Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P01275 (glucagon)
 26,492 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Severe resistance to subcutaneous insulin but sensitivity to intravenous insulin persisted for 15 months in a 17-year-old diabetic girl. Heat-labile insulin-degrading activity was present in the patient's ketotic sera and in the 100,000 g fraction (soluble fraction) of adipose tissue. Serum-degrading activity was not inhibited by N-ethylmaleimide. The soluble fraction also degraded glucagon and B chain but not growth hormone or myoglobin. It was inhibited by incubation with the patient's nonketotic sera, normal sera, or Trasylol. Glutathione-insulin-transhydrogenase (GIT) activity was 66% of normal. The biopsy of adipose tissue at remission showed a normal level of insulin- and glucagon-degrading activity. The activity was eluted from Sephadex G200 as a single peak and had properties consistent with those of the insulin-specific protease (ISP). The increased degrading activity present during insulin resistance had properties not shared with ISP, suggesting the presence of an uncharacterized protease.
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PMID:Insulin resistance caused by massive degradation of subcutaneous insulin. 10 40

Results obtained with Cerasi & Luft's method and OGTT in subjects with a historical, clinical and laboratory suspicion of dysmetabolism were compared. It was found that: 1) obese subjects showed increased blood sugar and insulinase areas by comparison with normal controls; 2) subjects of normal weight displayed: a) a mean increase in blood sugar areas by comparison with normal controls; b) less evident changes in blood insulin areas; in these subjects, it was also noted that, c) an early-phase secretion irregularity detected with Cerasi & Luft's method did not involve changes in the oral loading pattern displayed by subjects classed as normal by means of such method; d) in subjects classed as "chemical diabetics" by OGTT, the response to glucagon after venous loading was defective. In border-line cases, early-phase changes were observed in the venous curve after oral glucose, whereas the response to glucagone remained efficient. It is felt that OGTT is an effective means in the diagnosis of infantile dysmetabolism. Attention is also drawn to the possibilities offered by the method of Cerasi & Luft in the detailed and specific appraisal of insulin secretion.
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PMID:[Comparative evaluation of the results obtained with the Cerasi and Luft method and the OGGT in children]. 99 83

Haemoglobin damaged by exposure of red blood cells to oxidants is rapidly degraded by a proteolytic pathway which does not require ATP [Fagan, Waxman & Goldberg (1986) J. Biol. Chem. 261, 5705-5713]. By fractionating erythrocyte lysates, we have purified two proteases which hydrolyse oxidatively damaged haemoglobin (Ox-Hb). One protease hydrolysed small fluorogenic substrates in addition to Ox-Hb. Its molecular mass was approximately 700 kDa and it consisted of several subunits ranging in size from 22 to 30 kDa. This enzyme may be related to the high-molecular-mass multicatalytic proteinase previously isolated from a variety of tissue and cell types. The other Ox-Hb-degrading activity had an apparent molecular mass of 400 kDa on gel filtration, a subunit size of 110 kDa and an isoelectric point between 4.5 and 5.0. This protease also hydrolysed the small polypeptides insulin and glucagon, as well as other large proteins such as lysozyme. Insulin blocked the degradation of Ox-Hb and Ox-Hb blocked the hydrolysis of insulin by the purified protease. Thiol reagents and metal chelators strongly inhibited the hydrolysis of both Ox-Hb and insulin, whereas inhibitors of serine, aspartic and thiol proteases had little effect. These properties suggest that the Ox-Hb-degrading activity purified from rabbit erythrocytes is the cytosolic insulin-degrading enzyme that is believed to play a role in the metabolism of insulin in several tissues. We propose that this enzyme may also function as a key component in a cytoplasmic degradative pathway responsible for removing proteins damaged by oxidants.
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PMID:Purification of a protease in red blood cells that degrades oxidatively damaged haemoglobin. 187 13

