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)

Cathepsins M and B from rabbit liver lysosomes were separated by chromatography on Ultrogel AcA34 at low ionic strength and purified to homogeneity, and their catalytic and molecular properties were compared. Cathepsin M was relatively inactive with synthetic peptide substrates. Thus, it hydrolyzed benzoyl arginine naphthylamide at only one-fifth the rate observed with cathepsin B, and no activity was detected with Gly-Phe naphthylamide which is a relatively good substrate for cathepsin B. On the other hand, cathepsin M exhibited a preference for protein substrates. It was more active than cathepsin B in catalyzing the inactivation of the following enzymes: rabbit muscle or liver fructose-1,6-bisphosphate aldolases, rabbit liver fructose-1,6-bisphosphatase and pyruvate kinase, yeast glucose-6-phosphate dehydrogenase, and rabbit muscle glyceraldehyde-3-phosphate dehydrogenase. With glucagon as substrate, both enzymes showed similar peptidyl dipeptidase activities with some minor differences in peptide bond specificity. Cathepsins M and B are similar in size, with apparent molecular weights of 30,200 for cathepsin M and 28,800 for cathepsin B, and in amino acid composition and carbohydrate content. Each contains approximately 2-3 equivalents/mol glucosamine, 3 equivalents/mol mannose, and no fucose or galactosamine. They also show similar microheterogeneity in sodium dodecylsulfate-gel electrophoresis and isoelectric focusing; this microheterogeneity is probably related to differences in glycosylation. Extensive homology in primary structure for the two proteins was indicated by the similar patterns of peptides formed on digestion with trypsin.
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PMID:Purification and properties of rabbit liver cathepsin M and cathepsin B. 406 7

Bovine glucagon, a polypeptide of 29 amino acids, was immunogenic in guinea pigs. The immunologic determinants of glucagon were investigated using isolated tryptic peptides of the hormone. Antibodies from virtually all of more than two dozen animals had specificity primarily for the amino-terminal heptadecapeptide (NM) and showed little or no binding with the carboxy-terminal undeca- and dodecapeptides (C). The smallest synthetic peptide of a series initiated at residue 16 which measurably bound antibody comprised residues 5-16 of glucagon. In cellular immune assays, both NM and C elicited delayed cutaneous reactions and inhibited the migration of peritoneal cells from immune animals. However, only intact glucagon and its C fragment stimulated lymphoid cells to synthesize DNA. While glucagon was somewhat more active than C, the addition of NM to C did not enhance its transforming activity. The smallest synthetic carboxy-terminal peptide with discernible transforming activity comprised residues 19-29 of glucagon. In both native and synthetic C peptide preparations, the undecapeptide was generally more active than the dodecapeptide, although cells from different animals gave different response patterns. The difference between the two is the presence of arginine at the amino-terminus of the peptide chain. Thus, the recognition specificity of populations of antigen-reactive cells from different animals displays a variation which is at least superficially analogous to that of populations of antibody molecules. In limited experiments using NM and C peptides as immunogens, neither gave rise to delayed hypersensitivity or to glucagon-binding circulating antibody, following a regimen which invariably provoked these responses when glucagon itself served as the immunogen. These results indicate that glucagon was cleaved by trypsin along functional lines into two parts, one of which housed the major antigenic determinant and the other of which carried the major immunogenic determinant, and they are highly compatible with a two-cell mechanism of immune induction. An apparent dissociation between the capacity to provoke delayed hypersensitivity reactions and to transform antigen-reactive cells in culture was observed.
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PMID:The functional dissection of an antigen molecule: specificity of humoral and cellular immune responses to glucagon. 410 86

