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)

When incubated in vitro, isolated rat liver low-density vesicles degrade endocytosed insulin intraluminally. The rate of intravesicular degradation suggests that this pathway contributes significantly to insulin degradation in vivo. The vesicles can be selectively disrupted with digitonin at concentrations that abolish the latency of NADH pyrophosphatase, with minimal effect on the cisternal Golgi marker, galactosyl transferase. The results suggest that latent NADH pyrophosphatase may act as a marker enzyme for the vesicles within which insulin is degraded. The possible role of insulin-glucagon protease, a candidate enzyme for insulin degradation by the liver, was investigated. The activity of latent insulin-glucagon protease associated with low-density vesicles is sufficient to account for the rate of intravesicular proteolysis. However, the rate of intravesicular proteolysis is insensitive to membrane-permeant thiol reagents under conditions which strongly inhibit insulin-glucagon protease. This shows that insulin-glucagon protease is not rate-limiting for insulin degradation by these vesicles, and is unlikely to be involved in the regulation of degradation. After disruption with Brij, internalized insulin remains associated with the membrane. Degradation is not inhibited by addition of excess unlabelled insulin to the medium, and occurs more rapidly than the degradation of an equal activity of iodo-insulin added to the disrupted membranes. This implies that degradation of endocytosed insulin occurs while it is still bound to the inner surface of the vesicles. When bacitracin is coinjected with iodo-insulin, it inhibits degradation of internalized insulin both by intact and Brij-disrupted vesicles, but not the degradation of added exogenous insulin, confirming that degradation is membrane-associated, and that it does not require the release of insulin into free solution.
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PMID:Characterization of insulin degradation by rat-liver low-density vesicles. 354 5

Previous studies have shown that neutral thiopeptidase (E.C.3.4.22.11, insulinase) degrades (processes) insulin with a high affinity (Km = 30 X 10(-9) M). In the current studies, insulin was subjected to digestion with a highly purified rat liver neutral thiopeptidase and the peptides generated were separated by HPLC using a C8 column. With the use of structural analysis (which included the determination of amino terminal residues and amino acid composition), the major product was identified as a peptide containing portions of both chains of insulin, A1 to A13 and B1 to B9 having two disulfide bonds, an interchain disulfide bond and presumably the intra-A chain disulfide bond as well. Examination of insulin-like biological activity using a primary cultured hepatocyte test system showed that the fragment promoted neither short-term (alpha-aminoisobutyric acid uptake) nor long-term (glycogen synthesis) bioactivities of insulin.
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PMID:Identification of an insulin fragment produced by an insulin degrading enzyme, neutral thiopeptidase. 355 82

The activity of the insulin-degrading enzyme neutral cysteine proteinase (EC 3.4.22.11, insulinase) was studied in adipose tissue and in liver of nondiabetic, streptozotocin-diabetic, and insulin-treated diabetic rats. Proteinase activity was found to be significantly decreased during diabetes and was restored to near normal levels in both tissues following insulin treatment. The insulin-mediated increase of proteinase activity in both tissues was partially or completely blocked by actinomycin D (an inhibitor of RNA synthesis) and by cyclohexamide (an inhibitor of protein synthesis). Kinetic analysis showed that the changes in proteinase activity of both liver and adipose tissues were accompanied by a change in Vmax (i.e., maximal enzyme activity) without a change in Km (i.e., substrate affinity). These data indicate that insulin functions as an inducer for neutral cysteine proteinase in both tissues. These alterations in the proteinase activity paralleled the alterations in the activity of a second insulin-degrading enzyme, glutathione-insulin transhydrogenase in adipose tissue (this paper) and in liver (previously published papers) under the same physiological conditions.
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PMID:Insulin-degrading neutral cysteine proteinase activity of adipose tissue and liver of nondiabetic, streptozotocin-diabetic, and insulin-treated diabetic rats. 355 42

