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)

The prominent protein phosphatases involved in liver glycogen metabolism are the AMD (ATP, Mg-dependent, type-1) and PCS (polycation-stimulated, type-2A) phosphatases. The glycogen synthase phosphatase activity, measured from the rate of activation of liver glycogen synthase, is virtually accounted for by AMD phosphatases; the bulk of the activity belongs to the glycogen-bound protein phosphatase G and a small part is present in the cytosol. The major part of the phosphorylase phosphatase activity present in the post-mitochondrial supernatant is shared by protein phosphatase G and cytosolic enzymes, and a minor part belongs to a microsomal AMD phosphatase. In the liver cytosol, the phosphorylase phosphatase activity is about equally distributed between AMD and PCS phosphatases. Studies in vivo as well as on isolated, perfused livers have shown that glucagon (which raises the level of cyclic AMP) as well as vasopressin (which increases the cytosolic Ca2+ concentration) decrease the phosphorylase phosphatase activity in liver extract or cytosol (filtered through Sephadex G-25) by about 25% within a few minutes. These effects were not additive, and the activity of glycogen synthase phosphatase was not affected. Conversely, insulin as well as glucose increased both phosphatase activities by about 25%, and these effects were additive. Vanadate mimicked the effect of insulin on the perfused liver. All the activity changes were only observed when the assays were performed at high tissue concentration. Upon subcellular fractionation all the effects were well expressed in the cytosol, but not in the particulate fraction (glycogen and microsomes). However, quantitatively the hormonal responses were largely lost during the fractionation procedure; they could be restored by recombination of the liver cytosol from a hormone-treated rat with the particulate fraction from either a treated or an untreated animal. It appears that the effects of glucagon, insulin and glucose are mediated by cytosolic, transferable effectors of the Vmax of protein phosphatases. These effectors are eluted in the void volume of a Sephadex G-25 column. Rats of the gsd/gsd strain, which have a genetic deficiency of hepatic phosphorylase kinase, responded to an injection of insulin plus glucose with a normal increase in the cytosolic phosphorylase phosphatase activity. In contrast, they failed to respond to glucagon as well as vasopressin. A transient 80% inhibition of the phosphorylase phosphatase activity could be induced in vitro in a concentrate liver cytosol from Wistar rats upon addition of MgATP.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Short-term hormonal control of protein phosphatases involved in hepatic glycogen metabolism. 216 98

The intravenous administration of glucagon to anesthetized rats resulted within 5 min in a 20% drop in the hepatic phosphorylase phosphatase activity, as measured in a post-mitochondrial supernatant at low dilution, but it did not affect the activity of glycogensynthase phosphatase. On the other hand, the injection of insulin plus glucose caused increases by about 35% in both phosphatase activities. Upon subcellular fractionation these effects were recovered in the cytosol, but not in the glycogen/microsomal fraction. However, activity changes in the latter fraction were observed after recombination with the liver cytosol from a hormone-treated animal. Preincubation of the liver cytosol with modulator protein (a specific inhibitor of type-1 protein phosphatases) cancelled the activity changes induced by insulin plus glucose. No hormonal effects on hepatic protein phosphatase activities were observed when the fractions were either diluted an additional 10-fold or pretreated with trypsin. An acute hormonal regulation of protein phosphatases could also be demonstrated in the perfused liver. When added to the perfusion medium, glucose as well as insulin increased the cytosolic protein phosphatase activities by about 25%. Their effect was additive, irrespective of the order of addition. On the other hand, the addition of glucagon and/or vasopressin resulted in a 20% drop in the phosphorylase phosphatase activity. The presence of glucagon did not interfere with the effectiveness of insulin, and vice versa. The changes in the phosphorylase phosphatase activities induced by glucagon, insulin, and glucose represented changes in the Vmax only. We propose that the acute control of the hepatic glycogen synthase phosphatase and phosphorylase phosphatase activities is mediated by transferable, cytosolic effector(s).
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PMID:Acute regulation of hepatic protein phosphatases by glucagon, insulin, and glucose. 284 53

