Properties of the Insulin Receptor of Isolated Fat Cell Membranes

The interaction of 125 I-insulin with crude membrane preparations from isolated fat cells has many properties in common with the interaction of insulin with biologically significant receptors in intact fat cells. Specific binding of 125 I-insulin to the membranes is a saturable process with respect to insulin and to membranes, and native insulin competes for binding in a way expected from the biological identity of the 2 molecules. Reduced and desoctapeptide insulins do not compete with 125 I-insulin for binding, and desalanine insulin is indistinguishable from native insulin. Proinsulin binds to the membranes with an affinity which is 20 times less than that of native insulin. Insulin in the incubation medium containing fat cell membranes is not significantly destroyed under the conditions used in the binding assay. The 125 I-insulin-membrane complex can be dissociated by acid, and the insulin thus released from this complex is similar to native insulin by several physical properties, by its ability to bind to membranes, and by its ability to stimulate glucose oxidation in isolated fat cells. The rate constants of insulin-membrane association (8.5 x 10 6 mole -1 sec -1 ) and dissociation (4.2 x 10 -4 sec -1 ) have been measured independently, and the dissociation constant (5 x 10 -11 m ) based on these rate constants is similar to that (7.5 x 10 -11 m ) calculated separately from equilibrium data. These constants are similar to those calculated for the interaction of insulin with intact fat cells. Measurements of the rate constants at various temperatures indicate that binding is much tighter at lower temperatures because the decrease in dissociation rate is disproportionately greater than the decrease in association rate. Thermodynamic calculations reveal a Δ F of -14 kcal mole -1 , a Δ H of -28 kcal mole -1 , a Δ S of -45 cal mole -1 deg -1 . Binding of 125 I-insulin to membranes is unaffected by the nature of the buffer used or by a number of different ionic species, heavy metals, or by metal-complexing agents. The optimum pH for binding occurs sharply at about 7.5, and the pH range over which binding occurs is rather narrow. A sharp thermal transition, with a midpoint at 53°, results in irreversible inactivation of binding activity. Insulin binding to membranes is markedly affected by the ionic strength of the medium. Increasing concentrations of NaCl up to 2 m cause a dramatic (6-fold) increase in insulin binding. This probably results from the a