Peavy D E, Abram J D, Frank B H, Duckworth W C
Endocrinology. 1984 May;114(5):1818-24. doi: 10.1210/endo-114-5-1818.
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.
利用分离的大鼠脂肪细胞研究了单位点单碘化胰岛素的受体结合特性和生物活性。用乳过氧化物酶法对猪胰岛素进行碘化，通过高效液相色谱法分离出四种单碘衍生物（碘代酪氨酸 - A14、碘代酪氨酸 - A19、碘代酪氨酸 - B16和碘代酪氨酸 - B26）。制备了放射性125I标记和非放射性127I标记的碘胰岛素。在竞争性结合实验中，每种125I标记的碘异构体在15℃下与其同源的127I标记的碘异构体竞争受体结合，B26、A14和B16碘异构体的50%抑制示踪剂结合所需的同源物浓度分别为1.67±0.06、2.14±0.23和2.35±0.27×10（-9）M（平均值±标准误；n = 3）。使用双位点模型对同源竞争曲线进行Scatchard分析表明，每种异构体能够结合的特定位点总数或其与低亲和力位点结合的亲和力没有差异。然而，与（A14）-或（B16）碘异构体相比，观察到（B26）碘胰岛素与高亲和力位点结合的亲和力显著更高（P < 0.05）（B26、A14和B16的Kd值分别为1.09±0.18、2.09±0.40和2.24±0.15×10（-9）M）。在37℃下培养的脂肪细胞对异构体的降解速率与其受体结合亲和力成正比。基于刺激葡萄糖氧化或发挥抗脂解作用的能力，还评估了碘胰岛素的生物活性。与（A14）-或（B16）碘胰岛素相比，127I标记的（B26）碘异构体在两种测定中均表现出最大效力，这与其对受体结合的较高表观亲和力一致。直接比较了未修饰的天然胰岛素和127I标记的（A14）-碘胰岛素的受体结合活性和生物效力。在两种类型的测定中，每种都获得了相同的结果，表明（A14）碘胰岛素是分离的脂肪细胞中胰岛素的有效示踪剂。我们得出结论，在分离的大鼠脂肪细胞中，（B26）碘胰岛素比未标记的胰岛素或其他碘化衍生物具有更大的活性。（A14）碘胰岛素与天然胰岛素无区别；（B16）碘胰岛素活性略有降低，而（A19）碘胰岛素的活性则显著降低。