The subcellular site where insulin is degraded by rat hepatocytes in vivo is controversial. While several potential insulin-degrading enzyme systems, each with its own characteristic cellular location, are known to exist in the liver, questions remain about which of them participates in the degradation of physiologic doses of insulin. These studies examine the proteases that degrade physiologic doses of [125I]-insulin in vivo to determine (1) when and where initial degradation occurs, and (2) which of the potential degradative enzymes is active. Following injection into the mesenteric veins of male rats, intact [125I]-insulin and its labeled degradation products were analysed by reverse-phase high-performance liquid chromatography (RP-HPLC) of biopsy homogenates. [125I]-insulin was rapidly degraded in vivo; the t 1/2 of degradation was approximately 2.7 minutes. To test for extracellular protease activity, an isolated perfused liver system was employed. [125I]-insulin (or [125I]-glucagon) uptake was controlled by changing the temperature of the perfusion medium. Five minutes after [125I]-insulin injection, surface-bound label was recovered in an acidic (pH 3.5) wash. In perfusion at 15 degrees C, both the internalization and degradation of [125I]-insulin were inhibited; 7.2% of unbound hormone was degraded and 5.1% of surface-bound insulin was degraded. Only 11.4% of unbound insulin and 17.4% of surface-bound insulin were degraded at 35 degrees C. In contrast, 95.5% of unbound glucagon and 89.9% of surface-bound glucagon were degraded at 35 degrees C. Thus, although glucagon degradation occurs at the sinusoidal plasmalemma of perfused livers, the same membrane does not mediate the rapid degradation of insulin observed in vivo. Analysis of the RP-HPLC [125I]-insulin elution profiles from liver biopsy homogenates, and comparison of them to profiles produced by purified proteases, suggested that insulin protease is responsible for most hepatic degradation of physiologic doses of insulin. Some cathepsin D-like activity was also observed in vivo, confirming that two pathways exist for insulin metabolism. The time course over which insulin was degraded was more rapid than previous studies in vitro would have predicted. This suggests that more insulin was receptor-bound at the time of its initial degradation, and that the active protease was soluble and was introduced into endocytic peripheral endosomes within seconds after their formation.
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PMID:[125I]-insulin metabolism by the rat liver in vivo: evidence that a neutral thiol-protease mediates rapid intracellular insulin degradation. 240 25

We find, contrary to previous reports, that substantial cleavage of glucagon by insulin proteinase occurs at only one region, namely the double-basic sequence -Arg17-Arg18-. Cleavage takes place almost exclusively between these two residues, liberating fragments glucagon-(1-17) and glucagon-(18-29). Others have shown that the fragment glucagon-(19-29) is 1000-fold more efficient compared with intact glucagon, at inhibiting the Ca2+-activated and Mg2+-dependent ATPase activity and the Ca2+ pump of liver plasma membranes. We show that this fragment is not liberated in detectable quantities by our insulin proteinase preparation. On the other hand, others have shown that glucagon-(18-29), though less active than glucagon-(19-29), was still 100-fold more active than glucagon itself in the above-mentioned system. Our observations represent the first demonstration of the release by insulin proteinase of a hormone fragment having enhanced activity, although it has yet to be shown that the activity of this fragment is important in vivo. Since the formation of glucagon-(19-29) from glucagon-(18-29) would involve merely removal of Arg18, a second enzyme might exist to provide the more active fragment.
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PMID:Insulin proteinase liberates from glucagon a fragment known to have enhanced activity against Ca2+ + Mg2+-dependent ATPase. 297 45

RIN-m cells, cultured from a rat insulinoma, not only bind and secrete but also degrade insulin (Diabetes 1982; 31:521-31). The insulin-degrading activity resides in the cytosol and is similar to the insulin-specific proteases previously described in muscle and other tissues. It has an apparent Km of 0.15 microM for porcine insulin in crude cell-free extracts, a competitive inhibition constant for proinsulin that is close to the Km, and a lower but measurable affinity for glucagon. The enzyme is inactive at pHs below 6.0, indicating that it is not lysosomal, is completely inhibited by N-ethylmaleimide, and exhibits apparent competitive inhibition constants (microM) for the following peptides: desoctapeptide insulin, 0.043; guinea pig insulin, 0.048; proinsulin, 0.64; insulin B-chain, 1.17; glucagon, 7.0; and cyclic somatostatin, 8.6. Highly active insulin-degrading activity was found using cell suspensions of 22 cloned and 8 subcloned cell lines derived from RIN-m as well as 11 other continuous cell lines derived from a variety of nonislet tissues of rat, mouse, and human origin. Homogenates of the original rat islet tumor and cytosol of normal rat islets also contained insulin-degrading activity. Although insulin protease is present in a variety of tissues, it may have an additional regulatory function in cells that are actively synthesizing, storing, and secreting insulin.
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PMID:Cytosolic insulin-degrading activity in islet-derived tumor cell lines and in normal rat islets. 298 50

