THE JOURNAL OF BIOLOGICAL CHEMISTRY 252, No. 6, Issue of March 25, pp. 2095-2100, Prmted in U.S.A
April 12, 1976, and in revised form, August 19, 1976)
B. FAGUET the Department of Medicine, Medical College of Georgia, Augusta,Georgia 30902
The interactions of phytohemagglutinin (PHA) with normal human lymphocytes were studied utilizing radioiodinated leukoagglutinin (“$I-LPHA) over a concentration spectrum encompassing the entire range of lymphocyte metbinding was temperature-, pH-, abolic responses. “jI-LPHA and time-dependent. Ligand association was rapid with at,,* of 3 to 5 min, reaching steady state in30 min at 22”. Receptor specificity was demonstrated by the high receptor affinity for ‘Z”I-LPHA and by quantitative inhibition of lz51-LPHA binding with LPHA and 9-LPHA but not with concanaUnder our experimental valin A or bovine serum albumin. conditions there was no measurable degradation of 12jIshedding of lZSI-LPHA receptors or LPHA and no detectable receptor. ““I-LPHA complexes. Equilibriumstudies of YLPHA interactions with specific lymphocyte membrane receptors generated a complex curvilinear Scatchard plot. This, added to progressive deceleration of the dissociation reaction inversely proportional to receptor occupancy by “jI-LPHA, reflects changing receptor affinity for the ligand and suggests site-site interactions of the negative cooperativity type. These interactions whichappear to be common to all lymphocyte subpopulations, preclude accurate calculation of lymphocyte binding capacity for lz51-LPHA and of physically meaningful affinity constants. Although the fate and role of a small fraction of apparently nondissociable ““I-LPHA remains to be elucidated, occupancy-dependent receptor affinity for lZ51-LPHA, dissociation of receptor. lz51-LPHA complexes, retention ofbinding properties by cell-exposed ““I-LPHA, and the large numbers of spare surface receptors for lzSI-LPHA might represent important mechanisms for modulating cell activation by ““I-LPHA.
(b) inhibition of ligand binding by various competitive agents (lo), and (c) the use of carrier-bound lectins (11) and the study of the resulting changes induced on lymphocyte metabolic responses led to therecognition of the importance of membrane phenomena in cell activation. However, the introduction of more direct and versatile methodology using radiolabeled lectins (4, 6, 12) was necessary to characterize the comand the kinetics of receptor-lectin interacplex mechanisms tions. The location (13, 141, and nature (15, 16) of these recepkinetics (3, 4, 17) with ligands, tors and their interactionhowever, remain poorly understood. The delineation of these parameters should be of relevance to our understanding of antigen-mediated cellular activation and may contribute to of alterations of cell-mediated immunity the characterization associated with a growing number of diseases (3,12, 18,19). The present studies were undertaken to examine in detail the interactions of PHA’ with specificreceptors on the surface of human lymphocytes. The use of radiolabeled LPHA enabled precise measurements of the dynamics of these interactions.
MATERIALS AND METHODS
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In vitro lymphocyte transformation by plant lectins is a particularly suitable model to study mechanisms of cell activation. Its major advantages beingthe ready availability of lymphocyte suspensions of great purity (1,2), the possibility of studying lymphocyte membrane receptor-ligand interactions using radiolabeled pure mitogenic proteins (3, 41, and the unique opportunity to monitor physicochemical (5, 6) and metabolic changes these interactions engender, including DNA synthesis and cell mitosis (7, 8). Disturbance of the receptor-ligand...