Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Tryptophan 2,3-dioxygenase [EC 1.13.11.11] in primary cultures of adult rat hepatocytes was induced 3-4 fold by 1 microM dexamethasone and 6-7 fold by dexamethasone plus glucagon (0.1 microM). Changes of the enzyme activity, amount of enzyme, measured by immunotitration, and rate of enzyme synthesis, assayed by measurement of [3H]leucine incorporation into the enzyme protein, were closely correlated. Furthermore, in a reticulocyte lysate system for cell-free protein synthesis, mRNA of the enzyme was translated to the protein corresponding to the subunit of tryptophan 2,3-dioxygenase, which was identified by SDS-polyacrylamide gel electrophoresis. The activity of translatable mRNA of the enzyme was increased more than 10-fold by dexamethasone and its final content in total mRNA was 0.34%. Glucagon alone did not increase mRNA activity, but dexamethasone plus glucagon increased mRNA activity to twice that with dexamethasone alone, the maximal content of the mRNA being 0.77% of the total mRNA content 12 h after addition of hormones. Insulin (0.1 microM) caused 75% inhibition of the maximum increase of mRNA activity of the enzyme induced by dexamethasone and glucagon. Epinephrine (10 microM) also caused 58% inhibition of the maximum increase. Insulin and epinephrine also suppressed increase of mRNA of tryptophan 2,3-dioxygenase induced by dexamethasone alone. Therefore, dexamethasone alone or together with glucagon stimulated transcription of tryptophan 2,3-dioxygenase increasing its mRNA and enzyme synthesis in hepatocytes. Conversely, insulin and epinephrine suppressed these increases of mRNA synthesis and thus decreased enzyme synthesis.
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PMID:Hormonal regulation of translatable mRNA of tryptophan 2,3-dioxygenase in primary cultures of adult rat hepatocytes. 636 Oct 14

Rat hearts were perfused for long periods in the presence of 14C-labeled amino acids. From these hearts, postheparin-effluent and a tissue homogenate containing lipoprotein lipase and neutral lipase, respectively, were derived. Lipolytic activity and 14C-labeled protein in both preparations were characterized by affinity chromatography, immunoprecipitation and SDS-polyacrylamide gel electrophoresis. Lipase activity and 14C-labeled protein co-eluted from heparin-Sepharose 4B at 1.2 M NaCl and were inhibited and precipitated by preincubation with anti-lipoprotein lipase gamma-globulins. Gel electrophoresis of both preparations showed the presence of 14C-labeled protein with a molecular weight of 35 000. These data strongly suggest similarity between lipoprotein lipase and neutral lipase and their possible precursor-product relationship and indicate that during perfusion continuous synthesis, secretion and vascular binding of lipase molecules occur. Cycloheximide perfusion induced a dramatic decrease of lipoprotein lipase and neutral lipase activity, indicating a half-life of less than 90 min for both enzymes. Tunicamycin present during perfusion also induced a drop in lipoprotein lipase and tissue neutral lipase activity, indicating that glycosylation is necessary for secretion of lipoprotein lipase. Long-term perfusion of rat hearts in the presence of norepinephrine, glucagon or tyrosine leads to reciprocal alterations in lipoprotein lipase and neutral lipase activities, i.e., lipoprotein lipase activity increased and neutral lipase activity decreased, whereas total lipase activity (lipoprotein lipase + neutral lipase) remained unaltered. During perfusion in the presence of insulin, no net change in lipase activities was observed. Also, insulin did not affect the glucagon-induced inverse effects on either lipase activity. The reciprocal changes in lipase activities occurring during norepinephrine perfusion were hampered by colchicine and propranolol, pointing towards beta-receptor and microtubular mediation of tissue lipase processing and endothelial binding. Our data suggest that the tissue flux and vascular binding of lipase protein may be important sites of hormonal regulation of lipoprotein lipase homeostasis.
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PMID:Effects of hormones, amino acids and specific inhibitors on rat heart heparin-releasable lipoprotein lipase and tissue neutral lipase activities during long-term perfusion. 637 31

