Fundamental studies on the insulin receptor in rabbit erythrocytes

The authors studied the binding of insulin to rabbit erythrocytes as a mode case in the hope of characterizing the physiologic role of the binding of insulin to receptor in both normal adults and patients. Specific binding sites for insulin were detected in rabbit erythrocytes. The characteristics of the binding were similar to those observed in other target tissues. The specific binding of 125I-labeled insulin was competitively inhibited by a small amount of unlabeled insulin and was completely inhibited by 1,000 ng/ml of unlabeled insulin. Glucagon, however, had no effect on the insulin binding to fat cells or liver membranes nor had it any effect on the binding of insulin to rabbit erythrocytes. Scatchard analysis of this binding reaction indicated two different binding sites with Ksub(aff)=3.2 x 108/M, Ksub(diss)=3.1 x 10-9M; Ksub(aff)=1.4 x 108/M, Ksub(diss)=7.1 x 10-9M respectively, and the binding capacities of each site were estimated at 0.011 ng/4 x 108 cells and 0.138 ng/4 x 108 cells. The binding of 125I-insulin to rabbit erythrocytes was a saturable function of the insulin concentration and was a linear function of cell concentration. The pH optimum for the reaction was 7.4 at 00C, the amount of insulin binding increased continuously under the reaction and this binding reaction reached a steady state after 10 to 15hr. On the other hand, the specific binding of insulin at higher temperatures showed maximal amounts after 20 to 30 min. and subsequently fell off at later time points. (auth.)