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)

An endopeptidase activity that cleaves glucagon, producing miniglucagon or glucagon (19-29), a Ca2+ pump inhibitory peptide, was isolated from rat liver membranes. The purified enzyme has a molecular mass of approximately 100 kDa and a pH optimum of approximately 8. It is inhibited by both sulfhydryl-blocking reagents and metal-chelating reagents and activated by thiol compounds. The partial N-terminal amino acid sequence of the 100-kDa protein does not correspond to any known protein. An antiserum was raised against a synthetic octapeptide corresponding to the N-terminal sequence. Immunoblot analysis of crude liver membranes revealed a single band at 100 kDa. Immunoreactivity was found in liver, pancreas, and heart, which are glucagon and miniglucagon target tissues, and in gastric mucosa and kidney. Low levels were detected in spleen, whereas immunoreactivity was undetectable in skeletal muscle and intestinal mucosa. The endopeptidase activity was inhibited by insulin, glucagon-like peptide-1, and glucagon-like peptide-1 (7-36) amide, whereas other peptides that contain dibasic sites had no effect on its activity, indicating that the endopeptidase does not display strict selectivity toward basic doublets.
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PMID:Endopeptidase from rat liver membranes, which generates miniglucagon from glucagon. 840 29

The post-secretory processing of the potent insulinotropic peptide hormone, GLP-1(7-36)amide, probably involves one or more of a small group of membrane-bound ectopeptidases. Reported here, is the characterisation of the endoproteolysis of human GLP-1(7-36)amide by the recombinant human form of neutral endopeptidase (NEP) 24.11, which is one of the best characterised and widely-distributed of ectopeptidases and is involved in the processing of other peptide hormones. The products of the limited endoproteolysis were characterised by mass and primary structure following fractionation using high performance liquid chromatography. The rate of this endoproteolysis by NEP 24.11 was estimated and compared to that of GLP-1(7-36)amide-related peptides. GLP-1(7-36)amide appears to be good substrate for NEP 24.11 with most, but not all potential target bonds being cleaved. Also, the structurally-related peptides, secretin and glucagon appear to be good substrates whereas GIP and exendin-4 are very poor substrates. That the GLP-1(7-36)amide super-agonist, exendin-4 is a poor substrate for NEP 24.11 is significant for the possible use of this peptide as a prototype for the development of clinically-useful peptide agonists. Further studies should reveal whether NEP 24.11 is important for the metabolic clearance of GLP-1(7-36)amide and will be highly relevant for the attempts to realise the suggested therapeutic value of GLP-1(7-36)amide.
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PMID:Characterisation of the processing by human neutral endopeptidase 24.11 of GLP-1(7-36) amide and comparison of the substrate specificity of the enzyme for other glucagon-like peptides. 857 27

This study concerns whether the pancreatic beta cell expresses cell-surface ectopeptidases that are capable of proteolysis of peptide hormones and neuropeptides that modify glucose-dependent insulin release. These biochemical investigations of the RINm5F cell line found that these cells express ectopeptidases. We have characterized the limited endoproteolysis of GLP-1 (7-36) amide that occurs in the presence of RINm5F plasma membranes. The products and the sensitivity to specific peptidase inhibitors of the proteolysis is characteristic of neutral endopeptidase (NEP) 24.11. Vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), amylin, glucagon, glucose-dependent insulinotropic polypeptide (GIP), and exendin-4 also undergo proteolysis in the presence of RIN cell membranes. NEP 24.11-activity in RIN cell membranes was confirmed using a specific fluorogenic assay, by histochemistry, and by comparison with the recombinant enzyme with respect to the kinetics of proteolysis of GLP-1 (7-36) amide and of a fluorogenic substrate. Specific fluorogenic assays revealed the presence of aminopeptidase N and the absence of aminopeptidase A and of dipeptidylpeptidase IV.
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PMID:Endoproteolysis of glucagon-like peptide (GLP)-1 (7-36) amide by ectopeptidases in RINm5F cells. 921 54

