C was fairly rapid (rate constant for association: 2.6 x 10⁵ M⁻¹ sec⁻¹), saturable, reversible and specifically inhibited by N-acetylgalactosamine (K_diss: 3 x 10⁻⁴ M) and unlabeled PHA. A Scatchard plot was curvilinear and gave evidence for 3.6 x 10⁵ binding sites per cell comprising 8.7 % of high affinity sites (K_diss: 3.7 x 10⁻⁹ M) and 91.3 % of lower affinity (K_diss: 1.4 x 10⁻⁷ M). About 20 % of the sites were occupied under culture conditions giving maximal transformation. Alternative explanations for the curvilinear plot included negative cooperative interactions and/or increase in affinity through multivalent interaction. Negative cooperativity was supported by the demonstration that free PHA promoted the dissociation of bound PHA. Binding was not affected by metabolic inhibitors, and binding to purified lymphocyte plasma membrane resembled that to whole cells. These results suggested that PHA binding to whole lymphocytes was not grossly influenced by “capping”, endocytosis and shedding.