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)

Acetylated derivatives of glucagon have been prepared by reacting this hormone under various conditions with acetic anhydride. They have been chemically characterized by the use of a 14C-labeled reagent, by peptide mapping techniques following hydrolysis by pronase and chymotrypsin, and by spectroscopy. Acetylation in sodium acetate (pH 5.5) results in a full substitution of the alpha-amino group of the N-terminal histidyl residue, but in a partial (about 0.3 acetyl group per residue) substitution of the epsilon-amino group of the lysyl residue 12. The monosubstituted (on the alpha-amino group) and the disubstituted (on both amino groups) acetylated components have been separated by chromatography on DEAE-cellulose and CM-cellulose. Acetylation in sodium bicarbonate (pH 8.0) results in a complete substitution of both amino groups and of the hydroxyl groups of the tyrosyl residues 10 and 13. Complete deacetylation of the O-acetyltyrosyl residues occurs upon treatment with hydroxyl-amine. Mono, di and tetraacetylglucagon are homogeneous when analyzed by disc gel electrophoresis; di and tetrasubstituted derivatives show an increased mobility towards the anode. 125I-labeled derivatives of acetylglucagon show higher distribution coefficients in the aqueous two-phase dextran/poly(ethylene glycol) system than do similar derivatives of glucagon. Acetylation decreases in parallel the ability of glucagon to stimulate the activity of adenylate cyclase and to bind to its receptors in liver cell membranes of the rat. The biological potencies of the mono, di and tetrasubstituted derivates are, respectively, about 10, 1 and 0.1% that of native glucagon. The binding properties of the material dissociated from the acetylglucagon-receptor complex suggest that the reduction in biological activity results from a decrease in the intrinsic affinity of the modified glucagon for the receptors, as well as from the presence of small amounts of residual, unreacted glucagon. Studies with 125I-labeled derivatives of glucagon indicate that acetylation decreases the rate of association and increases the rate of dissociation of the hormone-receptor complex.
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PMID:Acetylglucagon: preparation and characterization. 0 Dec 70

The effects of the alpha-adrenergic agonist phenylephrine on the levels of adenosine 3':5'-monophosphate (cAMP) and the activity of the cAMP-dependent protein kinase in isolated rat liver parenchymal cells were studied. Cyclic AMP was very slightly (5 to 13%) increased in cells incubated with phenylephrine at a concentration (10(-5) M) which was maximally effective on glycogenolysis and gluconeogenesis. However, the increase was significant only at 5 min. Cyclic AMP levels with 10(-5) M phenylephrine measured at this time were reduced by the beta-adrenergic antagonist propranolol, but were unaffected by the alpha-blocker phenoxybenzamine, indicating that the elevation was due to weak beta activity of the agonist. When doses of glucagon, epinephrine, and phenylephrine which produced the same stimulation of glycogenolysis or gluconeogenesis were added to the same batches of cells, there were marked rises in cAMP with glucagon, minimal increases with epinephrine, and little or no changes with phenylephrine, indicating that the two catecholamine stimulated these processes largely by mechanisms not involving cAMP accumulation. DEAE-cellulose chromatography of homogenates of liver cells revealed two major peaks of cAMP-dependent protein kinase activity. These eluted at similar salt concentrations as the type I and II isozymes from rat heart. Optimal conditions for preservation of hormone effects on the activity of the enzyme in the cells were determined. High concentrations of phenylephrine (10(-5) M and 10(-4) M) produced a small increase (10 tp 16%) in the activity ratio (-cAMP/+cAMP) of the enzyme. This was abolished by propranolol, but not by phenoxybenzamine, indicating that it was due to weak beta activity of the agonist. The increase in the activity ratio of the kinase with 10(-5) M phenylephrine was much smaller than that produced by a glycogenolytically equivalent dose of glucagon. The changes in protein kinase induced by phenylephrine and the blockers and by glucagon were thus consistent with those in cAMP. Theophylline and 1-methyl-3-isobutylxanthine, which inhibit cAMP phosphodiesterase, potentiated the effects of phenylephrine on glycogenolysis and gluconeogenesis. The potentiations were blocked by phenoxybenzamine, but not by propranolol. Methylisobutylxanthine increased the levels of cAMP and enhanced the activation of protein kinase in cells incubated with phenylephrine. These effects were diminished or abolished by propanolol, but were unaffected by phenoxybenzamine. It is concluded from these data that alpha-adrenergic activation of glycogenolysis and gluconeogenesis in isolated rat liver parenchymal cells occurs by mechanisms not involving an increase in total cellular cAMP or activation of the cAMP-dependent protein kinase. The results also show that phosphodiesterase inhibitors potentiate alpha-adrenergic actions in hepatocytes mainly by a mechanism(s) not involving a rise in cAMP.
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PMID:Studies on the alpha-andrenergic activation of hepatic glucose output. II. Investigation of the roles of adenosine 3':5'-monophosphate and adenosine 3':5'-monophosphate-dependent protein kinase in the actions of phenylephrine in isolated hepatocytes. 0 57

