Evidence that insulin receptor from human placenta has a high affinity for only one molecule of insulin.
Insulin receptor, partially purified from human placenta by chromatography on wheat germ agglutinin, was shown, by means of double probe labeling, to bind only one molecule of insulin with a high affinity. In the double probe labeling protocol used, 125I-insulin (probe 1) was affinity cross-linked to its receptor in the presence of an excess of unlabeled N epsilon B29-biotinylinsulin (probe 2). The ability of succinylavidin to bind to receptor-linked probe 2 and alter the electrophoretic mobility of the cross-linked complex (during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate) was used to determine the amount of receptor which was cross-linked to both probes relative to that which was cross-linked to only probe 1. The fraction of receptor bound to two molecules of insulin prior to cross-linking was estimated from the cross-linking efficiency and the yield of receptor cross-linked to both probes relative to the yield of receptor cross-linked only to probe 1. The low fraction of receptor bound to both probes in the presence of high concentrations of probe 2 indicated that the affinity of the receptor for a second molecule of insulin was approximately 100 times less than that for the first and that in the range of insulin concentrations (less than 20 nM) usually used to determine the stoichiometry for the interaction between receptor and insulin, more than 80% of the receptor molecules should be bound to only one molecule of insulin. This knowledge of how insulin receptor interacts with insulin was shown to be important for proper determination of receptor purity, interpretation of curvilinear Scatchard plots, and interpretation of the insulin-enhanced rate of dissociation of receptor-bound insulin.
D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D005260:Female; D006801:Humans; D007106:Immune Sera; D007074:Immunoglobulin G; D007328:Insulin / Q000378:metabolism*; D007700:Kinetics; D037102:Lectins; D008433:Mathematics; D008954:Models, Biological; D008970:Molecular Weight; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy; D011972:Receptor, Insulin / Q000302:isolation & purification / Q000378:metabolism*; D014909:Wheat Germ Agglutinins
D007106:Immune Sera; D007074:Immunoglobulin G; D007328:Insulin; D037102:Lectins; D014909:Wheat Germ Agglutinins; D011972:Receptor, Insulin