The kinetic properties of the insulin receptor were studied in solution after its purification to homogeneity.Dissociation of "I-labeled insulin at a 1:50 dilution was not first order; unlabeled insulin at physiological concentrations accelerated the dissociation rate with a maximal effect at "17 nM. At higher concentrations, the unlabeled insulin slowed the dissociation rate. Maximal acceleration was seen at pH 8. The purification to homogeneity (1-3) and the cloning and expression (4, 5) of the insulin receptor, as well as the availability of monoclonal antibodies (6-12), have recently provided exciting means to investigate the exact mechanisms underlying the complex kinetics of insulin receptor binding. It is now well established that insulin binding departs markedly from simple reversible mass action kinetics (13)(14)(15)(16)(17)(18)(19)(20)(21)(22). Scatchard plots are curvilinear in most systems studied, and the dissociation is not first order. Furthermore, dissociation of 125I-labeled insulin (125I-insulin) at an "infinite" dilution is accelerated in the presence of unlabeled insulin. These basic findings, as well as the effects of numerous structural modifications of the insulin molecule (16,(18)(19)(20) and more recent studies with monoclonal antibodies (7,8,11,12,22), have led to the view (most explicitly developed in refs. 20 and 22) that the insulin-receptor complex can shift reversibly between at least three interconvertible states by site-site interactions, which depend on the nature of the ligand occupying neighboring sites. We distinguished the initial, "empty" state with an apparent affinity denoted KAE; a lower affinity state, Kf, characterized by a faster dissociation rate constant, induced by increased insulin occupancy-the phenomenon known as "negative cooperativity"-and a higher affinity state, which was denoted "Ksu.r " with a much slower dissociation rate.Although arguments have been raised against the negative cooperativity hypothesis (23-25), no alternative model has been proposed that is as compatible as the three-state model outlined above with a variety of experimental findings using either insulin analogs, monoclonal antibodies, or alterations ofreceptors in clinical states, and with more recent studies of alterations in the dimeric structure of the receptor (see ref. 22 for review). While we believe that the experimental evidence favors the site-site interactions model, it is clear that ultimate proof will have to come from the actual demonstration by crystallographic or other physicochemical methods, of alternative conformations of the purified receptor in various liganded states, or by appropriate changes in receptor structure and kinetics generated by site-directed mutagenesis.As a first step in that direction, we have now characterized in detail the kinetic behavior in solution of the insulin receptor purified to homogeneity from human placenta. One of us previously demonstrated that Scatchard plots of 125I-insulin binding to this purified preparation are curvilinear (1...
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