1. A neutral peptidase, previously shown to be located in the brush border of the proximal tubule, and assayed by its ability to hydrolyse [(125)I]iodoinsulin B chain was purified from rabbit kidney. 2. The starting material for the purification was a microsomal pellet prepared from a homogenate of cortical tissue. The membrane-bound enzymes were solubilized by treatment with toluene and trypsin. About half the neutral peptidase activity was released by this treatment in a form that no longer sedimented with the microsomal pellet and which penetrated polyacrylamide gels when subjected to disc electrophoresis. Other treatments with detergents or proteolytic enzymes either inactivated the peptidase or failed to convert it into a genuinely soluble form. 3. Chromatography with successive columns of Sephadex G-200, DEAE-cellulose and hydroxyl-apatite yielded an enzyme that was free of other brush-border peptidase activities and which was homogeneous on disc electrophoresis and ultracentrifugation. 4. The purified enzyme attacked [(125)I]iodoglucagon at a rate comparable with that for [(125)I]iodoinsulin B chain. It did not appear to attack proteins (insulin, albumin and casein) that had been similarly iodinated. 5. Unlabelled insulin B chain and unlabelled glucagon were substantially hydrolysed by the endopeptidase, whereas insulin and albumin released only trivial amounts of ninhydrin-reacting material. The resistance of insulin to attack by endopeptidase, even after prolonged incubation, was confirmed by biological and immunoassay. 6. The specificity of the peptidase was determined by analysis of the products after incubating unlabelled insulin B chain, and some oligopeptide substrates, including pentagastrin, with the enzyme. All of the bonds readily cleaved were those involving the alpha-amino group of hydrophobic residues, i.e. x-Leu-, x-Val-, x-Tyr-, x-Phe- and x-Met-, provided that the residues were not C-terminal. 7. The enzyme showed only endopeptidase activity. Substrates suitable for aminopeptidases, carboxypeptidases or esterases were not attacked.
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PMID:The purification and specificity of a neutral endopeptidase from rabbit kidney brush border. 442 92

We have developed a reverse-phase HPLC method to purify 125I-labeled products resulting from the chloramine-T-based iodination of glucagon and have used the products [(125I)iodoTyr13]glucagon, [(125I)iodoTyr10,13]glucagon, and [(125I)iodoTyr10]glucagon) to study the receptor binding of glucagon and the cell-mediated metabolism of the hormone by isolated canine hepatocytes. The extent of binding of the three labeled glucagons to cell receptors differed at steady state (8.5, 11.9, and 12.6% of the three peptides, respectively, becoming cell-associated), but each of the labeled glucagons approached steady state binding at the same rate. Further, unlabeled glucagon competed for the binding of each of the labeled peptides in parallel under steady state conditions, and each of the peptides showed potent activity in inhibiting [14C]fructose incorporation into glycogen. Gel filtration of the acetic acid-extracted, cell-associated products of radiolabeled glucagon binding revealed 10-20% of the material as a shoulder on the descending limb of the peak of hormone for each of the three labeled peptides. Trypsin digestion of the lower molecular weight peptide derived from [(125I)iodoTyr13]glucagon resulted in a fragment containing residues 13 to 17 as the only detectable radiolabeled product. On the other hand, trypsin digestion of the analogous peptide derived from [(125I)iodoTyr10]glucagon revealed, in addition to the radiolabeled fragment containing residues 1 to 12, a major fragment identified by radiosequence analysis to contain residues 4 to 12 and a minor fragment identified to contain residues 7 to 12. We conclude that (a) notwithstanding apparent differences in affinities exhibited by [(125I)iodoTyr13]glucagon, [(125I)iodoTyr10,13]glucagon, and [(125I)iodoTyr10]glucagon for binding to canine hepatocytes, the interactions of all three peptides with the glucagon receptor are functionally equivalent, and (b) the cell-mediated metabolism of receptor-bound glucagon involves the formation of hormone-derived peptides in which the biologically important NH2-terminal region of the hormone has been modified by limited proteolytic cleavage.
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PMID:Receptor binding and cell-mediated metabolism of [125I]monoiodoglucagon by isolated canine hepatocytes. 608 19

