A comparison of direct and indirect techniques using ferritin-conjugated ligands for the localization of concanavalin A binding sites on isolated hepatocyte plasma membranes.

Abstract

Concanavalin A binding sites have been localized on isolated plasma membranes both by a direct technique involving ferritin-concanavalin A and by an indirect technique in which membranes were treated successively with concanavalin A, rabbit anti-concanavalin A, and ferritin-conjugated sheep anti-rabbit F(ab')2. Binding studies showed that, at saturation, less than 25% of the concanavalin A binding sites were accessible to ferritin-concanavalin A. The decreased binding was apparently related to steric factors, since membranes saturated with the conjugated ligand were able to bind additional concanavalin A, and since the conjugated ligand, once bound to the membrane, caused the same inhibition of the membrane-bound enzyme 5'-nucleotidase as concanavalin A. Nonspecific binding sites accounted for 10% of the total binding of ferritin-concanavalin A and were localized mainly on the cytoplasmic side of the membrane, whereas specific sites were on the external side. The indirect technique, which was expected to increase the binding of ferritin-conjugate to the membrane, resulted in the binding of ferritin to less than 15% of the concanavalin A binding sites, and did not decrease the nonspecific binding.