Exendin-4, a 39-amino acid (AA) peptide, is a long-acting agonist at the glucagon-like peptide-1 (GLP-1) receptor. Consequently, it may be preferable to GLP-1 as a long-term treatment for type 2 diabetes mellitus. Exendin-4 (Ex-4), unlike GLP-1, is not degraded by dipeptidyl peptidase IV (DPP IV), is less susceptible to degradation by neutral endopeptidase, and possesses a nine-AA C-terminal sequence absent from GLP-1. Here we examine the importance of these nine AAs for biological activity of Ex-4, a sequence of truncated Ex-4 analogs, and native GLP-1 and GLP-1 analogs to which all or parts of the C-terminal sequence have been added. We found that removing these AAs from Ex-4 to produce Ex (1-30) reduced the affinity for the GLP-1 receptor (GLP-1R) relative to Ex-4 (IC50: Ex-4, 3.22+/-0.9 nM; Ex (1-30), 32+/-5.8 nM) but made it comparable to that of GLP-1 (IC50: 44.9+/-3.2 nM). The addition of this nine-AA sequence to GLP-1 improved the affinity of both GLP-1 and the DPP IV resistant analog GLP-1 8-glycine for the GLP-1 receptor (IC50: GLP-1 Gly8 [GG], 220+/-23 nM; GLP-1 Gly8 Ex (31-39), 74+/-11 nM). Observations of the cAMP response in an insulinoma cell line show a similar trend for biological activity.
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PMID:The importance of the nine-amino acid C-terminal sequence of exendin-4 for binding to the GLP-1 receptor and for biological activity. 1283 4

We have investigated the proteolytic mechanisms of glucagon degradation within hepatic endosomes at neutral pH before lumen acidification. Hepatic endosomes incubated at neutral pH rapidly degraded native glucagon into 13 intermediate products, one of which corresponded to the bioactive fragment glucagon-(19-29) (miniglucagon). The serine protease inhibitor phenylmethylsulfonyl fluoride as well as the nonspecific protease inhibitor bacitracin inhibited the endosomal degradation of glucagon at pH 7. In purified endosomal fractions, miniglucagon endopeptidase was undetectable as evaluated by immunoblotting, and immunoprecipitation with antibodies to insulin-degrading enzyme, cathepsins B and D, or furin failed to remove the endosomal neutral glucagonase activity. Incubation of endosomal fractions and [125I]iodoglucagon with the zero-length bifunctional cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide resulted in specific labeling of a 170-kDa polypeptide. The labeling was completely inhibited by unlabeled glucagon (IC50 value, 5 x 10-7 m) and bacitracin (IC50 value, 1 microg/ml), suggesting that it may correspond to a bacitracin-sensitive glucagon-degrading enzyme. Treatment of the 125I-labeled 170-kDa cross-linked polypeptide with N-glycanase demonstrated that the cross-linked complex contained approximately 30 kDa of N-linked oligosaccharides. Specific cross-linking of the 170-kDa polypeptide was also observed using [125I]Tyr12-miniglucagon as the radioligand. Together, these data suggest that the 170-kDa glycoprotein represents a novel glucagon-degrading activity that could mediate glucagon proteolysis within endosomes before the acidification step and generate the bioactive (19-29) miniglucagon peptide.
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PMID:Endosomal proteolysis of glucagon at neutral pH generates the bioactive degradation product miniglucagon-(19-29). 1295 81

