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)

Insulin and glucagon degrading proteinase (EC 3.4.23.5) purified from rat liver cytosol was characterized using radioiodinated insulin and glucagon as substrates. Maximum activity for breakdown of both hormones was found at pH 8.1. Thiol blocking reagents as well as indole derivatives inhibit the proteinase, whereas pepstatin, leupeptin, bestatin, elastatinal, antipain, chymostatin and phosphoramidon do not have any effect. Although the Km values and maximal velocities of insulin and glucagon breakdown deviate strongly from each other, the specificity constants (kcat/Km) for both substrates are nearly identical. The insulin and glucagon degrading proteinase, known as a thiol-dependent enzyme, was found to be also a metallo enzyme. Chelating agents, such as EDTA, EGTA, bipyridine and o-phenanthroline show a concentration dependent inhibition. The strongest inhibitor found was o-phenanthroline. Zn++, Co++, Mn++, and to a smaller extent Cd++ and Fe++, are capable of preventing the o-phenanthroline mediated inhibition. Removal of the protein-bound metal(s) results in a nearly total and irreversible loss of enzymatic activity.
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PMID:The insulin and glucagon degrading proteinase of rat liver: a metal-dependent enzyme. 637 97

In red cell lysates, three soluble proteases hydrolyze insulin at pH 8.5. One of these enzymes was purified to homogeneity by conventional chromatographic techniques. It appears to be a metalloprotease since it is inhibited by EDTA, o-phenanthroline, and 8-hydroxyquinoline, the metal-depleted enzyme can be reactivated by micromolar levels of Zn2+, Co2+, or Mn2+, and it is not inhibited by reagents specific for carboxyl, serine or thiol proteases. This enzyme has an apparent molecular weight of 300,000 +/- 25,000, and electrophoresis in sodium dodecyl sulfate indicates a single band with an Mr = 115,000 +/- 10,000. End group analysis and automated Edman degradation of the products of proteolysis showed that it is an endoprotease which cleaves on the NH2-terminal side of large hydrophobic amino acids. Although various small polypeptides with Mr = 2300-3500 are hydrolyzed (e.g. insulin chains, glucagon, and calcitonin), a variety of larger proteins are not degraded (e.g. casein and globin). The latter proteins, however, are converted to substrates for the metalloprotease by digestion with the ATP-stimulated endoprotease from erythrocytes. Thus, the metalloprotease may play a role in the ATP-dependent pathway for degrading proteins with abnormal structures and could account in part for the o-phenanthroline sensitivity of this process. A similar enzyme is found in humans, rabbits, and rats and is cytosolic in all tissues which have been examined including erythrocytes, reticulocytes, liver, kidney, brain, and skeletal muscle.
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PMID:A high molecular weight metalloendoprotease from the cytosol of mammalian cells. 640 23

Carbamylcholine produced a concentration-dependent stimulation of labelling of phosphatidylinositol and phosphatidic acid in rat islets of Langerhans following preincubation with 32PO43(-). The time course of these effects suggested that the initial action of carbamylcholine was to stimulate phosphatidic acid production, presumably by causing hydrolysis of phosphatidylinositol. This conclusion was substantiated by experiments in which islet phospholipids were pre-labelled with [3H]arachidonic acid. Under these conditions, carbamylcholine caused a loss of radioactivity from phosphatidylinositol, together with an increase in labelling of phosphatidic acid. The effects of carbamylcholine on islet phospholipid labelling were not dependent upon the presence of added Ca2+, but were abolished by EDTA and by atropine. An apparent stimulation of phosphatidylinositol and phosphatidic acid metabolism was also induced by cholecystokinin-pancreozymin, whereas glucagon, arginine, glibenclamide and thyrotropin had no significant effect. The data suggest that enhanced activity of the so-called phosphatidylinositol cycle may be an important event in regulating secretory activity of islets in response to certain neurotransmitter and hormonal stimuli. Furthermore, the results are compatible with the hypothesis that increased phospholipid metabolism may play a role in the modulation of ionic fluxes during stimulation by such agents.
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PMID:Phosphatidylinositol and phosphatidic acid metabolism in rat pancreatic islets in response to neurotransmitter and hormonal stimuli. 640 24

