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Query: UNIPROT:P01275 (glucagon)
 26,492 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Examination of insulin and glucagon degradation by rat kidney subcellular fractions revealed that most degrading activity was localized to the 100 000 X g pellet and 100 000 X g supernatant fractions. Further characterization of the degrading activities of the 100 000 X g pellet and supernatant suggested that three types of enzymatic activity were present at neutral pH. From the cytosol an enzyme with characteristics of the insulin glucagon protease of skeletal muscle was purified. This enzyme appeared to be responsible for insulin degradation by the kidney at physiological insulin concentrations. This enzyme also contributed to glucagon degradation but was not the most active mechanism for this. In the 100 000 X g pellet at least two separate enzymatic activities were present. One of these had properties consistent with those described for glutathione insulin transhydrogenase and appeared to be responsible for insulin degradation at high insulin concentration. The other enzyme was associated with the brush border and had properties consistent with the brush border neutral protease. This enzyme appeared responsible for glucagon degradation at both low and high substrate concentrations. An apparent marked synergism between the 100 000 X g pellet and the 100 000 X g supernatant was noted for insulin degradation at physiological insulin concentrations. Pellet glucagon-degrading activity and soluble insulin-degrading activity were necessary for this. The mechanism was found to be limited insulin degradation by the soluble enzyme resulting in both trichloroacetic acid-precipitable trichloroacetic acid-soluble fragments followed by further degradtion of the fragments by the glucagon-degrading enzyme resulting in an additional increase in trichloroacetic acid-soluble products.
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PMID:Insulin and glucagon degradation by the kidney. II. Characterization of the mechanisms at neutral pH. 0 6

The binding of labeled insulin to dissociated R3230AC mammary adenocarcinoma cells from diabetic and intact rats was investigated in vitro. At 20 degrees, specific binding (total binding minus binding in the presence of 1000-fold excess or 10(-6) M unlabeled insulin) reached a plateau at 45 to 60 min and was directly related to the number of cells used. Degradation of labeled insulin, as measured by trichloroacetic acid precipitation, was related to the number of cells used, was not prevented by trasylol or phenylmethylsulfonyl fluoride (general proteolytic enzyme inhibitors), but was prevented by addition of 1 to 2% bovine serum albumin to the incubation medium. Specificity of insulisulin, and desoctapeptide insulin were capable of competing for insulin binding in an order of potency related to their relative biological activity; prolactin and glucagon were unable to compete for insulin binding. Scatchard analysis of the binding data demonstrated a curvilinear-plot; specific binding (over the concentration range of 10(-11) to 10(-10) M insulin) showed a high affinity (Kd approximately 1 to 3 X 10(-10) M), and the estimated number of sites was greater in tumors from diabetic animals than in tumors from intact animals or intact animals given insulin prior to sacrifice. Reversibility of insulin binding was studied by dissociation experiments; dissociation was enhanced in the presence of added unlabeled insulin compared to dissociation examined under conditions of "infinite" dilutions only. Maximum dissociation of bound insulin was observed in the presence of 10(-7) M unlabeled insulin, with less of an effect at lower or higher concentrations of added insulin (no effect seen at 10(-10) M insulin). Two techniques were investigated for separating cells from unbound labeled insulin; the procedure using centrifugation was found to be more efficient. Thus, in the R3230AC mammary adenocarcinoma, data obtained on saturability, reversibility, and specificity of insulin binding indicate the existence of an insulin receptor with properties similar to those found in normal cells.
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PMID:Identification and characterization of the insulin receptor in the R3230AC mammary adenocarcinoma of the rat. 13 40

