Abstract
The interactions of phytohemagglutinin (PHA) with normal human lymphocytes were studied utilizing radioiodinated leukoagglutinin (125I-LPHA) over a concentration spectrum encompassing the entire range of lymphocyte metabolic responses. 125I-LPHA binding was temperature-, pH-, and time-dependent. Ligand association was rapid with a t1/2 of 3 to 5 min, reaching steady state in 30 min at 22 degrees. Receptor specificity was demonstrated by the high receptor affinity for 125I-LPHA and by quantitative inhibition of 125I-LPHA binding with LPHA and 127I-LPHA but not with concanavalin A or bovine serum albumin. Under our experimental conditions there was no measurable degradation of 125I-LPHA and no detectable shedding of 125I-LPHA receptors or receptor-125I-LPHA complexes. Equilibrium studies of 125I-LPHA 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 125I-LPHA, reflects changing receptor affinity for the ligand and suggests site-site interactions of the negative cooperativity type. These interactions which appear to be common to all lymphocyte subpopulations, preclude accurate calculation of lymphocyte binding capacity for 125I-LPHA and of physically meaningful affinity constants. Although the fate and role of a small fraction of apparently nondissociable 125I-LPHA remains to be elucidated, occupancy-dependent receptor affinity for 125I-LPHA, dissociation of receptor-125I-LPHA complexes, retention of binding properties by cell-exposed 125I-LPHA, and the large numbers of spare surface receptors for 125I-LPHA might represent important mechanisms for modulating cell activation by 125I-LPHA.
Highlights
Over a concentration spectrum encompassing the entire range of lymphocyte metabolic responses. “jI-LPHA binding was temperature, pH, and time-dependent
The excellent reproducibility of ““I-LPHA binding to lymphocyte surface receptors with intraexperimental variation of less than 5%, the absence of demonstrable receptor shedding or degradation of receptor-ligand during the interaction process, the ease of separation of bound from unbound ligand, and the opportunity to study subsequent physicochemical, biological, and metabolic changes induced makes this an almost ideal system for receptor studies
We have studied in detail the kinetics of lymphocyte receptor-LPHA
Summary
Over a concentration spectrum encompassing the entire range of lymphocyte metabolic responses. “jI-LPHA binding was temperature-, pH-, and time-dependent. 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 which appear to be common to all lymphocyte subpopulations, preclude accurate calculation of lymphocyte binding capacity for lz51-LPHA and of physically meaningful affinity constants. Its major advantages being the ready availability of lymphocyte suspensions of great purity [1,2], the possibility of studying lymphocyte membrane receptor-ligand interactions using radiolabeled pure mitogenic proteins
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