ATP-citrate lyase and acetyl-CoA carboxylase purified from lactating rat mammary gland are phosphorylated stoichiometrically by the calmodulin-dependent multiprotein kinase from rabbit skeletal muscle. The reactions are completely dependent on the presence of both Ca2+ and calmodulin. ATP-citrate lyase and acetyl-CoA carboxylase are also phosphorylated stoichiometrically by the Ca2+- and phospholipid-dependent protein kinase (protein kinase C) purified from bovine brain. Phosphorylation of these substrates is stimulated 6-fold and 40-fold respectively by Ca2+ and phosphatidylserine. The calmodulin-dependent and phospholipid-dependent protein kinases phosphorylate the same serine residue on ATP-citrate lyase that is phosphorylated by cyclic-AMP-dependent protein kinase. The sequence of the tryptic peptide containing this site on the mammary enzyme is identical with the sequence of the peptide containing the site on ATP-citrate lyase that is phosphorylated in isolated hepatocytes in response to insulin and/or glucagon. The calmodulin-dependent, phospholipid-dependent and cyclic-AMP-dependent protein kinases phosphorylate distinct sites on acetyl-CoA carboxylase. However, one of the three phosphorylated tryptic peptides derived from enzyme treated with the phospholipid-dependent kinase is identical with the major phosphopeptide (T1) derived from enzyme treated with cyclic-AMP-dependent protein kinase. Phosphorylation of acetyl-CoA carboxylase by the phospholipid-dependent protein kinase inactivates acetyl-CoA carboxylase in a similar manner to cyclic-AMP-dependent protein kinase. With either protein kinase slightly greater phosphorylation and inactivation is seen after pretreatment of acetyl-CoA carboxylase with protein phosphatase-2A, but the effects of the protein phosphatase treatment are not completely reversed. Inactivation by the phospholipid-dependent protein kinase is Ca2+- and phospholipid-dependent, is reversed by protein phosphatase-2A, and correlates with the degree of phosphorylation. The relevance of these findings to insulin- and growth-factor-promoted phosphorylation of ATP-citrate lyase and acetyl-CoA carboxylase in intact cells is discussed.
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PMID:Characterization of the phosphorylation of rat mammary ATP-citrate lyase and acetyl-CoA carboxylase by Ca2+ and calmodulin-dependent multiprotein kinase and Ca2+ and phospholipid-dependent protein kinase. 287 35

Acetyl-CoA carboxylase purified from isolated hepatocytes is activated dramatically by protein phosphatase treatment, concomitant with a reduction of the phosphate content from 3.7 to 1.1 mol/subunit. Glucagon treatment of the cells produces a further inactivation of the enzyme that is totally reversed by phosphatase treatment, and is associated with an increase in phosphate content of 0.8 mol/subunit, distributed in two peptides which contain the sites phosphorylated in vitro by the cyclic AMP-dependent and AMP-activated protein kinases. Sequencing of these peptides shows that the low activity of acetyl-CoA carboxylase is due to phosphorylation by the AMP-activated protein kinase, and not cyclic AMP-dependent protein kinase, even after glucagon treatment.
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PMID:The low activity of acetyl-CoA carboxylase in basal and glucagon-stimulated hepatocytes is due to phosphorylation by the AMP-activated protein kinase and not cyclic AMP-dependent protein kinase. 289 86

Hepatic glycogen metabolism was investigated in genetically diabetic C57BL/KsJ-db/db mice during their development. Initially, the development of obesity, hyperglycemia, hyperinsulinemia, and hyperglucagonemia in these mice was examined, which illustrated that the diabetes progressed normally. Little difference in hepatic glycogen concentrations was observed, averaging approximately 50 and 60 mg/g liver in diabetic (db/db) and control heterozygote (db/+) mice, respectively. Glycogen synthase activity (total and a-form) was significantly elevated by 5 wk in the diabetic mice relative to controls and reached maximum levels (two-fold higher than controls) around 8-9 wk. This activity then slowly declined during the rest of the 15-wk period examined. Both phosphorylase a and total phosphorylase activities were also elevated by 5 wk, reaching levels twofold higher than controls. These activities did not decline at the end of this 15-wk period, but instead continued to slowly increase. Glycogen synthase a activity showed a positive correlation (r = 0.54, N = 144) with circulating levels of insulin, and a similar correlation was seen for phosphorylase a activity and plasma glucagon levels (r = 0.64, N = 72). Protein kinase and phosphoprotein phosphatase activities were also measured, but no differences were detected between diabetic and control mice. This longitudinal study clarifies some of the changes in hepatic glycogen metabolism that occur during the progression of diabetes in the db/db mouse and indicates a role for circulating insulin and glucagon concentrations on the steady-state activities of glycogen synthase and phosphorylase, respectively.
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PMID:Age-related changes in hepatic glycogen metabolism in the genetically diabetic (db/db) mouse. 298 86