The components of fat cell membranes responsible for the binding of insulin were solubilized by treatment with the nonionic detergent Triton X-100. By using a polyethylene glycol precipitation method to assay specific insulin binding, the soluble preparation was shown to have insulin-binding characteristics similar to those of intact fat cells. Further studies of this preparation by polyacrylamide gel electrophoresis in the presence of (125)I-labeled insulin demonstrated two distinct insulin binding activities, designated species I and II. The two species were separated by electrophoresis in the absence of iodo-labeled hormone and eluted from the gel. Scatchard analysis of the insulin binding data for species I showed a curvilinear plot with the initial portion having a K(d) of 1.3 x 10(-10) M. The Scatchard plot for species II was linear with a K(d) of 6.0 x 10(-9) M. Desoctapeptide insulin and glucagon failed to compete for the insulin-binding sites in both species whereas desalanine insulin was an effective competitor. High concentrations of proinsulin competed with the iodo-labeled hormone for binding to species I but not to species II. In the presence of a low concentration of (125)I-labeled insulin (0.3 nM) some species I activity appeared to be converted to species II activity; there was no evidence of interconversion between the two species in the absence of insulin. Neither species degraded insulin as measured by trichloroacetic acid precipitation or rebinding to intact fat cells. These findings indicate the existence in the adipocyte plasma membrane of two insulin-binding species that have distinct physicochemical properties.
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PMID:Insulin binding to solubilized material from fat cell membranes: evidence for two binding species. 27 28

Recently, evidence has been reported to suggest that human platelets like several other circulating blood cells may bind insulin. To examine whether human platelets contain specific insulin receptors, washed human platelets suspended in Hepes buffer were incubated at 24 degrees C with 125I-insulin in the presence and absence of unlabeled insulin and specific insulin binding was determined. Insulin binding by platelets increased progressively with time of incubation to reach a maximum at 3 h and was proportional to the number of platelets in the incubation mixture. Maximum insulin binding was observed at pH 8. Insulin degradation by platelets as assessed by TCA precipitability and reincubation studies was minimal. Scatchard analysis of the binding data and dissociation studies revealed evidence of negative cooperativity of the platelet insulin receptor. A high affinity dissociation constant of approximately equal to 3 X 10(9) M-1 was determined and the concentration of platelet insulin receptors was estimated as 25 binding sites/micron2 platelet surface area. Binding of 125I-insulin by platelets was inhibited by unlabeled porcine insulin and to a lesser extent by catfish insulin and porcine proinsulin but not by glucagon, prolactin, growth hormone, and thrombin. The findings indicate that human platelets contain specific insulin receptors. The significance of the platelet insulin receptor, particularly with respect to altered platelet function in diabetes mellitus, remains to be determined.
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PMID:Demonstration and partial characterization of insulin receptors in human platelets. 44 28

Incubation of hepatocytes with [32P]orthophosphate resulted in the incorporation of 32P into material that is precipitated by reaction with antibodies to ATP citrate lyase. The amount of radioactivity precipitated was decreased when unlabeled, purified ATP citrate lyase was added to extracts of hepatocytes that had been incubated with [32P]orthophosphate. Addition of glucagon to hepatocytes that had been preincubated with [32P]orthophosphate resulted in a 56% increase in acid-stable 32P in the trichloroacetic acid-insoluble portion of immunoprecipitates. Catalytic phosphate bound to ATP citrate lyase reaction with ATP and Mg2+ is acid-labile; thus, glucagon-dependent phosphorylation is distinguished from the catalytic phosphate. When hepatocytes were incubated in the absence of [32P]orthophosphate and extracted in a medium containing [gamma-32P]ATP, no acid-stable 32P was present in immunoprecipitates. This indicates that the incorporation into ATP citrate lyase of acid-stable phosphate occurs prior to extraction of the enzyme. Preliminary studies, using a procedure that allows for measurement of enzyme activity starting 1 min after beginning the extraction of lyase from hepatocytes, have shown no difference in lyase activity when hepatocytes are treated with or without glucagon.
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PMID:Phosphorylation of ATP citrate lyase in response to glucagon. 48 38