An insulin-degrading enzyme (IDE) was purified from the cytosol of human erythrocytes via the use of ammonium sulfate precipitation and chromatography on columns composed of DEAE-Sephadex, pentylagarose, hydroxylapatite, chromatofocusing resins, and Ultrogel AcA-34. The final preparation was purified greater than 50,000-fold and exhibited a single protein band of Mr = 110,000 on reduced sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Cross-linking of 125I-labeled insulin to the enzyme preparation labeled a protein of the same molecular weight, indicating that this band was in fact the enzyme. Intact insulin, insulin B chain, and glucagon inhibited this cross-linking half-maximally at concentrations of 0.1, 1, and 1.5 microM, respectively. Under nondenaturing conditions, the enzyme had an Mr = 300,000, suggesting that the enzyme may exist under physiological conditions as a dimer or timer. The purified enzyme was inhibited by both sulfhydrylmodifying reagents and chelating agents, indicating that a free thiol and metal were both required for the activity of the enzyme. The purified enzyme was found to degrade physiological concentrations of intact insulin more rapidly than insulin B chain, although at high substrate concentrations (greater than 1 microM) the enzyme degraded B chain to a greater extent. Additional characteristics of the enzyme were a pl of 5.2 and a pH optimum of 7.0. These properties of the red blood cell (RBC) enzyme were very similar to those reported for IDEs from other tissues. Moreover, a polyclonal antiserum to the IDE from skeletal muscle was found to recognize the RBC enzyme.
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PMID:Purification and characterization of insulin-degrading enzyme from human erythrocytes. 351 22

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

A mathematical model of normal regulation of carbohydrate metabolism by the pancreas endocrine apparatus is presented. In a numerical experiment the model imitated changed levels of sucrose, insulin glucagon and gastrointestinal hormones in the blood in response to the ingested 50 g of glucose. The model of normal regulation was damaged in the way which theoretically should result in diabetes development. Then an estimation was made to what extent the disturbances of carbohydrate metabolism characteristic of diabetes were reproduced by the changed model. It has been shown that disturbances specific for diabetes appear when the sensitivity of beta-cells to glucose stimulus or hyperproduction of glucagon decreased. No changes in the behaviour of blood glucose typical of diabetes were obtained in the model when a decrease of the sensitivity of insulin receptors due to hyperinsulinemia in insulin-dependent tissues was imitated, as well as an increased activity of liver insulinase or hyposecretion of gastrointestinal hormones. These results point to the necessity of further development of these hypotheses.
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PMID:[Checking of some hypotheses of the pathogenesis of diabetes mellitus by mathematical modeling]. 635 85

Insulin protease was purified 700-fold from rat liver homogenate by combined ultracentrifugation, ammonium sulfate fractionation, and glucagon-Sepharose-4B affinity chromatography. Optimum degradation of insulin was observed at pH 7.6 with the purified protease whose Km was 24 nM. The enzyme activity was inhibited completely by N-ethylmaleimide, p-hydroxymercuribenzoate, and heavy metals at 1 mM, whereas at the same concentration glutathione and mercaptoethanol stimulated the protease activity. These results indicate that the catabolic activity of the protease is sulfhydryl dependent. Furthermore, the activity of insulin protease was also enhanced by calcium and other divalent metal ions at a concentration of 1 mM. When supernatants, recovered from rat liver homogenates after centrifugation at 100,000g, were subjected to combined Sepharose 4B-insulin protease affinity chromatography and dialysis, a potent inhibitor of insulin protease was obtained which was heat stable. On the basis of kinetic studies, the inhibition of insulin degradation caused by this inhibitor was of the competitive type. Greater than 90% of the inhibitor activity was retained on dialysis with tubing with an inclusion limit of 3500 Da, whereas only 10% of this activity could be retained in dialysis tubing with an exclusion limit of 15,000 Da. These findings suggest that the insulin protease inhibitor is a low-molecular-weight protein. Analysis of homogenates from 13 different tissues of the rat showed that the highest levels of insulin protease inhibitor activity were associated with those tissues which have the highest capacity to degrade insulin. These data suggest that insulin protease and insulin protease inhibitor may be an important natural regulatory mechanism of insulin activity.
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PMID:Partial purification and characterization of insulin protease and its intracellular inhibitor from rat liver. 636 62


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