Active Insulin Degrading Enzyme (IDE)

Insulin Degrading Enzyme (IDE) is an evolutionarily conserved 110-kDa zinc metalloprotease. It has been described principally as a cytosolic enzyme but is also found in multiple cellular compartments including endosomes, peroxisomes, mitochondria, the cell surface and in secreted form. IDE is a major enzyme responsible for insulin degradation. In addition to insulin, IDE degrades many targets including glucagon, atrial natriuretic peptide, and beta-amyloid peptide, regulates proteasomal degradation and other cell functions. In addition, IDE can degrade IGF2, thus affecting the concentration and activity of IGF2 in the cell. Thus a functional binding ELISA assay was conducted to detect the interaction of recombinant human IDE and recombinant rabbit IGF2. Briefly, IDE was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to IGF2-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-IDE pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody for 1h at 37℃, wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50 µL stop solution to the wells and read at 450/630 nm immediately. The binding activity of recombinant human IDE and recombinant rabbit IGF2 was shown in Figure 1, the EC50 for this effect is 0.08 ug/mL.