Binding of phytohemagglutinin to porcine lymphocyte receptors. Time-course studies and comparative binding isotherms using whole cells or cellular receptors incorporated into phospholipid vesicles

We have studied the binding of 125I-labeled phytohemagglutinin (PHA) to porcine splenic lymphocytes (107 cells∙mL−1) at 37 °C. Our data show that PHA binding is dependent on the incubation period and that a maximum quantity of 4.53 ± 0.13 μg is bound when 200 μg of PHA is added. The binding process is rapid and saturation can readily be achieved in less than 2 min. These observations suggest, in accordance with the mass-action principle, that the binding equilibrium can be rapidly displaced towards PHA–receptor complex formation when sufficient amounts of PHA are added. Our data further suggest that receptor sites are exposed at the cell surface and that putative cryptic receptor sites are unlikely to play a major role in the initial part of the binding process. We have studied this aspect by comparing Scatchard plots for PHA binding to receptors in intact lymphocytes and to lymphocyte-derived receptors incorporated into liposomes. In one case, partially purified plasma membranes were solubilized and incorporated into phosphatidylcholine–phosphatidylserine (PC–PS) vesicles prepared by detergent dialysis. In another case, PHA-receptor glycoproteins were purified by affinity chromatography and reassembled into PC–PS vesicles, using the same technique. Scatchard plot analyses showed nonlinear profiles with a concavity turned upward for PHA binding to receptors of intact cells or of PC–PS vesicles with plasma membranes, and a concavity turned downward for vesicles with purified glycoproteins. These observations suggest that PHA-receptor environment plays a determining role in the binding process of the lectin. Numerical data from binding experiments were obtained by computer-assisted nonlinear regression analysis, using a one ligand–two sites model. The (apparent) high-affinity constant (K1) for intact lymphocytes or partially purified plasma membrane components reassembled into liposomes was 4.6 × 107 M−1 and the (apparent) low-affinity constants (K2) were 4.4 × 106 ± 1.5 × 106 M−1 (intact lymphocytes) and 1.7 × 106 ± 0.6 × 106 M−1 (plasma membranes constituents reassembled into liposomes). The value obtained for reconstituted PHA-glycoprotein receptors (K) was 0.70 × 107 ± 0.10 × 107 M−1. The apparent number of sites was n1 = 0.19 × 106 ± 0.05 × 106 (high affinity) and n2 = 2.50 × 106 ± 0.50 × 106 (low affinity) sites per intact lymphocyte cell. In the case of the reconstituted systems, the number of sites per microgram protein of the PC–PS vesicles were n1 = 0.79 × 1010 ± 0.18 × 1010 and n2 = 31.8 × 1010 ± 8.5 × 1010 for the partially purified plasma membrane and n = 26.6 × 1010 ± 2.9 × 1010 for the purified PHA receptor glycoproteins. Data presented here are discussed in terms of the lymphocyte activation model of Prujansky and colleagues, which suggests that positively cooperative lectin binding is a sine qua non condition for lymphocyte activation.