Solubilization of myocardial adenylate cyclase abolished responsiveness to glucagon and catecholamines, two of the hormones which activate the membrane-bound enzyme. Adenylate cyclase freed of detergent by DEAE-cellulose chromatography continues to remain unresponsive to hormone stimulation. However, adding purified bovine brain phospholipids--phosphotidylserine and monophosphatidylinositol--restored responsiveness to glucagon and catecholamines, respectively. 125-i-glucagon binding appeared to be independent of phospholipid, since equal binding was observed in the presence or absence of detergent and in the presence or absence of phospholipids. Chromatography of the solubilized preparation on Sephadex G-100 WAS CHARACTERIZED BY 125-I-glucagon binding and fluoride-stimulatable adenylate cyclase activity appearing in the fractions consistent with the void volume, suggesting a molecular weight greater than 100,000 for the receptor-adenylate cyclase complex. Prior incubation of the binding peak with 125-I-glucagon and rechromatography of the bound glucagon on Sephadex G-100 shifted its elution to a later fraction consistent with a smaller-molecular-weight peak. The molecular weight of this material was 24,000 to 28,000, as determined by SDS polyacrylamide gel electrophoresis. The latter findings are consistent with a dissociable receptor site for glucagon on myocardial adenylate cyclase.
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PMID:Glucagon and adenylate cyclase: binding studies and requirements for activation. 16 84

The protein-bound cyclic AMP and the activity of cytosolic protein kinases in the presence and absence of cyclic AMP were determined in rat liver up to 2h after injection of glucagon. On the basis of the different salt-sensitivities of the activated cyclic AMP-dependent proteinkinases I and II, an activation of protein kinase II restricted to the high cyclic AMP concentrations present in the first 30 min after hormone injection was found. Essentially the same result was obtained by chromatographic analysis on DEAE-cellulose of liver cytosol from untreated rats and from rats killed at 2 and 60 min after glucagon injection. Protein kinase II activation was only detected at 2 min after injection. In contrast, the cyclic AMP-dependent protein kinase I was found to be nearly totally activated at 2 min and to be still almost as active at 60 min after the hormone stimulus, whereas the amount of bound cyclic AMP and the activation of total cytosolic protein kinases had fallen to two-thirds of their maximal values during this time period. A third cyclic AMP-independent protein kinase, which co-chromatographed with protein kinase type II, could be clearly distinguished from the two cyclic AMP-dependent kinases by use of the heat-stable inhibitor from bovine muscle, which totally inhibited the cyclic AMP-dependent enzymes, but stimulated the cyclic AMP-independent protein kinase.
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PMID:Differential activation of type-I and type-II adenosine 3':5'-cyclic monophosphate-dependent protein kinases in liver of glucagon-treated rats. 20 61

Because in the dog, the gastric fundus contains the largest amount of glucagon immunoreactivity (IRG), the IRG of mucosal scrapes of 105 canine stomachs was extracted by acid-ethanol and then precipitated by ether-ethanol. The IRG recovered was measured by antisera 30K, specific for glucagon and K-4023, which cross-reacts with glucagon-like immunoreactivity. Extracts of mucosa of stomach fundus were further purified by gel filtration on Bio-Gel P-30 in 3M acetic acid. One pooled fraction corresponding to marker pancreatic glucagon in its elution volume was then gel-filtered on Bio-Gel P-30 in 0.05 M NH(4)HCO(3) and yielded one IRG peak, which, however, showed three immunoreactive components on polyacrylamide disc gel electrophoresis in urea. In addition, antiserum K-4023 reacted more strongly with that peak than antiserum 30K indicating the presence of glucagon-like immunoreactivity in this fraction. Subsequent ion-exchange column chromatography on DEAE-Sephadex A-25 and then CM-Bio-Gel A allowed purification to a single protein band on disc gel electrophoresis reacting equally to both antisera 30K and K-4023. 1.5 mug of purified gastric glucagon was obtained and its biological effects were compared to those of pancreatic glucagon in isolated rat hepatocytes. When immuno-equivalent amounts (300-2,500 pg/ml) of either type of glucagon were used, the same biological responses with respect to glycogenolysis and gluconeogenesis as well as urea, lactate, and pyruvate production were observed. Liver cyclic AMP was also raised to the same extent by either one of these hormones. We conclude that this moiety of gastric IRG is apparently identical to pancreatic glucagon because (a) their molecular weights, elution properties in ion exchange chromatography, and their electrophoretic mobility are indistinguishable and (b) both hormones elicited identical biological effects in isolated rat hepatocytes.
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PMID:Identical biological effects of pancreatic glucagon and a purified moiety of canine gastric immunoreactive glucagon. 42 72