A method has been developed for routine high yield separation of canalicular (cLPM) from basolateral (blLPM) liver plasma membrane vesicles of rat liver. Using a combination of rate zonal floatation (TZ-28 zonal rotor, Sorvall) and high speed centrifugation through discontinuous sucrose gradients, 9-16 mg of cLPM and 15-28 mg of blLPM protein can be isolated in 1 d. cLPM are free of the basolateral markers Na+/K+-ATPase and glucagon-stimulatable adenylate cyclase activities, but are highly enriched with respect to homogenate in the "canalicular marker" enzyme activities leucylnaphthylamidase (48-fold), gamma-glutamyl-transpeptidase (60-fold), 5'-nucleotidase (64-fold), alkaline phosphatase (71-fold), Mg++-ATPase (83-fold), and alkaline phosphodiesterase I (116-fold). In contrast, blLPM are 34-fold enriched in Na+/K+-ATPase activity, exhibit considerable glucagon-stimulatable adenylate cyclase activity, and demonstrate a 4- to 15-fold increase over homogenate in the various "canalicular markers." cLPM have a twofold higher content of sialic acids, cholesterol; and sphingomyelin compared with blLPM. At least three canalicular-(130,000, 100,000, and 58,000 mol wt) and several basolateral-specific protein bands have been detected after SDS PAGE of the two LPM subfractions. Specifically, the immunoglobin A-binding secretory component is restricted to blLPM as demonstrated by immunochemical techniques. These data indicate virtually complete separation of basolateral from canalicular LPM and demonstrate multiple functional and compositional polarity between the two surface domains of hepatocytes.
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PMID:Structural and functional polarity of canalicular and basolateral plasma membrane vesicles isolated in high yield from rat liver. 669 96

Polyadenylated RNA extracted from anglerfish islets was translated in a wheat germ cell-free system containing [35S]methionine in the presence and absence of microsomal membranes prepared from a canine pancreas. Labeled translation products were analyzed by immunoprecipitation with an antiserum to porcine glucagon, followed by electrophoresis of the translation products and immunoprecipitated proteins on SDS polyacrylamide gels. In the absence of microsomal membranes two proteins of Mr = 14,500 and Mr = 12,500 were specifically immunoprecipitated with antiglucagon serum. Addition of microsomal membranes to the translation reactions resulted in a diminution of the labeled protein of Mr = 14,500 and a marked increase in the immunoreactive protein of Mr = 12,500. The protein of Mr = 12,500 was resistant to degradation by proteolytic enzymes added to translation reactions, indicating that it was segregated within microsomal vesicles. These results are consistent with synthesis of anglerfish islet glucagon in the form of a pre-prohormonal precursor (Mr = 14,500) containing a leader sequence that is cotranslationally cleaved from the protein by enzymes associated with microsomal membranes to produce a smaller intermediate prohormonal precursor (Mr = 12,500) of pancreatic glucagon (Mr = 3500).
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PMID:Glucagon precursors identified by immunoprecipitation of products of cell-free translation of messenger RNA. 699 42

To further investigate the regulation of glucagon biosynthesis in mammalian A2-cells, we have studied the incorporation of [3H]-tryptophan into acid alcohol extracts of isolated pancreatic islets of guinea pig and mouse. Gel chromatography on Sephadex G-50 indicated that labelled proteins, migrating either with the void volume (peak I) or in region (peak II) between the void volume and the insulin marker, were formed during a 6h incubation of the islets. However, a period of at least two days in tissue culture was required before the islets showed any significant accumulation of labelled protein eluting in a position corresponding to that of pancreatic glucagon (peak III). Addition of glucose (16.7 mM) enhanced the incorporation into all chromatograph fractions during the culture period. Binding of gel chromatographed proteins Sepharose coupled anti-glucagon antibodies indicated that both guinea pig and mouse islets contained only small amounts of labelled, immunoreactive proteins eluting with either peak I or peak II. However, proteins eluting with peak III contained 6-8 times more lbelled, immunoreactive material than any of the other peaks. Total glucagon immunoreactivity was abundant in peaks I and II but less evident in peak II. The results of pulse-chase experiments provided no convincing evidence for a precursor-product relationship between larger proteins and glucagon. However, the heterogeneity of the putative precursor pool, as evidenced both by SDS-polyacrylamide gel electrophoresis and by the low immune binding, might have masked a conversion process. The combined data show that glucagon is, indeed, synthesized in isolated islets of guinea-pig and mouse, but that this process occurs slowly.
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PMID:Glucagon biosynthesis in isolated pancreatic islets of mice and guinea pigs. 699 3

