Yonezawa K, Yokono K, Shii K, Hari J, Yaso S, Sakamoto T, Kawase Y, Akiyama H, Taketomi S, Baba S
Second Department of Internal Medicine, Kobe University School of Medicine, Japan.
Endocrinology. 1989 Jan;124(1):496-504. doi: 10.1210/endo-124-1-496.
We previously reported on the degradation of monocomponent porcine insulin by affinity-purified pig skeletal muscle insulin-degrading enzyme (IDE) and on the detection and HPLC separation of the initial degradation product (peak VI). Using relatively high concentration of insulin, peak VI appeared rapidly at 30 sec of incubation, whereas other peaks were not detected within 5 min of incubation. Performate oxidation studies suggested that peak VI is composed of a cleaved A-chain and an intact B-chain. To assess whether the initial degradation product of insulin generated by IDE preserves biological properties, we analyzed several insulin-like activities of peak VI. It had a hypoglycemic effect on rats. In vitro, it bound to the insulin receptors of rat adipocytes and stimulated glucose oxidation there. It also strengthened insulin receptor kinase activity in insulin receptors from rat liver and human placenta. Its biological potency, however, was 1/40th to 1/160th that of insulin itself. This is probably due to reduced affinity for the insulin receptor, since it had 2.5% of insulin's ability to both bind to the receptor and stimulate glucose oxidation. Moreover, peak VI had all of insulin's agonistic effect on glucose oxidation when used at a higher concentration. On the other hand, cross-linking analysis suggested that peak VI preserves almost the same affinity for IDE as does insulin. These results suggest that pig skeletal muscle IDE may cleave peptide bonds within the A-chain early in insulin degradation, generating peak VI; this then serves as the next substrate of IDE while exerting reduced insulin-like activity, and peak VI is converted to several relatively low mol wt products.
我们之前报道了亲和纯化的猪骨骼肌胰岛素降解酶（IDE）对单组分猪胰岛素的降解作用，以及初始降解产物（峰VI）的检测和高效液相色谱分离。使用相对高浓度的胰岛素时，在孵育30秒时峰VI迅速出现，而在孵育5分钟内未检测到其他峰。过甲酸氧化研究表明，峰VI由裂解的A链和完整的B链组成。为了评估IDE产生的胰岛素初始降解产物是否保留生物学特性，我们分析了峰VI的几种胰岛素样活性。它对大鼠有降血糖作用。在体外，它与大鼠脂肪细胞的胰岛素受体结合并刺激那里的葡萄糖氧化。它还增强了大鼠肝脏和人胎盘胰岛素受体中的胰岛素受体激酶活性。然而，其生物学效力仅为胰岛素本身的1/40至1/160。这可能是由于对胰岛素受体的亲和力降低，因为它结合受体和刺激葡萄糖氧化的能力仅为胰岛素的2.5%。此外，当以较高浓度使用时，峰VI对葡萄糖氧化具有胰岛素的所有激动作用。另一方面，交联分析表明，峰VI对IDE的亲和力与胰岛素几乎相同。这些结果表明，猪骨骼肌IDE可能在胰岛素降解早期切割A链内的肽键，产生峰VI；然后峰VI作为IDE的下一个底物，同时发挥降低的胰岛素样活性，并且峰VI被转化为几种相对低分子量的产物。
