Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.56 (insulin-degrading enzyme)
 737 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Covalent linkage of 125I-insulin to a cytosolic insulin-degrading enzyme. 388 82

Although previous studies from this and other laboratories have extensively characterized insulin degrading activity in animal tissues, little information has been available on insulin responsive human tissues. The present study describes the insulin degrading activity in skeletal muscle from normal human subjects. Fractionation of a sucrose homogenate of skeletal muscle demonstrated that 97% of the total neutral insulin degrading activity was in the 100 000 x g supernatant with no detectable glutathione-insulin transhydrogenase activity. The 100000 x g pellet contained 85% of the total acid protease activity and all the glutathione-insulin transhydrogenase activity. The soluble insulin degrading activity was purified 1400-fold by ammonium sulfate fractionation, molecular exclusion, ion-exchange and affinity chromatography. Enzymatic activity was determined by measuring an increase in trichloroacetic acid-soluble products of the 125I-labeled hormone substrates. The purified enzyme showed marked proteolytic specificity for insulin with a Km of 1.63 X 10(-7)M (+/-0.32) and was competitively inhibited by proinsulin and glucagon with Ki values of 2.1 X 10(-6)M and 4.0 X 10(-6)M, respectively. This insulin protease exhibited a pH optimum between 7 and 8, a molecular weight of 120000 and was capable of degrading glucagon. Inhibition studies demonstrated that a sulfhydryl group is essential for activity. Molecular exclusion chromatography of [125I]insulin degraded products revealed a time-dependent increase in degradation products with molecular weights intermediate between intact insulin and iodotyrosine. These studies demonstrate that the major enzymatic system responsible for insulin degrading activity is a soluble cysteine protease capable of rapidly metabolizing insulin under physiologic conditions.
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PMID:Insulin degradation by human skeletal muscle. 675 60

Insulin-degrading enzyme (IDE), a nonlysosomal metalloprotease involved in metabolizing internalized insulin, has catalytic properties that have been strongly conserved through evolution. Two major properties distinguish IDE from the prototypic metalloprotease thermolysin. 1) It is inhibited by cysteine protease inhibitors as well as metalloprotease inhibitors; 2) it contains an inversion of the HEXXH active site motif of thermolysin, where the histidines coordinate zinc and the glutamate participates in catalysis. Furthermore, cysteine is adjacent to the glutamate residue (HXCEH) in human, rat, and Drosophila IDE, although it is not conserved in their close homologue, Escherichia coli protease III. This cysteine has been postulated to mediate the differential sensitivity of IDE and protease III to cysteine protease inhibitors and chelators. The role of the cysteine in IDE catalysis and inhibitor sensitivity was examined by mutating Cys110 to glycine or serine. To determine whether glutamate in this unusual motif participates in catalysis, we mutated Glu111 to aspartate, valine, or glutamine. Vectors containing wild type or mutant enzymes were transfected into COS cells, and expression was confirmed by Western blotting. Although the glutamate mutants were devoid of insulin degrading activity, the cysteine mutants were indistinguishable from wild type enzyme in both catalytic activity and sensitivity to inhibitors. The loss of activity in the glutamate mutants was not due to gross alterations in tertiary structure, as shown by retention of the ability to bind substrate and by conservative and nonconservative mutation of a neighboring residue with no apparent effect on catalysis. These results demonstrate that the conserved glutamate in the zinc-binding site of human insulin-degrading enzyme is a major catalytic residue, while a conserved cysteine in this region is not essential for catalysis or inhibitor sensitivity.
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PMID:Functional analysis of conserved residues in the active site of insulin-degrading enzyme. 810 41

The malaria parasite Plasmodium falciparum degrades hemoglobin in its acidic food vacuole for use as a major nutrient source. A novel metallopeptidase activity, falcilysin, was purified from food vacuoles and characterized. Falcilysin appears to function downstream of the aspartic proteases plasmepsins I and II and the cysteine protease falcipain in the hemoglobin proteolytic pathway. It is unable to cleave hemoglobin or denatured globin but readily destroys peptide fragments of hemoglobin. Falcilysin cleavage sites along the alpha and beta chains of hemoglobin are polar in character, with charged residues located in the P1 and/or P4' positions. In contrast, plasmepsins I and II and falcipain prefer hydrophobic residues around the scissile bond. The gene encoding falcilysin has been cloned. Its coding sequence exhibits features characteristic of clan ME family M16 metallopeptidases, including an "inverted" HXXEH active site motif. Falcilysin shares primary structural features with M16 family members such as insulysin, mitochondrial processing peptidase, nardilysin, and pitrilysin as well as with data base hypothetical proteins that are potential M16 family members. The characterization of falcilysin increases our understanding of hemoglobin catabolism in P. falciparum and the unusual M16 family of metallopeptidases.
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PMID:Identification and characterization of falcilysin, a metallopeptidase involved in hemoglobin catabolism within the malaria parasite Plasmodium falciparum. 1054 84

The S. cerevisiae genome encodes two M16A enzymes: Axl1p and Ste23p. Of the two, Ste23p shares significantly higher sequence identity with M16A enzymes from other species, including mammalian insulin-degrading enzymes (IDEs). In this study, recombinant Ste23p and R. norvegicus IDE (RnIDE) were isolated from E. coli, and their enzymatic properties compared. Ste23p was found to cleave established RnIDE substrates, including the amyloid-beta peptide (Abeta1-40) and insulin B-chain. A novel internally quenched fluorogenic substrate (Abz-SEKKDNYIIKGV-nitroY-OH) based on the polypeptide sequence of the yeast P2 a-factor mating propheromone was determined to be a suitable substrate for both Ste23p and RnIDE, and was used to conduct comparative enzymological studies. Both enzymes were most active at 37 degrees C, in alkaline buffers and in high salt environments. In addition, the proteolytic activities of both enzymes towards the fluorogenic substrate were inhibited by metal chelators, thiol modifiers, inhibitors of cysteine protease activity and insulin. Characteristics of STE23 expression were also evaluated. Our analysis indicates that the 5' terminus of the STE23 gene has been mischaracterized, with the physiologically relevant initiator corresponding to residue M53 of the publicly annotated protein sequence. Finally, we demonstrate that, unlike haploid-specific Axl1p, Ste23p is expressed in both haploid and diploid cell types. Our study presents the first comprehensive biochemical analysis of a yeast M16A enzyme, and provides evidence that S. cerevisiae Ste23p has enzymatic properties that are highly consistent with mammalian IDEs and other M16A enzymes.
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PMID:Yeast Ste23p shares functional similarities with mammalian insulin-degrading enzymes. 1975 Apr 77