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)

Further modification of Neville's method of preparation of rat liver plasma membrane has been made in order to study glucagon-sensitive adenylate cyclase. This modified method introduces two additional steps to the Neville procedures. One involves saving an intermediate layer above the pellet following 1500g centrifugation. The other adds a centrifugation step at 20,000g. The improved method increases the yield of membrane protein by 5-fold and increases forskolin, glucagon, and 5'-guanyl imidodiphosphate stimulated activity of adenylate cyclase by 2.8-fold. A 13-fold increase in the yield of total adenylate cyclase activity above the current method was obtained. The membrane adenylate cyclase and its hormone sensitivity was stable in liquid nitrogen for at least 8 months. This modified method appears to be useful in preparing a better yield and quality of rat liver plasma membrane from given starting hepatic tissue for studies of glucagon-sensitive adenylate cyclase.
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PMID:Preparation of rat liver plasma membrane with respect to glucagon-sensitive adenylate cyclase. 376 66

The photoaffinity crosslinker hydroxysuccinimidyl-p-azidobenzoate was used to attach (125)I-labeled glucagon covalently to a rat liver membrane protein of M(r) approximately 53,000. Membranes that had been incubated with (125)I-labeled glucagon were treated in the dark with hydroxysuccinimidyl-p-azidobenzoate, and a covalent complex was then formed by irradiation with ultraviolet light. Characteristics of (125)I-labeled glucagon binding and covalent attachment to the M(r) 53,000 peptide were consistent with this peptide being a component of the glucagon receptor involved in the activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. Binding and covalent attachment of (125)I-labeled glucagon to the M(r) 53,000 peptide were inhibited by glucagon concentrations that were within the dose-response curve for adenylate cyclase activation, and GTP specifically decreased the photoaffinity crosslinking of (125)I-labeled glucagon to the M(r) 53,000 peptides. Insulin did not compete for the photoaffinity crosslinking of (125)I-labeled glucagon. The same technique of photoaffinity crosslinking that covalently attached (125)I-labeled glucagon to the M(r) 53,000 peptide with an efficiency of 1-2% can be used to attach (125)I-labeled insulin covalently to a M(r) 125,000 peptide with an efficiency of approximately 10%. This peptide has been shown to be a subunit of the high-affinity insulin-binding site in rat liver membranes. The technique of photoaffinity crosslinking with agents like hydroxysuccinimidyl-p-azidobenzoate provides a rapid, simple method of covalently attaching ligands to their putative receptors. Photoaffinity crosslinking does not require chemical modification of the labeled ligand and has a less stringent requirement for specific reactive groups than the commonly used bifunctional crosslinking reagents.
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PMID:Identification of the glucagon receptor in rat liver membranes by photoaffinity crosslinking. 626 79

Specific binding sites for secretin have been identified in rat fundic membranes, using 125I-secretin. The binding was saturable, reversible, time and temperature dependent. Optimal pH for binding was around 7-7.5. Scatchard plots were compatible with the existence of 2 classes of receptors; the first class with a high affinity for secretin (apparent Kd of 4 x 10(-10) M) and a low binding capacity (150 fmol per mg membrane protein, i.e., 4,500 high affinity sites/cell) and a class of receptors with a lower affinity (Kd of 3 x 10(-9) M) and a higher binding capacity (580 fmol per mg membrane protein i.e., 17,400 sites/cell). Glucagon, gastric inhibitory polypeptide and somatostatin had no-effect on secretin binding. In contrast, VIP inhibited 125I-secretin binding and stimulated adenylate cyclase activity, in both cases at a 200-times lower potency than secretin (ID50 and Ka = 2 x 10(-7) M VIP). The properties of these secretin receptors strongly support the concept that secretin acts as a regulatory peptide on the rat gastric epithelium.
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PMID:Secretin binding sites coupled with adenylate cyclase in rat fundic membranes. 628 94