The conversion of proglucagon and proinsulin by secretory granules isolated from both prelabeled and unlabeled anglerfish islets was investigated. Either granules isolated from tissue labeled with [3H]tryptophan and [14C]isoleucine or [35S]cysteine, or lysed granules from unlabeled tissue to which exogenously labeled prohormones had been added were incubated under various conditions. Acetic acid extracts of these granule preparations were analyzed for prohormone and hormone content by gel filtration. Both prelabeled and lysed, unlabeled secretory granules converted radiolabeled precursor peptides (Mr 8,000-15,000) to labeled insulin and glucagon. The accuracy of the cleavage process was established by demonstrating comigration of products obtained from in vitro cleavage with insulin and glucagon extracted from intact islets using electrophoresis and high-pressure liquid chromatography (HPLC). The pH optimum for granule-mediated conversion was found to be in the range of pH 4.5-5.5. Conversion of both proglucagon and proinsulin by secretory granules was significantly inhibited in the presence of antipain, leupeptin, p-chloromercuribenzoate (PCMB) or dithiodipyridine (DDP) but not chloroquine, diisopropyl fluorophosphate, EDTA, p-nitrophenyl guanidinobenzoate, soybean trypsin inhibitor, or N-p-tosyl-L-lysine chloromethyl ketone HCl. The inhibitory action of PCMB and DDP was reversed in the presence of dithiothreitol. Both membranous and soluble components of the secretory granules possessed significant converting activity. HPLC and electrophoretic analysis of cleavage products demonstrated that the converting activities of the membranous and soluble components were indistinguishable. The amount of inhibition of proinsulin and proglucagon conversion caused by 600 micrograms/ml porcine proinsulin was significantly lower than that caused by the same concentration of unlabeled anglerfish precursor peptides. These results indicate that the proinsulin and proglucagon converting enzyme(s) in the anglerfish pancreatic islet is a unique intracellular thiol proteinase(s) that may be granule membrane-associated and may require the presence of prohormone sequences in addition to the dibasic residues at cleavage sites for substrate recognition and/or binding.
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PMID:Characterization of proinsulin- and proglucagon-converting activities in isolated islet secretory granules. 702 70

1. Injection of rats with glucagon leads to an increased effective activity of glutaminase in subsequently isolated liver mitochondria. 2. This effect of glucagon is manifested as a decreased requirement of glutaminase for phosphate in the presence of HCO3-. The HCO3--concentration-dependence is unchanged. 3. The effect of glucagon is lost on disruption of the mitochondria. 4. In accordance with previous reports, incubation of mitochondria in hypo-osmotic media also increases the effective activity of glutaminase. Glucagon increases glutamine hydrolysis at intermediate osmolarities of the suspending medium, but does not affect glutaminase activity when it is already maximally activated by hypo-osmotic conditions. 5. From this and previous work, it seems that hypo-osmotic incubation conditions, EDTA and glucagon may all activate glutaminase by a common mechanism. It is postulated that this mechanism involves modification of the interaction of glutaminase with the mitochondrial inner membrane.
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PMID:Increased activity of phosphate-dependent glutaminase in liver mitochondria as a result of glucagon treatment of rats. 730 82

