Receptor binding and biological activity of specifically labeled [125I]- and [127I]monoiodoinsulin isomers in isolated rat adipocytes.

The receptor binding characteristics and biological activity of single site monoiodinated insulin was investigated using isolated rat adipocytes. Pork insulin was iodinated by the lactoperoxidase method, and each of the four monoiodo derivatives (iodotyr-A14, iodotyr-A19, iodotyr-B16, and iodotyr-B26) was isolated by high performance liquid chromatography. Both radioactive 125I-labeled and nonradioactive 127I-labeled iodoinsulins were prepared. In competitive binding experiments in which each 125I-labeled iodoisomer competed for receptor binding at 15 C with its homologous 127I-labeled iodoisomer, the concentrations of homolog required for 50% inhibition of tracer binding were 1.67 +/- 0.06, 2.14 +/- 0.23, and 2.35 +/- 0.27 X 10(-9) M for the B26, A14, and B16 iodoisomers, respectively (mean +/- SEM; n = 3). Scatchard analysis of the homologous competition curves using a two-site model indicated that there was no difference in the total number of specific sites to which each isomer could bind or in their affinity for binding to the low affinity site. However, a significantly (P less than 0.05) higher affinity for (B26)iodoinsulin binding to the high affinity site was observed compared to either the (A14)- or (B16)iodoisomers (Kd values of 1.09 +/- 0.18, 2.09 +/- 0.40, and 2.24 +/- 0.15 X 10(-9) M for B26, A14, and B16, respectively). Degradation of the isomers by adipocytes incubated at 37 C occurred at a rate proportional to their receptor binding affinity. The biological activity of iodoinsulins was also evaluated, based on the ability either to stimulate glucose oxidation or to exert an antilipolytic effect. The 127I-labeled (B26)iodoisomer exhibited the greatest potency in both assays compared to either (A14)- or (B16)iodoinsulins, consistent with its higher apparent affinity for receptor binding. The receptor-binding activity and biological potency of unmodified native insulin and 127I-labeled (A14)-iodoinsulin were directly compared. Identical results were obtained for each in both types of assay, suggesting that (A14)iodoinsulin is a valid tracer for insulin with isolated adipocytes. We conclude that in isolated rat adipocytes, (B26)iodoinsulin has greater activity than either unlabeled insulin or the other iodinated derivatives. (A14)Iodoinsulin is indistinguishable from native insulin; (B16)iodoinsulin has slightly reduced activity, while that of (A19)iodoinsulin is considerably reduced.