Glucagon has a short plasma t(1/2) in vivo, with renal extraction playing a major role in its elimination. Glucagon is degraded by neutral endopeptidase (NEP) 24.11 in vitro, but the physiological relevance of NEP 24.11 in glucagon metabolism is unknown. Therefore, the influence of candoxatril, a selective NEP inhibitor, on plasma levels of endogenous and exogenous glucagon was examined in anesthetized pigs. Candoxatril increased endogenous glucagon concentrations, from 6.3 +/- 2.5 to 20.7 +/- 6.3 pmol/l [COOH-terminal (C)-RIA, P < 0.05]. During glucagon infusion, candoxatril increased the t(1/2) determined by C-RIA (from 3.0 +/- 0.5 to 17.0 +/- 2.5 min, P < 0.005) and midregion (M)-RIA (2.8 +/- 0.5 to 17.0 +/- 3.0 min, P < 0.01) and reduced metabolic clearance rates (MCR; 19.1 +/- 3.2 to 9.4 +/- 2.0 ml.kg(-1).min(-1), P < 0.02, C-RIA; 19.2 +/- 4.8 to 9.0 +/- 2.3 ml.kg(-1).min(-1), P < 0.05, M-RIA). However, neither t(1/2) nor MCR determined by NH2-terminal (N)-RIA were significantly affected (t(1/2), 2.7 +/- 0.4 to 4.5 +/- 1.6 min; MCR, 30.3 +/- 6.4 to 28.5 +/- 9.0 ml.kg(-1).min(-1)), suggesting that candoxatril had no effect on NH2-terminal degradation but leads to the accumulation of NH2-terminally truncated forms of glucagon. Determination of arteriovenous glucagon concentration differences revealed that renal glucagon extraction was reduced (but not eliminated) by candoxatril (from 40.4 +/- 3.8 to 18.6 +/- 4.1%, P < 0.02, C-RIA; 29.2 +/- 3.1 to 14.7 +/- 2.2%, P < 0.02, M-RIA; 26.5 +/- 4.0 to 19.7 +/- 3.5%, P < 0.06, N-RIA). Femoral extraction was reduced by candoxatril when determined by C-RIA (from 22.7 +/- 2.4 to 8.0 +/- 5.1%, P < 0.05) but was not changed significantly when determined using M- or N-RIAs (10.0 +/- 2.8 to 4.7 +/- 3.7%, M-RIA; 10.5 +/- 2.5 to 7.8 +/- 4.2%, N-RIA). This study provides evidence that NEP 24.11 is an important mediator of the degradation of both endogenous and exogenous glucagon in vivo.
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PMID:Neutral endopeptidase 24.11 is important for the degradation of both endogenous and exogenous glucagon in anesthetized pigs. 1512 40

Miniglucagon (MG), the C-terminal glucagon fragment, processed from glucagon by the MG-generating endopeptidase (MGE) at the Arg17-Arg18 dibasic site, displays biological effects opposite to that of the mother-hormone. This secondary processing occurs in the glucagon- and MG-producing alpha-cells of the islets of Langerhans and from circulating glucagon. We first characterized the enzymatic activities of MGE in culture media from glucagon and MG-secreting alphaTC1.6 cells as made of a metalloendoprotease and an aminopeptidase. We observed that glucagon is a substrate for N-arginine dibasic convertase (NRDc), a metalloendoprotease, and that aminopeptidase B cleaves in vitro the intermediate cleavage products sequentially, releasing mature MG. Furthermore, immunodepletion of either enzyme resulted in the disappearance of the majority of MGE activity from the culture medium. We found RNAs and proteins corresponding to both enzymes in different cell lines containing a MGE activity (mouse alphaTC1.6 cells, rat hepatic FaO, and rat pituitary GH4C1). Using confocal microscopy, we observed a granular immunostaining of both enzymes in the alphaTC1.6 and native rat alpha-cells from islets of Langerhans. By immunogold electron microscopy, both enzymes were found in the mature secretory granules of alpha-cells, close to their substrate (glucagon) and their product (MG). Finally, we found NRDc only in the fractions from perfused pancreas that contain glucagon and MG after stimulation by hypoglycemia. We conclude that MGE is composed of NRDc and aminopeptidase B acting sequentially, providing a molecular basis for this uncommon regulatory process, which should be now addressed in both physiological and pathophysiological situations.
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PMID:Miniglucagon (MG)-generating endopeptidase, which processes glucagon into MG, is composed of N-arginine dibasic convertase and aminopeptidase B. 1553 58

Conventionally, mesenchymal stem cells (MSC) are generated by plating cells from bone marrow (BM) or other sources into culture flasks and selecting plastic-adherent cells with fibroblastoid morphology. These cells express CD9, CD10, CD13, CD73, CD105, CD166, and other markers but show only a weak or no expression of the embryonic markers stage-specific embryonic antigen-4 (SSEA-4), Oct-4 and nanog-3. Using a novel protocol we prepared MSC from BM and non-amniotic placenta (PL) by culture of Ficoll-selected cells in gelatin-coated flasks in the presence of a serum-free, basic fibroblast growth factor (b-FGF)-containing medium that was originally designed for the expansion of human embryonic stem cells (ESC). MSC generated in gelatin-coated flasks in the presence of ESC medium revealed a four-to fivefold higher proliferation rate than conventionally prepared MSC which were grown in uncoated flasks in serum-containing medium. In contrast, the colony forming unit fibroblast number was only 1.5- to twofold increased in PL-MSC and not affected in BM-MSC. PL-MSC grown in ESC medium showed an increased surface expression of SSEA-4 and frizzled-9 (FZD-9), an increased Oct-4 and nestin mRNA expression, and an induced expression of nanog-3. BM-MSC showed an induced expression of FZD-9, nanog-3, and Oct-4. In contrast to PL-MSC, only BM-MSC expressed the MSC-specific W8B2 antigen. When cultured under appropriate conditions, these MSC gave rise to functional adipocytes and osteoblast-like cells (mesoderm), glucagon and insulin expressing pancreatic-like cells (endoderm), as well as cells expressing the neuronal markers neuron-specific enolase, glutamic acid decarboxylase-67 (GAD), or class III beta-tubulin, and the astrocyte marker glial fibrillary acidic protein (ectoderm). In conclusion, using a novel protocol we demonstrate that adult BM-and neonatal PL-derived MSC can be induced to express high levels of FZD-9, Oct-4, nanog-3, and nestin and are able of multi-lineage differentiation.
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PMID:Human placenta and bone marrow derived MSC cultured in serum-free, b-FGF-containing medium express cell surface frizzled-9 and SSEA-4 and give rise to multilineage differentiation. 1728 45

