Covalent linkage of 125I-insulin to a cytosolic insulin-degrading enzyme.
Cytosol extracts high in insulin-degrading activity were cross-linked to 125I-insulin with the bifunctional cross-linker disuccinimidyl suberate. With cytosols from either rat muscle, liver, kidney or brain or human erythrocytes, only a single protein (Mr = 110,000) was specifically labeled. Three different lines of evidence indicated that this labeled protein is insulin-degrading enzyme, a cysteine protease which accounts for most of the insulin-degrading activity in cell extracts. Firstly, the cross-linking of 125I-insulin to this protein is inhibited by unlabeled insulin over the same concentration range of insulin which inhibits degradation. Separated insulin A and B chain were less potent at inhibiting cross-linking, whereas bovine serum albumin and cytochrome c were without effect. Secondly, antibodies to purified insulin-degrading enzyme precipitated the labeled protein in parallel with their ability to precipitate the insulin-degrading activity of the extracts. Thirdly, when the insulin-degrading activity was purified 40,000-fold from erythrocytes, this Mr 110,000 protein co-purified. These results indicate that cross-linking 125I-insulin may be a convenient method for labeling the insulin-degrading enzyme.
D000818:Animals; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D007328:Insulin / Q000031:analogs & derivatives* / Q000378:metabolism; D007339:Insulysin / Q000378:metabolism*; D008297:Male; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D014018:Tissue Distribution
D007328:Insulin; C008147:insulin, iodo-; D010447:Peptide Hydrolases; D007339:Insulysin