Fetal bovine pancreas was extracted for glucagon using (A) ethanol-Hcl after trichloroacetic acid (TCA) treatment of the pancreas, (B) ethanol-HCl and (C) urea-acetic acid. Fractionation of the acetic acid soluble proteins vi Sephadex G-50 columns yielded glucagon immunoreactivity in the void volume, high molecular weight glucagon immunoreactivities (HMW-IRGs), "proglucagon" (approximately equal to 9 K delta), and true glucagon (3.5 K delta) regions. HMW-IRGs were obtainable using all three methods of extraction. The material obtained from the ethanol-HCl-TCA method appeared stable on Sephadex G-100 (1 M acetic acid) rechromatography. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis analysis showed immunoreactive species corresponding to approximately 40 K delta and approximately 12 K delta. HMW-IRGs did not bind to concanavalin A (Con A)-agarose. SDS-polyacrylamide gel electrophoresis of the Con A-agarose filtered IRG again showed a major immunoreactive peak of approximately 40 K delta. Dose-response RIA studies indicated that the HMW-IRGs from both the gel filtration and SDS-polyacrylamide gel experiments were immunochemically indistinguishable from glucagon. HMW-IRGs bind to antiglucagon antibody agarose, further indicating their reactivity towards glucagon antibodies. When HMW-IRGs are incubated with guanidinium hydrochloride and gel filtered in the same system, a significant fraction of HMW-IRG (representing up to 25% of the total IRG analysed) was found to resist disruption. Our data support the contention that a significant portion of the HMW-IRGs (molecular weight greater than 20 K delta) extracted from fetal bovine pancreas are composed of glucagon covalently linked to larger protein unit(s).
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PMID:Glucagon from the bovine fetal pancreas: chromatographic and electrophoretic characterizations of high molecular weight immunoreactive species. 700 59

Photoaffinity labeling techniques were used to identify insulin-binding components of the plasma membrane in insulin-responsive, monolayer-cultured hepatoma cells. The activated, photosensitive reagent, an n-hydroxysuccinimide ester of 4-azidobenzoic acid, was coupled with highly purified insulin, and the hormone derivative was subsequently iodinated, bound to cell surface receptors of intact H4 cells, and photoactivated. Ater dissolution of the cells, labeled proteins were analyzed by SDS/polyacrylamide gel electrophoresis under reducing conditions. The main labeled band exhibited an apparent molecular weight of 130,000. Two minor components of apparent mol wt 95,000 and 40,000 were also identified. Specific labeling of all 3 bands was inhibited by simultaneous incubation of the cells with native insulin, but not by the heterologous hormone, glucagon, prior to photoactivation. Binding of azidobenzoyl insulin to H4 cells was time-dependent, as was the correlated labeling of receptor components. Band-labeling by the photosensitive insulin derivative was totally light-dependent; spontaneous covalent linking of insulin and receptor was not observed. The labeled receptor-related proteins were not degraded by the cells under our experimental conditions.
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PMID:Photoaffinity labeling of the insulin receptor in H4 hepatoma cells: lack of cellular receptor processing. 701 74

The glycoproteinic nature of the insulin receptor was indicated using two different approaches: 1. [125I] insulin binding to soluble receptors from mouse liver was inhibited by digestion with beta-galactosidase or pretreatment with Ricinus communis I or concanavalin A. An other enzyme (neuraminidase) and lectins (wheat germ agglutinin, Dolichos biflorus) did not affect the binding reaction. These data confirmed that insulin directly interacts with the galactoglycoproteins of liver membranes. 2. The galactose oxidase-sodium boro[3H] hydride technique, previously used for labeling accessible membrane galactoglycoproteins, was again utilized to discern the components that interact with insulin. When liver membranes were equilibrated with 10-7 M insulin prior to labeling, the SDS gel radioactive profiles were specifically modified with two galactoglycoprotein of apparent molecular sizes 195 000 and 145 000, compatible with their participation in the insulin binding interaction. Membrane pretreatment with beta-galactosidase or Sophora japonica lectin reduced the labeling in most peaks, thus supporting the argument for labeling sensitivity. Preincubation of membranes with 10-7 M proinsulin slightly hindered labeling, while pretreatment with 10-7 M glucagon was ineffective, suggesting a specificity of the insulin effect. These data indicate that glycoprotein nature of the insulin receptor for two reasons: alteration of insulin binding after modification of the galactoglycoproteins, and alteration of galactoglycoprotein labeling after insulin binding. Two galactoglycoproteins, with apparent molecular weights 145 000 and 195 000, respectively, were identified and they are suggested to have insulin binding properties.
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PMID:Identification of liver cell membrane galactoglycoproteins involved in the process of insulin binding. 703 Mar 99