Previously, we reported that pancreatic acini have specific receptors for the insulin-like growth factors (IGF) I and II. We now report that the binding of 125I-labeled IGF II to mouse pancreatic acini is maximally increased by 100 nM insulin (51%) and is maximally reduced by 10 nM cholecystokinin octapeptide (CCK8) (34%), but is not affected by other regulatory peptides such as somatostatin or glucagon. Since many polypeptide hormones are internalized, we determined whether this regulation of IGF II binding occurred via a change in internalization. Acid washing or trypsinization has been shown to remove surface-bound hormone while the acid- or trypsin-resistant radioactivity represents internalized radioligand. Insulin increased and CCK8 decreased the internalization of IGF II as determined by these techniques. Studies of IGF II binding to acini at low temperature (15 degrees C) and binding to particulate fractions from acini were also consistent with the effect of insulin to increase and CCK8 to decrease the internalization of IGF II. When insulin and CCK8 were added together, the inhibitory effect of CCK8 predominated, indicating that CCK8 acted distal to the effect of insulin. Several lines of evidence suggest that this effect of CCK8 was via the CCK receptor and was mediated via a change in intracellular Ca2+: the effect of CCK8 on inhibiting IGF II binding was blocked by the cholecystokinin antagonist N2,O2'-dibutyryl cGMP; the cholinergic agent carbachol (1-100 microM), which acts through the muscarinic receptor to increase intracellular Ca2+, also inhibited IGF II binding; the Ca2+ ionophore A23187 (1-5 microM) mimicked the effects of CCK8 and carbachol. These data indicate, therefore, that CCK8 and possibly insulin may regulate the internalization of IGF II via intracellular Ca2+. Moreover, the data raise the possibility that alterations of hormone internalization may be a general phenomenon of hormone-hormone interaction.
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PMID:Effect of intracellular Ca2+ on insulin-like growth factor II. internalization into pancreatic acini. Roles of insulin and cholecystokinin. 609 32

Many small biologicaly active peptides are derived from larger precursor forms which fulfil a variety of roles in the synthesis, segregation and intracellular migration of secretory products. Limited proteolysis may occur at several stages during this process, giving rise to products that are either degraded (e.g. the prepeptides) or discharged coordinately from their cells of origin during exocytosis (e.g. insulin and C-peptide). Molecular defects have recently been found to occur at cleavage sites in proinsulin as well as in other proproteins, and these point mutations may, in some instances, be responsible for familial metabolic disorders. The nature and cell specificity of the proteolytic enzymes involved in the conversion of the various precursor forms remains unresolved. Recent studies in our laboratory have led to the identification of precursors of glucagon and somatostatin in rat islets of Langerhans. Analysis of tryptic maps of these precursors has shown that a trypsin-like enzyme would be sufficient to cleave the C-terminally located somatostatin sequence from its precursor (relative molecular mass 12,500), but that both trypsin-like and carboxypeptidase B-like enzymes would be necessary to cleave the internal glucagon sequence from its prohormone (relative molecular mass 18,000). Molecular cloning techniques have provided valuable new approaches to analysing the structures of a variety of precursor forms, including those for insulin, gastrin, growth hormone, adrenocorticotropic hormone and the endorphins, and in the future will undoubtedly shed more light on the structures of their chromosomal genes, the mechanisms regulating their expression, and their evolutionary origins.
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PMID:Formation of biologically active peptides. 610 30

Using a new model of a reversible pancreatic fistula which allows the long-term-investigation under nearly physiological conditions on the unrestrained dog, we tested the effect of somatostatin (50 micrograms), calcitonin (4 micrograms), glucagon (1 microgram), and prostaglandin E1 (150 micrograms) on the exocrine pancreatic function in 45 experiments over a period of 13 h: SST inhibits the basal as well as the secretin or CCK-stimulated secretion: calcitonin shows inhibition of the stimulated secretion only; glucagon blocks the secretin-stimulated pancreatic function; and PGE1 reduces the bicarbonate concentration and trypsin output in secretin stimulation, but in one of the two series it stimulates the basal secretion.
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PMID:The effect of SST, glucagon, calcitonin and PGE1 on exocrine pancreatic secretion in the unrestrained dog in long-term experiments. 611 65