Plant cathepsin B-like cysteine protease (CBCP) plays a role in disease resistance and in protein remobilization during germination. The ability of animal cathepsin B to function as a dipeptidyl carboxypeptidase has been attributed to the presence of a dihistidine (His110-His111) motif in the occluding loop, which represents a unique structure of cathepsin B. However, a dihistidine motif is not present in the predicted sequence of the occluding loop of plant CBCP, as determined from cDNA sequence analysis, and the loop is shorter. In an effort to investigate the enzymatic properties of plant CBCP, which possesses the unusual occluding loop, we have purified CBCP from the cotyledons of daikon radish (Raphanus sativus) by chromatography through Sephacryl S-200, DEAE-cellulose, hydroxyapatite and organomercurial-Sepharose. The molecular mass of the enzyme was estimated to be 28 kDa by SDS/PAGE under reducing conditions. The best synthetic substrate for CBCP was t-butyloxycarbonyl Leu-Arg-Arg-4-methylcoumaryl 7-amide, as is the case with human cathepsin B. However, the endopeptidase activity of CBCP towards glucagon and adrenocorticotropic hormone showed broad cleavage specificity. Human cathepsin B preferentially cleaves model peptides via its dipeptidyl carboxypeptidase activity, whereas daikon CBCP displays both endopeptidase and exopeptidase activities. In addition, CBCP was found to display carboxymonopeptidase activity against the substrate o-aminobenzoyl-Phe-Arg-Phe(4-NO(2)). Daikon CBCP is less sensitive (1/7000) to CA-074 than human cathepsin B. Expression analysis of CBCP at the protein and RNA levels indicated that daikon CBCP activity in cotyledons is regulated by post-transcriptional events during germination.
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PMID:Purification and characterization of cathepsin B-like cysteine protease from cotyledons of daikon radish, Raphanus sativus. 1895 67

Oxyntomodulin (Oxm) is an intestinal peptide that inhibits food intake and body weight in rodents and humans. These studies used peptide analogs to study aspects of structure and function of Oxm, and the sensitivity of parts of the Oxm sequence to degradation. Analogs of Oxm were synthesized and studied using receptor binding and degradation studies in vitro. Their effects on food intake and conditioned taste avoidance were measured in vivo in rodents. Oxm breakdown by the enzyme dipeptidyl peptidase IV (DPPIV) was demonstrated in vitro and in vivo. In vitro degradation was reduced and in vivo bioactivity increased by inhibitors of DPPIV. Modifications to the N terminus of Oxm modulated binding to the glucagon-like peptide (GLP)-1 receptor and degradation by DPPIV. Modifications to the midsection of Oxm modulated binding to the GLP-1 receptor and degradation by neutral endopeptidase. These modifications also altered bioactivity in vivo. The C-terminal octapeptide of Oxm was shown to contribute to the properties of Oxm in vitro and in vivo but was not alone sufficient for the effects of the peptide. Elongation and acylation of the C terminus of Oxm altered GLP-1 receptor binding and duration of action in vivo, which may be due to changes in peptide clearance. An Oxm analog was developed with enhanced pharmaceutical characteristics, with greater potency and longevity with respect to effects on food intake. These studies suggest that Oxm is a potential target for antiobesity drug design.
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PMID:Investigation of structure-activity relationships of Oxyntomodulin (Oxm) using Oxm analogs. 1907 79


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