A tumor antigen isolated from the cytosol of a methylcholanthrene-induced sarcoma (Meth A) has been purified to homogeneity by the criteria of two-dimensional gel analysis and NH2- and COOH-terminal sequencing. The purified antigen has a mol. wt of 82,000 by SDS gel electrophoresis. However, the apparent mol. mass of the antigen was found to be 71,600 and 67,700 by gel filtration chromatography and sedimentation analysis, respectively. It is not a glycoprotein, possesses an acidic isoelectric point (6.0) and exists as dimeric and monomeric species. The dimer is not held together by disulfide bonds. The purified protein retains its ability to induce transplantation immunity in syngeneic hosts when challenged with Meth A sarcomas. Chemical analyses of the NH2- and COOH-termini gave the following sequences: NH2-PKPINVRVTTMDAELEFAIQPN and IDE(F,A)EM-COOH, respectively.
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PMID:Characterization of a chemically homogeneous tumor antigen from a methylcholanthrene-induced sarcoma, Meth A. 374 14

We have studied insulin degrading activity (IDA) in cultured human fibroblasts and assessed the effect of various inhibitors of insulin processing on IDA. To evaluate the role of three enzymes of insulin degradation (neutral protease, microsomal glutathione insulin transhydrogenase, and lysosomal acid protease), we subfractionated homogenized fibroblasts into membrane (and nuclei) cytosol, mitochondria, microsomes, and lysosomes. Greater than 90% of IDA was found to be present in the cytosolar fraction containing neutral protease. IDA in intact fibroblasts was completely inhibited by 1 mM N-ethylmaleimide and partially by 0.5 mM dansylcadaverine (75%), 0.5 mM chloroquine (48%), 1 mg/ml bacitracin (32%) and Trasylol (30%). Lidocaine (5 mM) and glucagon (10(-6)M) exhibited about 15% inhibition with minimal inhibition (7%) by nonsuppressible insulin-like activity. Study of similar inhibitors on subfractionated components indicated inhibition of cytosolar enzyme by N-ethylmaleimide (100%), glucagon (30%), chloroquine (41%), nonsuppressible insulin-like activity (30%), Lidocaine (25%), dansylcadaverine (16%), and bacitracin (11%). Incubation of ammonium sulfate-fractionated cytosolar enzyme at 37 C with A14-125I-insulin resulted in generation of two intermediate peaks as early as 1 min. These peaks could be identified by HPLC but not by molecular sieve chromatography. These intermediates exhibited less immunoprecipitability with antiinsulin antibody and receptor binding with liver membrane preparations than intact insulin. Further incubation of A14-125I-insulin with the cytosolar enzyme(s) resulted in reduction of these peaks as well as insulin and formation of 125Iodotyrosine peak. We conclude that human fibroblast is capable of metabolizing cell-associated A14-125I-insulin in a time- and temperature-dependent manner. This process is inhibited by various inhibitors of insulin processing. The bulk of IDA consists of soluble neutral protease(s) with properties similar to other more purified neutral insulin protease preparations. This fraction, similar to the intact fibroblast degrades insulin to two intermediates with similar molecular weight to that of intact insulin but with more hydrophilicity and less binding affinity to antiinsulin antibody and liver membrane than intact insulin.
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PMID:Characterization of insulin-degrading activity of intact and subcellular components of human fibroblasts. 388 99

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

The effect of calcium on insulin degradation by intact or homogenized skeletal muscle, by skeletal muscle cytosol, and by partially purified skeletal muscle insulin-degrading protease activity was examined. After a 15-min lag phase, intact soleus muscles degraded [125I]insulin to trichloroacetic acid-soluble products in a time-dependent fashion. Degradation was accelerated by the addition of calcium (greater than or equal to 1 mM), such that maximal stimulation (2-fold) was obtained with 10 or 25 mM calcium. Calcium stimulated insulin degradation by skeletal muscle homogenate and by cytosol in a nearly identical manner. Furthermore, after inactivation of the purified skeletal muscle, insulin-degrading protease by dialysis against EDTA, this enzyme was reactivated fully (greater than 80%) by a 100 microM concentration of free Ca2+. These observations identify a previously unrecognized but important influence of calcium on cellular insulin handling and provide further evidence for a major role of the calcium-activated enzyme, insulin protease, in cellular insulin degradation.
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PMID:The calcium dependence of insulin degradation by rat skeletal muscle. 392 38