A periplasmic insulin-cleaving proteinase (ICP), purified to its electrophoretic homogeneity in the SDS-PAGE from the Gram-negative bacterium Acinetobacter calcoaceticus, was examined and compared in its properties with the protease III (protease Pi, pitrilysin, EC 3.4.99.44) of Escherichia coli and the insulin-destroying proteinase (IDE, insulinase, EC 3.4.99.45) from eucaryotes. The enzyme was proven to be a metalloprotease like protease III and IDE, as was shown by the inhibitory effects exerted by EDTA and o-phenanthroline. Furthermore, dialysis against EDTA and o-phenanthroline led to a complete loss of activity, which could be restored by addition of Co2+, and, to a lesser extent, but at a lower metal ion concentration by Zn2+. Similar to protease III and IDE, ICP prefers the cleavage of small polypeptides (insulin, insulin B-chain, glucagon) to the cleavage of proteins (casein, human serum albumin, globin) and was inactive against synthetic amino acid derivates (esters, p-nitranilides, and furoylacroleyl substrates) of subtilisin, thermolysin, trypsin, and chymotrypsin. The peptide-bond-specificity of the ICP in the cleavage of the oxidized insulin B-chain was investigated and the results were compared to the specificity of protease III of E. coli, IDE, protease-24,11, and thermolysin. Cleavage sites in the oxidized insulin B-chain generated by ICP are Asn3-Gln4, His10-Leu11, Ala14-Leu15, Leu17-Val18, Gly23-Phe24, Phe24-Phe25, and Phe25-Tyr26. Principally, ICP cleaves between hydrophobic amino acids and amides. The ICP shares one of the only two cleavage sites with the protease III and four sites with the IDE.
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PMID:A periplasmic insulin-cleaving proteinase (ICP) from Acinetobacter calcoaceticus sharing properties with protease III from Escherichia coli and IDE from eucaryotes. 773 84

The acidic glucagon-degrading activity of hepatic endosomes has been attributed to membrane-bound forms of cathepsins B and D. Endosomal lysates processed full-length nonradiolabeled glucagon to 32 different peptides that were identified by amino acid analysis and full-length sequencing. These indicated C-terminal carboxypeptidase, endopeptidase as well as N-terminal tripeptidyl-aminopeptidase activities in endosomes. Glucagon proteolysis was inhibited 95% by E-64 and pepstatin A, inhibitors of cathepsins B and D, respectively. This was confirmed by the pH 6-dependent chemical cross-linking of [125I]iodoglucagon to a polypeptide of 30 kDa, which was immunodepleted by polyclonal anti-cathepsin B antibody, and the removal of greater than 80% of glucagon-degrading activity by polyclonal antibodies to cathepsins B and D. By similar criteria, insulin-degrading enzyme was ruled out as a candidate enzyme for endosomal proteolysis of glucagon. Lysosomal contamination was unlikely since all forms of cathepsin B in endosomes, i.e. the major 45-kDa inactive precursor as well as the lesser amounts of the 32- and 28-kDa active forms, were tightly bound to endosomal membranes. Furthermore the mature 29-kDa single-chain and 22-kDa heavy-chain forms of cathepsin L were undetectable in endosomes, although high levels of the 37-kDa proform were observed. Membrane association of the cathepsins B and D was not to the mannose 6-phosphate receptor since association was unaffected by mannose 6-phosphate and/or EDTA, thereby indicating a distinct endosomal receptor. Hence, a pool of active cathepsins B and D as well as a poorly defined tripeptidyl aminopeptidase is maintained in endosomes by selective membrane retention. These hydrolases degrade glucagon internalized into liver parenchyma early in endocytosis.
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PMID:Proteolysis of glucagon within hepatic endosomes by membrane-associated cathepsins B and D. 779 82