Endosomes have recently been identified as one major site of glucagon degradation in intact rat liver. In this study, a cell-free system has been used to assess the role of ATP-dependent acidification in endosomal glucagon degradation and identify the glucagon products generated. Percoll gradient fractionation of Golgi-endosomal fractions prepared 10-30 min after injection of [125I]iodoglucagon showed a time-dependent shift of the radioactivity towards high densities. Regardless of time, the radioactivity was less precipitable by trichloroacetic acid (Cl3Ac) at high densities than at low densities. Chloroquine treatment slightly increased the density shift of the radioactivity and decreased its Cl3Ac-precipitability throughout the gradient. Incubation of endosomal fractions containing [125I]iodoglucagon in 0.15 M-KCl at 30 degrees C resulted in a time- and pH-dependent generation of Cl3Ac-soluble radioactivity, with a maximum at pH 4 (t1/2, 7 min). At pH 5, 1,10-phenanthroline, bacitracin and p-chloromercuribenzoic acid partially inhibited [125I]iodoglucagon degradation. At pH 6-7, ATP stimulated [125I]iodoglucagon degradation by 5-10-fold and caused endosomal acidification as judged from Acridine Orange uptake. The effects of ATP were inhibited by chloroquine, monensin, N-ethylmaleimide and dansylcadaverine. Poly(ethylene glycol) (PEG) precipitation of the radioactivity associated with endosomes showed that lowering the pH below 5.5 caused dissociation of the glucagon-receptor complex, and that, regardless of incubation conditions, all degraded [125I]iodoglucagon diffused extraluminally. On h.p.l.c., at least three products less hydrophobic than [125I]iodoglucagon were identified in incubation mixtures along with monoiodotyrosine. Radiosequence analysis of the products revealed one major cleavage located C-terminally to Tyr-13 and two minor cleavages affecting Thr-5-Phe-6 and Phe-6-Thr-7 bonds. It is concluded that glucagon degradation in liver endosomes is functionally linked to ATP-dependent endosomal acidification and involves several cleavages in the glucagon sequence.
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PMID:Degradation of glucagon in isolated liver endosomes. ATP-dependence and partial characterization of degradation products. 174 49

The renal catabolism of [125I]glucagon-like peptide 1 (GLP-1) and [125I]glucagon-like peptide 2 (GLP-2) has been studied both in vivo, by the disappearance of these peptides from the plasma of bilaterally nephrectomized (BNX), ureteral-ligated (BUL) or normal rats, and in vitro, analyzing their catabolism by the isolated, perfused rat kidney. Results from in vivo studies demonstrated that half-disappearance time for both peptides was lower in controls than in BUL rats, and this value in BUL rats was not significantly different from that in BNX rats. In addition, metabolic clearance rate of GLP-1 was higher in control rats than in the other two groups of animals. Urinary clearance rate of both peptides was negligible. In isolated kidney experiments, values for organ clearance of both [125I]GLP-1 and [125I]GLP-2 were similar to those of inulin clearance, which represents the glomerular filtration rate. Urinary clearance of trichloroacetic acid precipitable radioactivity represented less than 1% of total clearance. In conclusion, these results demonstrate a significant role for the kidney in the plasma removal of [125I]GLP-1 and [125I]GLP-2 by a mechanism that involves glomerular filtration and tubular catabolism.
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PMID:Renal catabolism of human glucagon-like peptides 1 and 2. 208 1

The uptake and processing of glucagon into liver endosomes were studied in vivo by subcellular fractionation. After injection of [[125I]iodo-Tyr10]glucagon and [[125I]iodo-Tyr13]glucagon to rats, the uptake of radioactivity into the liver was maximum at 2 min (6% of the dose/g of tissue). On differential centrifugation, the radioactivity in the homogenate was recovered mainly in the nuclear (N), microsomal (P) and supernatant (S) fractions, with maxima at 5, 10 and 40 min, respectively; recovery of radioactivity in the mitochondrial-lysosomal (ML) fraction did not exceed 6% and was maximal at 20 min. On density-gradient centrifugation, the radioactivity associated first (2-10 min) with plasma membranes and then (10-40 min) with Golgi-endosomal (GE) fractions, with 2-5-fold and 20-150-fold enrichments respectively. Subfractionation of the GE fractions showed that, unlike the Golgi marker galactosyltransferase, the radioactivity was density-shifted by diaminobenzidine cytochemistry. Subfractionation of the ML fraction isolated at 40 min showed that more than half of the radioactivity was recovered at lower densities than the lysosomal marker acid phosphatase. Throughout the time of study, the [125I]iodoglucagon associated with the P, PM and GE fractions remained at least 80-90% trichloroacetic acid (TCA)-precipitable, whereas that associated with other fractions, especially the S fraction, became progressively TCA-soluble. On gel filtration and h.p.l.c., the small amount of degraded [125I]iodoglucagon associated with GE fractions was found to consist of monoiodotyrosine. Chloroquine treatment of [125I]iodoglucagon-injected rats caused a moderate but significant increase in the late recovery of radioactivity in the ML, P and GE fractions, but had little effect on the association of the ML radioactivity with acid-phosphatase-containing structures. Chloroquine treatment also led to a paradoxical decrease in the TCA-precipitability of the radioactivity associated with the P and GE fractions. Upon h.p.l.c. analysis of GE extracts of chloroquine-treated rats, at least four degradation products less hydrophobic than intact [125I]iodoglucagon were identified. Radio-sequence analysis of four of these products revealed three cleavages, affecting bonds Ser2-Gln3, Thr5-Phe6 and Phe6-Thr7. When GE fractions containing internalized [125I]iodoglucagon were incubated in iso-osmotic KCl at 30 degrees C, a rapid generation of TCA-soluble products was observed, with a maximum at pH 4. We conclude that endosomes are a major site at which internalized glucagon is degraded, endosomal acidification being required for optimum degradation.
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PMID:Fate of injected glucagon taken up by rat liver in vivo. Degradation of internalized ligand in the endosomal compartment. 226 96