A marked resistance to the stimulatory action of insulin on glucose metabolism has previously been shown in guinea pig, compared to rat, adipose tissue and isolated adipocytes. The mechanism of insulin resistance in isolated guinea pig adipocytes has, therefore, been examined by measuring 125I-insulin binding, the stimulatory effect of insulin on 3-0-methylglucose transport and on lipogenesis from [3-3H]glucose, the inhibitory effect of insulin on glucagon-stimulated glycerol release, and the translocation of glucose transporters in response to insulin. The translocation of glucose transporters was assessed by measuring the distribution of specific D-glucose-inhibitable [3H]cytochalasin B binding sites among the plasma, and high and low density microsomal membrane fractions prepared by differential centrifugation from basal and insulin-stimulated cells. At a glucose concentration (0.5 mM) where transport is thought to be rate-limiting for metabolism, insulin stimulates lipogenesis from 30 to 80 fmol/cell/90 min in guinea pig cells and from 25 to 380 fmol/cell/90 min in rat cells with half-maximal effects at approximately 100 pM in both cell types. Insulin similarly stimulates 3-O-methylglucose transport from 0.40 to 0.70 fmol/cell/min and from 0.24 to 3.60 fmol/cell/min in guinea pig and rat fat cells, respectively. Nevertheless, guinea pig cells bind more insulin per cell than rat cells, and insulin fully inhibits glucagon-stimulated glycerol release. In addition, the differences between guinea pig and rat cells in the stimulatory effect of insulin on lipogenesis and 3-O-methylglucose transport cannot be explained by the greater cell size of the former compared to the latter (0.18 and 0.09 micrograms of lipid/cell, respectively). However, the number of glucose transporters in the low density microsomal membrane fraction prepared from basal guinea pig cells is markedly reduced compared to that from rat fat cells (12 and 70 pmol/mg of membrane protein, respectively) and the translocation of intracellular glucose transporters to the plasma membrane fraction in response to insulin is correspondingly reduced. These results suggest that guinea pig adipocytes are markedly resistant to the stimulatory action of insulin on glucose transport and that this resistance is the consequence of a relative depletion in the number of intracellular glucose transporters.
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PMID:Proposed mechanism of insulin-resistant glucose transport in the isolated guinea pig adipocyte. Small intracellular pool of glucose transporters. 634 23

Specific binding sites for vasoactive intestinal peptide were characterized in plasma membranes from rat intestinal epithelial cells. At 30 degrees C, the interaction of 125I-labelled peptide with intestinal membranes was rapid, reversible, specific and saturable. At equilibrium, the binding of 125I-labelled peptide was competitively inhibted by native peptide in the 3 . 10(-11)--3 . 10-(7) M range concentration. Scatchard analysis of binding data suggested the presence of two distinct classes of vasoactive intestinal peptide binding sites: a class with a high affinity (Kd = 0.28 nM) and a low capacity (0.8 pmol peptide/mg membrane protein) and a class with a low affininty (Kd = 152 nM) and a high capacity (161 pmol peptide/mg membrane protein). Secretin competitively inhibited binding of 125I-labelled peptide but its potency was 1/1000 that of native peptide. Glucagon and the gastric inhibitory peptide were ineffective. The guanine nucleotides, GTP and Gpp(NH)p inhibited markedly the interaction of 125I-labelled peptide with its binding sites, by increasing the rate of dissociation of peptide bound to membranes. The other nucleotides triphosphate tested (ATP, ITP, UTP, CTP) were also effective in inhibiting binding of 125I-labelled peptide to membranes but their potencies were 1/100--1/1000 that of guanine nucleotides. The specificity and affinity of the vasoactive intestinal peptide-binding sites in plasma membranes prepared from rat intestinal epithelial cells, which is in agreement with an adenylate cyclase highly sensitive to the peptide recently characterized in these membranes (Amiranoff, B., Laburthe, M., Dupont, C. and Rosselin, G. (1978) Biochim. Biophys. Acta 544, 474--481) further argue for a physiological role of the peptide in the regulation of intestinal epithelial function.
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PMID:Characterization of specific binding sites for vasoactive intestinal peptide in rat intestinal epithelial cell membranes. 735 Sep 25

We have found [125I]glucagon-like peptide (GLP)-1(7-36)amide specific binding activity in rat liver and isolated hepatocyte plasma membranes, with an M(r) of approximately 63,000, estimated by cross-linking and SDS-PAGE. The specific binding was time- and membrane protein concentration-dependent, and equally displaced by unlabelled GLP-1(7-36)amide and by GLP-1(1-36)amide, achieving its ID50 at 3 x 10(-9) M of the peptides. GLP-1(7-36)amide did not modify the basal or the glucagon (10(-8) M)-stimulated adenylate cyclase in the hepatocyte plasma membranes. These data, together with our previous findings of a potent glycogenic effect of GLP-1(7-36)amide in isolated rat hepatocytes, led us to postulate that the insulin-like effects of this peptide on glucose liver metabolism could be mediated by a type of receptor probably different from that described for GLP-1 in pancreatic B-cells or, alternatively, by the same receptor which, in this tissue as well as in muscle, uses a different transduction system.
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PMID:Glucagon-like peptide-1 binding to rat hepatic membranes. 756 16