In order to study the cellular mechanisms involved in peptide YY (PYY) and truncated glucagon-like peptide 1 (TGLP1) release, a model of rat intestinal cells dispersed with collagenase/EDTA and enriched for L-cells by counterflow elutriation was developed. Elutriation significantly increased in the harvested cells the concentration of PYY (828 +/- 97 vs 151 +/- 16 fmol/10(6) cells) and TGLP1 (1,094 +/- 109 vs 167 +/- 20 fmol/10(6) cells), and brought the contribution of L-cells to 4-5% of the total cell population. Forskolin (1-10 microM) and dibutyryl cyclic AMP (dbcAMP, 1-5 mM) increased over an 1-h period PYY and TGLP1 secretion, with a maximal rate at 5 microM forskolin (232% and 250% of basal, respectively) and at 5 mM dbcAMP (347% and 234% of basal, respectively). Furthermore, 3-isobutylmethyl xanthine (IBMX, 1 mM) increased PYY (226% of basal) and TGLP1 (198% of basal) secretion. A combination of both 10 microM forskolin and 1 mM IBMX stimulated in an additive manner PYY (389% of basal) and TGLP1 (393% of basal) secretion. TPA (12-0-tetradecanoylphorbol-13-acetate, 0.1-1 microM) dose-dependently increased the secretion of PYY and TGLP1 (maximal release at 328% and 326%, respectively), whereas 4 alpha-phorbol was ineffective. Ionomycin (1-5 microM) and thapsigargin (0.1-5 microM) produced a dose-dependent increase in PYY and TGLP1 release (272% and 337% of basal for 5 microM ionomycin; 342% and 339% of basal for 5 microM thapsigargin, respectively). At gel chromatography, the immunoreactive PYY and TGLP1 material in cell extracts and in release medium co-eluted with the respective synthetic peptides.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Secretion of peptide YY and truncated glucagon-like peptide 1 by isolated intestinal cells in rats]. 781 61

A general aminopeptidase (St-PepN) was purified from an intracellular extract of Streptococcus salivarius ssp. thermophilus CNRZ 302 by ion-exchange chromatography and hydrophobic interaction chromatography. Gel electrophoresis of the purified enzyme in denaturing or nondenaturating conditions showed a single protein band. The enzyme is a monomer with a molecular mass of 97 kDa. Its activity is maximal at pH 7 and 36 degrees C and is completely abolished by CuCl2 and ZnCl2. The enzyme is strongly inhibited by metal-chelating reagents, such as EDTA and o-phenanthroline, which suggests that St-PepN is a metalloenzyme. The enzyme showed activity toward p-nitroanilide derivatives or dipeptides and tripeptides and showed a preference for hydrophobic or basic amino acids at the N-terminal position. Longer peptide chains, such as the B-chain of insulin, glucagon, or peptides generated by the hydrolysis of caseins, were degraded, too. The sequence of the first 21 residues of the mature enzyme was determined and showed high homology with that of the aminopeptidase PepN isolated from Lactococcus lactis ssp. cremoris Wg2. The properties of the enzyme are compared with those of corresponding enzymes of other species of lactic acid bacteria.
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PMID:Purification and characterization of a general aminopeptidase (St-PepN) from Streptococcus salivarius ssp. thermophilus CNRZ 302. 783 77

The metabolism of glucagon-like peptide-1 (GLP-1) has not been studied in detail, but it is known to be rapidly cleared from the circulation. Measurement by RIA is hampered by the fact that most antisera are side-viewing or C-terminally directed, and recognize both intact GLP-1 and biologically inactive. N-terminally truncated fragments. Using high pressure liquid chromatography in combination with RIAs, methodology allowing specific determination of both intact GLP-1 and its metabolites was developed. Human plasma was shown to degrade GLP-1-(7-36)amide, forming an N-terminally truncated peptide with a t1/2 of 20.4 +/- 1.4 min at 37 C (n = 6). This was unaffected by EDTA or aprotinin. Inhibitors of dipeptidyl peptidase-IV or low temperature (4 C) completely prevented formation of the metabolite, which was confirmed to be GLP-1-(9-36)amide by mass spectrometry and sequence analysis. High pressure liquid chromatography revealed the concentration of GLP-1-(9-36)amide to be 53.5 +/- 13.7% of the concentration of endogenous intact GLP-1 in the fasted state, which increased to 130.8 +/- 10.0% (P < 0.01; n = 6) 1 h postprandially. Metabolism at the C-terminus was not observed. This study suggests that dipeptidyl peptidase-IV is the primary mechanism for GLP-1 degradation in human plasma in vitro and may have a role in inactivating the peptide in vivo.
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PMID:Degradation of glucagon-like peptide-1 by human plasma in vitro yields an N-terminally truncated peptide that is a major endogenous metabolite in vivo. 788 56


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