The expressed catalytic activity of liver microsomal HMG CoA reductase, the limiting enzyme in cholesterol synthesis, is reversibly diminished by phosphorylation in vitro. In intact hepatocytes the expressed activity of HMG CoA reductase is enhanced by incubation of cells with insulin, and diminished by treatment with glucagon or with mevalonate. In the latter situations the level of total reductase activity falls following initial inactivation (phosphorylation) of the enzyme. This observation suggested that the phosphorylated form of HMG CoA reductase is more sensitive to proteolysis. HMG CoA reductase is a 97,000 dalton (97 K) integral protein of the endoplasmic reticulum with a cytosolic domain that includes the catalytic site and serine residues that may be reversibly phosphorylated. In vitro the Ca2+-activated proteolytic enzyme, calpain, generates two catalytically-active fragments: a membrane bound 62 K and a soluble 53 K form of the enzyme which are quantified by specific immunoblot procedures. Cleavage of the native 97 K HMG CoA reductase is enhanced by pretreatment (inactivation) of microsomes with ATP (Mg2+) and liver reductase kinase compared to microsomes pretreated with protein phosphatase. This is reflected in a loss of 97 K reductase and an increase in the soluble 53 K form of the enzyme. Degradation of HMG CoA reductase in hepatocytes is partially blocked by lysosomotropic agents and insulin. A steady state model for the turnover of proteins subject to reversible phosphorylation has been developed which recognizes fractional degradative rate constants for the phosphorylated and dephosphorylated species.
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PMID:Phosphorylation state of HMG CoA reductase affects its catalytic activity and degradation. 302 50

The effects of amiloride and of natural aliphatic polyamines on basal and hormone-stimulated protein phosphorylations in hepatocytes were studied. Cells isolated from adult rats were incubated in suspension with (32P)-orthophosphate, in the absence or presence of the effectors at varying concentrations and for different times; hepatocytes were then exposed to various hormones for 10 min. Phosphoproteins contained in total cell lysates were analyzed by one- and two-dimensional gel electrophoresis and autoradiography. Amiloride and spermine (the most effective amine) decreased the basal level of phosphorylation of proteins of 46, 34 and 22 kDal, and increased that of 18 kDal and 93 kDal proteins. These effects were maximal with external concentrations of 1 mM and 7.5-10 mM amiloride and spermine, respectively. They were detectable after a lag period of about 10 min and reached a plateau after 45 min. Pretreatment of cells with these effectors almost completely prevented stimulation of the phosphorylation of the 46 and 34 kDal proteins by insulin. In contrast, the effects of vasopressin on the same proteins were only partly inhibited, whereas those of glucagon appeared largely unaffected. The major effect observed in intact cells (i.e., decreased phosphorylation) could be reproduced in a cell-free system where no kinase activity persisted. Amiloride or spermine added directly to cell extracts strongly accelerated the dephosphorylation of 46 kDal protein and also of the 61 kDal protein identified as pyruvate kinase. Furthermore, restoration of the activity of this enzyme occurred concomitantly with dephosphorylation of the 61 kDal protein, an observation supporting the notion that amiloride and spermine could activate a phosphoprotein phosphatase.
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PMID:Insulin regulation of protein phosphorylation in hepatocytes. Studies using two effectors: amiloride and natural aliphatic polyamines. 390 16

Acetyl-CoA carboxylase has been purified from lactating rat mammary gland using a combination of ammonium sulphate and poly(ethyleneglycol) precipitations. The enzyme was purified from 35--70-fold with a yield of over 50%, the exact figures being difficult to estimate because of activation of the enzyme that occurs during the preparation. The preparation was homogeneous by the criterion of polyacrylamide gel electrophoresis in sodium dodecyl sulphate and had a single subunit of molecular weight 240,000, containing 1.02 +/- 0.04 molecules of biotin and 3.1 +/- 1.7 molecules of alkali-labile phosphate per subunit. The purified enzyme was phosphorylated and inactivated rapidly when incubated in the presence of [gamma 32P]ATP and magnesium ions with the purified catalytic subunit of cyclic-AMP-dependent protein kinase from rabbit skeletal muscle. Both phosphorylation and inactivation are blocked by the heat-stable protein inhibitor of cyclic-AMP-dependent protein kinase, and can be reversed by incubation with purified protein phosphatase-1 from rabbit skeletal muscle. The inactivation by the protein kinase and reactivation by the protein phosphatase correlate with the near-stoichiometric phosphorylation and dephosphorylation of site(s) located in a single tryptic peptide. Phosphorylation does not affect the Km for substrates, but brings about a twofold decrease in V and a twofold increase in the apparent dissociation constant for the allosteric activator, citrate. We also present evidence that the activation of rabbit mammary acetyl-CoA carboxylase by protein phosphatase-1 described previously [Hardie and Cohen (1979) FEBS Lett. 103, 333-338] is due to dephosphorylation at site(s) which are not phosphorylated by either cyclic-AMP-dependent protein kinase or acetyl-CoA carboxylase kinase-2. These results suggest that the rapid inactivation of acetyl-CoA carboxylase, and hence fatty acid synthesis, by adrenaline in adipose tissue, or glucagon in the liver, is due to phosphorylation of the enzyme by cyclic-AMP-dependent protein kinase.
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PMID:Reversible phosphorylation and inactivation of acetyl-CoA carboxylase from lactating rat mammary gland by cyclic AMP-dependent protein kinase. 610 9

