Binding interaction studies of selected receptor subpopulations after partial cross-linking receptor-ligand complexes with a photoactivated heterobifunctional reagent.

Certain hormonal and nonhormonal binding systems such as the leukoagglutinin-lymphocyte model exhibit complex receptor-ligand interactions that result in nonlinear Scatchard plots. Such plots are interpreted as indicating either homogeneous negatively interacting binding sites or heterogeneous sites with different and fixed affinity. We assessed the validity of these interpretations in our system by conjugating the ligand to a photoactivated heterobifunctional agent and cross-linking the conjugate to a subset of receptors before studying the binding interactions of non-cross-linked sites. Conjugation did not qualitatively or quantitatively affect the binding properties of the ligand. Cross-linking was specific, efficient, and stable and had no effect on irrelevant surface receptors. Cross-linking of only 3% of the total receptors resulted in 50% decreased ligand binding to high affinity sites consistent with a calculated inactivation of 85% and 2% of high and low affinity sites, respectively. Such preferential inactivation of high affinity sites in an unequivocal demonstration of binding sites heterogeneity in this system and shows a clear rejection of the homogeneous cooperation model.