Rat hepatic pyruvate kinase (type L) has been purified to homogeneity by a simple, rapid procedure involving DEAE-cellulose chromatography and elution from a blue Sepharose column. The enzyme was homogeneous by the criteria of sodium dodecyl sulfate disc gel electrophoresis, had a subunit molecular weight of 57,000, and a specific activity of 558 units/mg of protein at 30 degrees. In order to test whether the enzyme is phosphorylated in vivo, rats were injected with radioactive inorganic phosphate. Incorporation into pyruvate kinase was determined after purification of the enzyme to homogeneity as well as after specific immunoprecipitation of the enzyme from partially purified preparations. Sodium dodecyl sulfate disc gel electrophoresis revealed that 32P was incorporated into the enzyme in both cases. Glucagon administration in vivo resulted in a 200 to 300% increase in the incorporation of 32P into the enzyme which was correlated with an inhibition of enzyme activity and an elevation of hepatic levels of cyclic AMP. These results represent the first demonstration of in vivo phosphorylation of a hepatic glycolytic enzyme and strongly support the hypothesis that glucagon regulates pyruvate kinase activity, at least in part, by a phosphorylation mechanism.
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PMID:Stimulation of glucagon of in vivo phosphorylation of rat hepatic pyruvate kinase. 62 Nov 97

The effect of metabolic or hormonal status on CoA biosynthesis was studied by comparing the rates of incorporation of [14C]-panthothenate into CoA in fasted and glucose-fed rats. Rat hearts and livers were freeze-clamped 1.5 hours after intravenous injection of [14C] pantothenate. CoA, pantothenate, and other pathway intermediates were separated by chromatography of tissue extracts on DEAE cellulose paper. Compared to the fasted rats, rats forced-fed glucose 0.5 hours before pantothenate injection 69% lower incorporation of radioactivity into CoA in liver, a 69% lower specific radioactivity of liver CoA, a 63% lower specific radioactivity of liver mitochondrial CoA, and a 44% lower incorporation of radioactivity in CoA in heart. The accumulation of labeled pathway intermediates was negligible. The cysteine content of liver was equal for the two conditions. There was no difference in level of unacylated CoASH for fasted and glucose-fed rats, suggesting that hormonal effects on degree of CoA acylation are not involved in this regulator mechanism. The specific radioactivities and concentrations of pantothenate in heart or liver were also nearly equal for the two conditions, so the regulatory mechanism does not involve hormonal effects on pantothenate uptake into tissues. Thus, the effect of glucose-feeding (and related changes of metabolite and insulin-glucagon levels) on the incorporation o[14C] pantothenate into CoA is exerted at a locus on the biosynthetic and/or degradative pathway between pantothenate and CoA.
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PMID:The effect of metabolic state on incorportion of [14C] pantothenate into CoA in rat liver and heart. 64 1

Glucagon was iodinated with the lactoperoxidase method at pH 10.0 in the presence of propylene glycol using a substitution of 0.3 g-atom I/mol glucagon. Under these conditions the reactivity of the iodine to tyrosine at position 13 is found to be 4-fold that of the tyrosine at position 10. The amount of diiodotyrosine was less than one-twentieth that of the monoiodotyrosine at either tyrosine residue. Relatively pure monoiodo[125I]tyrosine-13-glucagon can be separated from other iodoglucagons by means of DEAE-chromatography. Such a homogeneous preparation with a known position of the iodine makes it possible to study a specific interaction between the monoiodoglucagon and the glucagon antisera or the glucagon receptor.
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PMID:Preparation of monoiodotyrosine-13-glucagon. 85 7

