The insulin-receptor interaction: is the kinetic approach for inferring negative-cooperative site-site interactions valid?

The dissociation of insulin from its receptor is reportedly enhanced when the dissociation is induced by dilution in the presence of insulin. This experiment is frequently conducted when curvilinear Scatchard plots of insulin binding are observed in order to infer negative cooperative site-site interactions amongst insulin receptors. However, when insulin binding to purified liver plasma membranes was measured at 15 degrees C in 50 mM Tris, pH 7.5 containing 0.1% bovine serum albumin and 100 U/ml bacitracin, the insulin binding data was characterised by a linear Scatchard plot and a Hill plot with a slope equal to unity. Thus, under the conditions of this binding assay, insulin apparently bound to a single non-interacting class of homogeneous binding sites. But, despite the apparent absence of cooperative interactions under these specific conditions, the dissociation of receptor-bound insulin was still enhanced when the dissociation of insulin from its receptor was induced by dilution in the presence of insulin. This result cast serious doubt on the validity of inferring negative-cooperative site-site interactions amongst insulin receptors based solely on the observation that the dissociation of receptor-bound insulin is enhanced by dilution in the presence of insulin.