In primary cultured monolayer hepatocytes of adult rats, insulin (1 x 10(-8) M) induced glucose-6-phosphate dehydrogenase [EC 1.1.1.49, G6PDH] several fold in 48 h. It also induced lipogenesis, measured as [1-14C]acetate incorporation in 2 h, in these cells. Of the various lipids, triglycerides and phospholipids were induced markedly, while cholesterol and its esters were not induced. The increase of G6PDH and lipogenesis were parallel. Glucagon, dibutyryl cyclic AMP, triiodothyronine, dexamethasone, epinephrine, isoproterenol, and dibutyryl cyclic GMP were also tested under similar conditions, but none of them caused significant induction of G6PDH or lipogenesis. Use of anti-G6PDH serum showed that induction of G6PDH by insulin was due to increase in the amount of enzyme protein. Insulin was found to increase the rate of synthesis of G6PDH about 3-fold. SDS-polyacrylamide gel electrophoresis of the immunoprecipitable protein revealed that besides G6PDH another radioactive fraction (Mr 37,000) was increased by insulin. This suggests that complete synthesis of G6PDH protein is slowed down in primary cultured hepatocytes and that an apparent nascent peptide of the enzyme accumulates. Although on long-term treatment (48 h), glucagon and dibutyryl cyclic AMP had no effect on lipogenesis, when added with [14C]acetate for 2 h they strongly inhibited lipogenesis. Significant inhibition of lipogenesis by short-term treatment with glucagon was seen even in cells with a high capacity for lipogenesis induced by long-term treatment with insulin. Insulin again stimulated lipogenesis in short-term treatment, but its effect was slight. It is concluded from these results that insulin exerts long-term stimulation of lipogenesis by inducing enzymes related to lipogenesis including G6PDH as well as causing slight stimulation by enhancing supply of substrate for lipogenesis. Glucagon seems to play a minor role in long-term control, but it causes short-term inhibition of lipogenesis.
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PMID:Hormonal regulations of glucose-6-phosphate dehydrogenase and lipogenesis in primary cultures of rat hepatocytes. 704 Mar 64

We investigated the influence of and interactions among pancreatic hormones on the secretion of insulin-like growth factor-I (IGF-I) and IGF-binding proteins (IG-FBPs) by treating primary hepatocytes from young male Long-Evans rats with insulin or glucagon in combination with rat GH (rGH). The concentration of IGF-I secreted into the medium was estimated by radioimmunoassay after formic acid-acetone cryoextraction, and secreted IGFBPs were analysed by Western ligand blot and immunoblot; accumulation of IGF-I mRNA was analysed by Northern blot. Both insulin (0.1-100 nmol/l) and rGH (0.5, 5 and 50 pmol/l) produced a dose-dependent stimulation of IGF-I secretion over a 24-h incubation period. In contrast, glucagon (0.1-100 nmol/l) inhibited IGF-I production in a dose-related manner. Glucagon (10 nmol/l) also inhibited IGF-I secretion stimulated by rGH (5 pmol/l) and insulin (10 nmol/l). Northern blot analysis of total RNA isolated from rat hepatocytes revealed that rGH (5 pmol/l) elevated IGF-I mRNA levels, glucagon (10 nmol/l) alone had no effect on this parameter, but glucagon significantly reduced IGF-I transcript accumulation in response to rGH. IGFBPs secreted by rat hepatocytes run in two molecular weight ranges on SDS-PAGE: approximately 25 kDa (IGFBP-4) and approximately 29-31 kDa (IGFBP-1 and -2); the predominant hormonally regulated IGFBP was identified as IGFBP-1. Insulin produced a dose-dependent inhibition of production of IGFBP-1, while glucagon was stimulatory; when given together at an equivalent concentration (1 nmol/l), the effects of insulin were dominant to glucagon on IGFBP-1. These observations provide support for significant opposite roles for the pancreatic hormones, insulin and glucagon, in the regulation of liver IGF-I and IGFBP-1 production. As the production of pancreatic hormones is influenced by nutritional status, these polypeptides may mediate the effects of changing nutritional state on the hormonal control of protein anabolism and glucose homeostasis by directly influencing the circulating level of liver-derived IGF-I and its binding proteins.
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PMID:Pancreatic hormones differentially regulate insulin-like growth factor (IGF)-I and IGF-binding protein production by primary rat hepatocytes. 752 61


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