The human glucagon receptor was expressed at high density in Drosophila Schneider 2 (S2) cells. Following selection with G418 and induction with CuSO4, the cells expressed the receptor at a level of 250 pmol/mg of membrane protein. The glucagon receptor was functionally coupled to increases in cyclic AMP in S2 cells. Protein immunoblotting with anti-peptide antibodies revealed the expressed receptor to have an apparent molecular mass of 48 kDa, consistent with low levels of glycosylation in this insect cell system. Binding of [fluorescein-Trp25]glucagon to S2 cells expressing the glucagon receptor was monitored as an increase in fluorescence anisotropy along with an increase in fluorescence intensity. Anisotropy data suggest that the mobility of the fluorescein is restricted when the ligand is bound to the receptor. Kinetic analysis indicates that the binding of glucagon to its receptor proceeds via a bimolecular interaction, with a forward rate constant that is several orders of magnitude slower than diffusion-controlled. These data would be consistent with a conformational change upon the binding of agonist to the receptor. The combination of [fluorescein-Trp25]glucagon with the S2 cell expression system should be useful for analyzing glucagon receptor structure and function.
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PMID:Interaction of [fluorescein-Trp25]glucagon with the human glucagon receptor expressed in Drosophila Schneider 2 cells. 759 63

We have found [125I]glucagon-like peptide-1(7-36)-amide-specific binding activity in rat skeletal muscle plasma membranes, with an estimated M(r) of 63,000 by cross-linking and SDS-PAGE. The specific binding was time and membrane protein concentration dependent, and displaceable by unlabeled GLP-1(7-36)-amide with an ID50 of 3 x 10(-9) M of the peptide; GLP-1(1-36)-amide also competed, whereas glucagon and insulin did not. GLP-1(7-36)-amide did not modify the basal adenylate cyclase activity in skeletal muscle plasma membranes. These data, together with our previous finding of a potent glycogenic effect of GLP-1(7-36)-amide in rat soleus muscle, and also in isolated hepatocytes, which was not accompanied by a rise in the cell cyclic AMP content, lead use to believe that the insulin-like effects of this peptide on glucose metabolism in the muscle could be mediated by a type of receptor somehow different to that described for GLP-1 in pancreatic B cells, where GLP-1 action is mediated by the cyclic AMP-adenylate cyclase system.
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PMID:Glucagon-like peptide-1 binding to rat skeletal muscle. 778 53

Specific binding of [125I]glucagon-like peptide-1(7-36)amide ([125I]GLP-1(7-36)amide) to solubilized rat adipose tissue membranes was found to be dependent on temperature, time, and membrane protein concentration and readily dissociated. GLP-1(1-36)amide, GLP-2, or glucagon (10(-6) M) did not compete with [125I]GLP-1(7-36)amide binding. Half-maximal binding was achieved with 8 x 10(-10) M unlabeled GLP-1(7-36)amide, and the Scatchard plot revealed the presence of high and low affinity binding sites with Kd values of approximately 0.6 and 20 nM, respectively. The binding capacity of [125I]GLP-1(7-36)amide was about 3 times higher than that of [125I]glucagon, while the high affinity Kd and the half-maximal binding of the two peptides were similar. The presence and abundance of GLP-1(7-36)amide receptors in fat tissue together with the previous findings that the peptide stimulates glycerol and cAMP production in rat adipocytes and stimulates fatty acid synthesis in explants of rat adipose tissue open the possibility that this insulinotropic intestinal peptide may also be involved in the regulation of lipid metabolism in health and disease.
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PMID:Presence and characterization of glucagon-like peptide-1(7-36) amide receptors in solubilized membranes of rat adipose tissue. 838 Mar 88

Sucrose density centrifugation was used to prepare a partially purified membrane fraction from the mammary glands of non-pregnant, pregnant and lactating sheep. The binding of 125I-labelled insulin-like growth factor-I (IGF-I) was dependent on membrane protein concentration, pH, time and temperature. The binding showed the characteristics of a type-1 IGF receptor, being displaced by IGF-I (median effective dose (ED50) 0.55 nmol/l), less effectively by IGF-II (ED50 8.8 nmol/l) and least effectively by insulin. Glucagon, ovine prolactin and ovine placental lactogen could not displace binding. A molecular weight of 135,000 was determined by affinity cross-linking using disuccinimidyl suberate; this was consistent with the reported size of the type-1 receptor alpha-subunit. Scatchard analysis was used to determine binding affinity and numbers of IGF-I-binding sites. A single class of high-affinity binding sites was found in all physiological states. In non-pregnant sheep and sheep at days 40, 75 and 110-120 of pregnancy and at term, the binding affinity was similar (apparent dissociation constant (Kd) 2.73 +/- 0.31 nmol/l, n = 22). In lactating sheep (weeks 1, 4 and 10), the binding affinity was significantly (P = 0.02) higher (Kd 0.77 +/- 0.06 nmol/l n = 9). Binding capacity was similar in non-pregnant and pregnant sheep (1005 +/- 113 fmol/mg, n = 19), but fell by parturition and remained low in lactation (570 +/- 52 fmol/mg membrane protein, n = 12). The results suggest that the mammary growth of pregnancy is not regulated at the level of the type-1 IGF receptor.
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PMID:Receptor binding of insulin-like growth factor-I to mammary microsomes from non-pregnant, pregnant and lactating sheep. 845 95


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