An ATP x Mg-dependent protein phosphatase (FC) was purified to near homogeneity from rabbit muscle. The enzyme was completely devoid of any spontaneous activity but could be activated by a protein activator (FA) in the presence of ATP and Mg ions. The inactive phosphatase migrated as a single protein band on sodium dodecyl sulfate-gel electrophoresis, and in discontinuous gel electrophoresis, where the potential phosphatase activity was located in the main protein band. The molecular weight determined by sodium dodecyl sulfate electrophoresis or by sucrose density centrifugation was found to be 70,000. FC migrated on gel filtration as a 140,000 molecular weight species. The activation by FA was not paralleled by an incorporation of [32P]-phosphate into the ATP x Mg-dependent phosphatase, and from the kinetics of activation a protein-protein interaction with ATP x Mg as a necessary factor, can be inferred as the mechanism of activation. After activation by FA and ATP X Mg, the purified enzyme had a specific activity of 10,000 units/mg of protein, and a Km for rabbit muscle phosphorylase a of approximately 1.0 mg/ml. The activated enzyme did not release [32P]phosphate from 32[-labeled rabbit muscle synthase b, prepared from glucagon-treated dogs. It did, however, remove all the 32P label from phosphorylase b kinase, autophosphorylated to the level of 2.0 mol/mol of 1.3 X 10(6) molecular weight.
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PMID:ATP x Mg-dependent protein phosphatase from rabbit skeletal muscle. I. Purification of the enzyme and its regulation by the interaction with an activating protein factor. 625 81

We have previously reported that the enzymic activity of rat liver-3-hydroxy-3-methyl-glutaryl-CoA reductase (NADPH) (HMG-CoA reductase) is modulated in vitro by a phosphorylation-dephosphorylation reaction sequence. The in vitro phosphorylation of HMG-CoA reductase was further studied by utilizing purified HMG-CoA reductase and reductase kinase. Analysis of 32P-labeled HMG-CoA reductase revealed 1 mol of phosphate per subunit. Purified [32P]HMG-CoA reductase could be dephosphorylated with phosphoprotein phosphatase. To demonstrate the in vivo phosphorylation, rats were injected with 32P and hepatic HMG-CoA reductase was isolated by immunoprecipitation and also by purification of the enzyme to homogeneity. Analysis of [32P]HMG-CoA reductase by sodium dodecyl sulfate gel electrophoresis revealed a single peak of radioactivity comigrating with HMG-CoA reductase. Administration of glucagon enhances the in vivo phosphorylation of both HMG-CoA reductase and reductase kinase. In response to glucagon, HMG-CoA reductase activity is decreased whereas reductase kinase activity is increased. These results support our concept that the enzymic activity of HMG-CoA reductase is modulated by a bicyclic cascade system involving phosphorylation-dephosphorylation. The enzymic activity of HMG-CoA reductase has also been shown to be modulated by cholesterol and mevalonolactone by both short-term and long-term mechanisms. The effects of cholesterol and mevalonolactone are twofold. Rapid inhibition of HMG-CoA reductase activity is due to increased phosphorylation of the enzyme; the long-term effect of HMG-CoA reductase is achieved by reduction in enzyme concentration by modulation of enzyme synthesis and/or degradation. Regulation of HMG-CoA reductase by mevalonolactone is of major importance in cellular metabolism because mevalonate serves as precursor for four separate metabolic pathways, including the formation of cholesterol, ubiquinone, dolichols, and isopentenyl tRNA.
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PMID:Modulation of rat liver 3-hydroxy-3-methylglutaryl-CoA reductase activity by reversible phosphorylation. 628 63


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