The occurrence of insulin receptors and biological responses to insulin has been investigated in trypsin-dissociated fetal rat brain cells maintained in culture for 8 days. Binding of [125I]insulin to brain cells in culture was time- and pH-dependent and 85--90% specific. Porcine insulin competed for [125I]insulin binding in a dose-dependent manner. Unrelated polypeptides, including angiotensin II, glucagon, bovine growth hormone, and bovine prolactin did not compete for [125I]insulin binding. The half-life of [125I]insulin dissociation from receptors at 24 degrees C was 15 min and a plot of In[B/Bo] vs time suggested two dissociated rate constants of 2.7 X 10(-4) sec-1 and 5.0 X 10(-5) sec-1. Scatchard analysis of the binding data gave a curvilinear plot which may indicate negative cooperativity or the occurrence of both high affinity (Ka = 2 X 10(11) M-1) and low affinity (Ka = 4 X 10(10) M-1) sites. Of the estimated total of 4.9 X 10(4) binding sites per cell, 28--30% appear to be high affinity sites. Incubation of cultures with insulin caused a time- and dose-dependent stimulation of [3H]thymidine and [3H]uridine incorporation into TCA-precipitable material. Maximum stimulation of thymidine incorporation (2--5-fold) occurred 11 h after incubation with 167 nM insulin. The same concentration of insulin caused a 2.2-fold increase in [3H]uridine incorporation in 2 h. These results indicate that cells cultured from rat brain contain specific insulin receptors capable of mediating effects of insulin on macromolecular synthesis in the central nervous system.
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PMID:Binding of [125I]insulin to specific receptors and stimulation of nucleotide incorporation in cells cultured from rat brain. 615 64

1. GTP, but not p[NH]ppG (guanosine 5'-[betagamma-imido]triphosphate), abolishes the sensitivity of glucagon-stimulated adenylate cyclase to the lipid-phase separations occurring in the outer half of the bilayer in liver plasma membranes from rat. 2. When either GTP or p[NH]ppG alone stimulate adenylate cyclase, the enzyme senses only those lipid-phase separations occurring in the inner half of the bilayer. 3. Trypsin treatment of intact hepatocytes has no effect on the basal, fluoride-, GTP- or p[NH]ppG-stimulated adenylate cyclase activity. However, (125)I-labelled-glucagon specific binding decays with a half-life matching that of the decay of glucagon-stimulated adenylate cyclase activity. 4. When GTP or p[NH]ppG are added to assays of glucagon-stimulated activity, the half-life of the trypsin-mediated decay of activity is substantially increased and the decay plots are no longer first-order. 5. Trypsin treatment of purified rat liver plasma membranes abolishes basal and all ligand-stimulated adenylate cyclase activity, and (125)I-labelled-glucagon specific binding. 6. Benzyl alcohol activates the GTP- and p[NH]ppG-stimulated activities in an identical fashion, whereas these activities are affected differently when glucagon is present in the assays. 7. We suggest that guanine nucleotides alter the mode of coupling between the receptor and catalytic unit. In the presence of glucagon and GTP, a complex of receptor, catalytic unit and nucleotide regulatory protein occurs as a transient intermediate, releasing a free unstable active catalytic unit. In the presence of p[NH]ppG and glucagon, the transient complex yields a relatively stable complex of the catalytic unit associated with a p[NH]ppG-bound nucleotide-regulatory protein.
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PMID:Guanosine 5'-triphosphate and guanosine 5'-[beta gamma-imido]triphosphate effect a collision coupling mechanism between the glucagon receptor and catalytic unit of adenylate cyclase. 624 58

[32P]ATP-citrate lyase phosphorylated by the cAMP-dependent protein kinase was partially digested by trypsin. Two tryptic 32P-labeled phosphopeptides containing more than 90% of the 32P radioactivity present on the phosphorylated enzyme were purified and found to have overlapping amino acid sequences around the same phosphorylated site (Thr-Ala-Ser(32P)-Phe-Ser-Glu-Ser-Arg). Tryptic digestion of 32P-labeled ATP-citrate lyase purified from 32P-labeled hepatocytes exposed to glucagon yielded a major 32P-labeled peptide of identical amino acid composition with that indicated above. Thus, the site on ATP-citrate lyase phosphorylated by the cAMP-dependent protein kinase in vitro resides on the same octapeptide as the site of glucagon-stimulated phosphorylation in intact hepatocytes.
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PMID:ATP-citrate lyase. Structure of a tryptic peptide containing the phosphorylation site directed by glucagon and the cAMP-dependent protein kinase. 626 53


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