Affinity purification of insulin-degrading enzyme and its endogenous inhibitor from rat liver.
A metallothiol protease called insulin-degrading enzyme (IDE) seems to be implicated in insulin metabolism to terminate the response of cells to hormone, as well as in other biological functions, including muscle differentiation, regulation of growth factor levels, and antigen processing. In order to obtain highly pure and biologically active IDE, we have developed an immunoaffinity method using a monoclonal antibody to this enzyme (9B12). When the cytosolic fraction of rat liver was first applied to a 9B12-coupled Affi-Gel 10 column, more than 97% of the insulin-degrading activity was absorbed. Among various kinds of buffers successfully eluting the enzyme, only the buffer with a high pH (pH 11) could retain the full biological activity of this enzyme. IDE was further purified via two steps of chromatography using Mono Q anion exchange and Superose 12 molecular sieve columns. The final preparation showed a single band at 110 kDa on reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In the eluate from the immunoaffinity column, the inhibitory activity associated with the enzyme was also observed. To better recover this endogenous inhibitor, heat-treated cytosolic fraction was fractionated by ammonium sulfate precipitation and applied to the immunoaffinity column on which IDE had been adsorbed. Then, IDE and its inhibitor could be co-eluted with pH 11 as a complex form. After heat treatment of this fraction, the inhibitor was further purified using the same series of chromatography as IDE to more than 20,000-fold; it showed a 14 kDa band on SDS-PAGE. It inhibited both the insulin degradation by IDE in a competitive manner and the cross-linking of 125I-insulin to IDE. Highly purified IDE and the endogenous inhibitor will be useful tools for better understanding the various biological functions of this enzyme.
Ogawa
W
W
Second Department of Internal Medicine, Kobe University School of Medicine, Japan.
Shii
K
K
Yonezawa
K
K
Baba
S
S
Yokono
K
K
D000818:Animals; D000911:Antibodies, Monoclonal; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D007339:Insulysin / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D007457:Iodine Radioisotopes; D008099:Liver / Q000201:enzymology* / Q000378:metabolism; D008970:Molecular Weight; D051381:Rats
D000911:Antibodies, Monoclonal; D007457:Iodine Radioisotopes; D007339:Insulysin