Earlier studies from this laboratory reported that, in rats bearing the Walker 256 carcinosarcoma, circulating insulin levels were significantly reduced relative to non-tumor-bearing rats. The present study extends this observation to include a significantly (P less than 0.01) reduced plasma level of glucagon in rats bearing the tumor for both 7 and 10 days. In order to determine if the tumor itself somehow plays a role in the degradation of these protein hormones, either cultured Walker 256 tumor cells (in the case of the insulin studies) or cells from freshly excised tumor (for the glucagon studies) were incubated with 125I-labeled insulin or glucagon. Following the incubation period, the amount of TCA-precipitable radio-label remaining in the incubation medium was markedly reduced after exposure to cells. This suggests that the tumor cells have the capability of degrading both insulin and glucagon. In a separate series of experiments, it was found that medium, in which freshly excised tumor cells had been incubated previously and then discarded, retained a substance which degraded 125I-labeled glucagon and that this degradation of glucagon could be blocked by co-incubation with aprotinin, a protease inhibitor.
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PMID:Degradation of insulin and glucagon by a factor associated with Walker 256 carcinosarcoma cells. 242 71

To evaluate the effect of glucagon degrading activity (GDA) on radioimmunoassay (RIA) of glucagon, I measured GDAs in plasma, serum, lysed red blood cells (RBC), and suspension of mononuclear cells and granulocytes. Serum levels of GDA in patients with various diseases were also examined. 1. Serum GDA values in normal subjects (control) measured by TCA precipitation method were 4.6 +/- 2.2% (mean +/- S.D.). GDA values in plasma treated with citrate and those in serum treated with aprotinin were not different from those in nontreated serum. GDAs in plasma treated either with EDTA and aprotinin or with EDTA alone were significantly lower than those in control serum, but the values in plasma treated with heparin were markedly higher than those in control serum. 2. GDAs in RBC lysate and suspension of mononuclear cells and granulocytes remained low up to the concentration of 23 X 10(4) RBC/microliters and 5000 mononuclear cells or granulocytes/microliters, respectively. GDAs in RBC lysate and mononuclear cells were markedly suppressed by the treatment with EDTA, whereas GDAs in granulocytes were inhibited by the treatment with aprotinin. 3. Markedly high values of GDA were obtained in serum of patients with pancreas diseases, liver diseases, renal diseases and hyperthyroidism. However, in four patients these elevated levels were restored to normal value after recovery from these disorders. The elevated GDA values in serum were suppressed to normal value by the addition of EDTA and aprotinin. 4. On Bio-Gel P-6 column chromatography, 125I-glucagon incubated with patient serum containing high GDA values revealed several peaks eluted after 125I-glucagon. 5. In healthy subjects, immunoreactive glucagon (IRG) levels in nontreated serum were not different from those in plasma treated either with EDTA and aprotinin or with heparin. 6. In patients showing high serum GDA levels, serum GDA levels were not significantly related to IRG levels in plasma treated with EDTA and aprotinin. These results indicate that a series of treatment of blood samples before assay: addition of EDTA and aprotinin to the blood samples, immediate separation of plasma from blood cells, and storage at 4 degrees C is recommended to avoid breakdown of glucagon by GDAs.
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PMID:[Glucagon degrading activity]. 247 36


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