1. An anionic and a cationic chymotrypsin (EC 3.4.21.1) were isolated from the pancreas glands of the moose (Alces alces) and elk (Cervus elaphus). The A and B chymotrypsins from each species were purified to homogeneity by (NH4)2SO4 fractionation, affinity chromatography on 4-phenylbutylamine-Sepharose and ion-exchange chromatography on DEAE- and CM-cellulose. 2. The molecular weight and pH optimum of each chymotrypsin were similar to those of the corresponding ox A and B chymotrypsins. 3. The substrate specificities of the chymotrypsins were investigated by digestion of glucagon and the oxidized B chain of insulin. The primary specificity of each chymotrypsin for aromatic amino acid residues was further established by determining the Km and kcat for the hydrolysis of a number of synthetic amino acid ester substrates. 4. The amino acid composition and total number of residues of moose and elk chymotrypsin A were similar to those of ox chymotrypsin A. An even greater similarity was observed among the B chymotrypsins of the three species. 5. The A chymotrypsins of moose and elk were fragmented to their constituent 'A', 'B' and 'C' polypeptide chains by succinylation (3-carboxypropionylation), reduction and alkylation of the native enzymes. In each case, the two major chains ('B' and 'C') were separated and isolated. By comparison of the amino acid compositions of moose, elk and oxy 'B' and 'C' chains, a greater difference was observed among the three A chymotrypsins than was suggested by the amino acid compositions of the native enzymes alone. 6. Peptides were isolated from the disulphide bridge and active-site regions of the A and B chymotrypsins of moose and elk by diagonal peptide-'mapping' techniques. From the amino acid compositions of the isolated peptides (assuming maximum homology) and from a comparison of diagonal peptide 'maps', there was established a high degree of primary-structure identity among the mooae, elk and ox chymotrypsins. Tentative sequences were deduced for the peptides isolated by diagonal peptide 'mapping'. 7. Details of the isolation procedures of the moose and elk chymotrypsins A and B and the amino acid analyses of some peptides obtained by diagonal peptide 'mapping' have been deposited as Supplementary Publication SUP 50064 (27 pages) at the British Library Lending Division, Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1976) 153, 5.
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PMID:Chymotrypsins from the deer (Cervidae) family. Isolation, partial characterization and primary-structure studies of chymotrypsins A and B from both moose (Alces alces) and elk (Cervus elaphus) pancreas. 94 18

Iodinated derivatives of glucagon containing an average of 1 to 5 g-atoms of 127I per mol have been prepared by reacting the hormone with increasing amounts of iodine monochloride. Their iodoamino acid composition has been determined by ion-exchange chromatography and electrophoresis, following hydrolysis by pronase. Iodination of the two tyrosyl residues occurs first and is nearly complete after addition of a 4-fold molar excess of ICl. Iodination of the single histidyl residue is a later event and does not exceed an average of one atom per residue. Hydrolysis of iodoglucagon by trypsin and subsequent separation of the iodotyrosyl peptides shows that iodine is equally distributed between tyrosyl residues 10 and 13. Crude iodoglucagon containing an average of 1 g-atom of iodine per mol has been resolved into several components of differing iodine content and iodoamino acid composition by chromatography on DEAE-cellulose. Monoiodoglucagon isolated by this procedure shows a single band when analyzed by polyacrylamide gel electrophoresis. Iodoglucagons containing an average of 1 to 4 g-atoms of iodine per mol are more potent than native glucagon in their ability to stimulate adenylate cyclase activity and to bind to glucagon receptors of liver cell membranes of the rat. The maximal increase in biological potency occurring upon iodination is about 5-fold with respect to adenylate cyclase activity, and 2-fold with respect to binding to receptors; tetra and triiodinated derivatives show, respectively, the highest potency. Similar effects occur whether inactivation by liver membranes is inhibited or not, indicating an enhancement in the intrinsic affinity of iodoglucagon for the receptors. Iodination beyong 4 g-atoms per mol slightly decreases the affinity of the hormone for adenylate cyclase and for the receptors. Iodination causes a 2-20 fold decrease in the ability of liver plasma membranes and of blood plasma to inactivate glucagon in vitro; these effects correlate with the degree of iodination. With liver microsomal membranes, a decrease in glucagon inactivation occurs only at iodine contents exceeding 4 g-atoms per mol, and lower degrees of iodination result in opposite effects. Monoiodination causes a 4-6-fold increase in the plasma concentration of glucagon within the first 18 min following a single intrvenous injection of the hormone to rats. More extensive iodination results, in addition, in a marked decrease in the rate of dissappearance of glucagon from the blood. The immunological reactivity of glucagon is little affected by monoidination, but strongly depressed by higher degrees of iodination...
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PMID:Iodoglucagon. Preparation and characterization. 114 Feb 1


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