A double-antibody radioimmunoassay for the insulin-degrading enzyme, glutathione-insulin transhydrogenase (GIT), has been developed with the use of rabbit antiserum against human liver GIT and [125I]-GIT. The method can determine as little as 32 fmol of GIT, thus allowing measurements in needle tissue biopsy samples and in plasma, which have not been possible with previous enzymatic procedures. Relative competition in the radioimmunoassay by unlabelled GITs purified from other sources are in agreement with homologies in GITs previously found using the enzymatic assay. No competition was observed with pork insulin, bovine ribonuclease, human albumin or human gamma-globulin, indicating that the radioimmunoassay is highly specific for GIT. Similar competition curves were observed for native GIT; active, reduced GIT; or for the inactive, S-(ethylsuccinimido) derivative of GIT. The radioimmunoassay thus measures total (active + inactive) GIT and permits determinations in the presence of materials which react with the active site and render the enzymatic methods unusable. Radioimmunoassay of plasma and extracts of liver, muscle and adipose tissues from diabetic and non-diabetic subjects showed parallel competition curves with standard purified human GIT indicating that GITs of non-diabetic and diabetic persons are immunologically very similar or identical. Concentrations of GIT in plasma determined by radioimmunoassay were significantly higher in diabetic than those in non-diabetic subjects (1620 +/- 80 versus 1070 +/- 30 fmol/l, p less than 0.001). Tissue GIT levels found by the radioimmunoassay as well as by the enzyme assay, both in non-diabetic and diabetic subjects, were highest in the liver, intermediate in the adipose tissue and lowest in the muscle.
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PMID:Insulin degradation: radioimmunoassay for glutathione-insulin transhydrogenase and its application. 393 Mar 32

A method has been described for the direct measurement of proinsulin in human plasma. The method makes use of an insulin-degrading enzyme designated "insulin-specific protease (ISP)", which is obtained from rat skeletal muscle. Under the conditions used, this enzyme rapidly degrades insulin and insulin-like polypeptides to nonimmunoassayable components, whereas proinsulin and proinsulin cleaved at position B(54,55) are not appreciably affected. The incubation of plasma with ISP results in the disappearance of insulin, but not proinsulin, as demonstrated by column chromatography. Immunoassay of the plasma, therefore, before and after incubation, determines the values for the total immunoreactive substance (TIR) and for immunoreactive proinsulin (IRP), respectively. The values obtained for proinsulin levels are reproducible and compare closely with the more complicated column fractionation methods. Proinsulin responses were studied in four normal subjects and one patient with an insulinoma after a glucose load. Fasting proinsulin levels varied widely in the normal subjects, and the levels rose more slowly than TIR levels after glucose. IRP levels in the patient with an insulinoma were very high and fell to normal after removal of the tumor. The ISP method, therefore, appears to be suitable for the direct, accurate, and rapid determination of proinsulin and proinsulin-like materials in human plasma.
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PMID:Direct measurement of proinsulin in human plasma by the use of an insulin-degrading enzyme. 432 76

Receptor-bound insulin is substrate for a degradation leading to the release of about half the cell-associated [125I]monoiodoinsulin as [125I]monoiodotyrosine. Classical lysosomal inhibitors of the amine type (cloroquine, methylamine and NH+4) only partly inhibited this receptor-mediated degradation. Leupeptin, which is very effective in other systems, was without any effect in the present system. The degradation could not be reduced by lowering the ATP content of the cells. Sulphydryl reagents strongly inhibited the degradation as has also been shown for the cytosolic insulin-specific protease. Microtubules and microfilaments are probably not involved since inhibitors of the cytoskeleton were without marked effects. It is suggested that in the rat adipocyte only a minor part of the receptor-mediated degradation of insulin takes place via the classical endocytotic lysosomal pathway.
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PMID:The mechanism of receptor-mediated degradation of insulin in isolated rat adipocytes: indirect evidence for a non-lysosomal pathway. 613 87


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