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: EC:4.6.1.1 (adenylate cyclase)
19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The properties associated with ligand interactions with membrane-bound beta adrenergic receptors prepared from L6 myoblasts were examined at five temperatures between 10 degrees and 30 degrees C. The interactions of antagonists with membrane-bound receptors were insensitive to temperature, whereas the interactions of agonists were temperature-dependent. The affinity constants for the low-affinity binding states of agonists (KL) decreased slightly with decreasing assay temperature. The small temperature-dependent changes in KL values were similar to the changes in Kd values observed in studies of the binding of agonists in the presence of GTP. The high-affinity dissociation constants (KH) for binding of full agonists to membrane-bound receptors in the absence of GTP were approximately 50-fold lower at 10 degrees than at 30 degrees C. The KH values for partial agonists also decreased with decreasing temperature, but the changes were smaller in magnitude. Thermodynamically, the binding of antagonists was primarily entropy-driven, whereas the binding of agonists was enthalpy-driven. The energetics of the low-affinity component of agonist binding to membrane-bound receptors were similar to the energetics for binding of agonists to membrane-bound receptors in the presence of GTP. Under these conditions, standard enthalpy change (delta H degree) and standard entropy change (delta S degree) values for binding of agonists were more negative than the corresponding values for binding of antagonists, possibly reflecting a conformational change in the receptor or an increased ordering of the lipids surrounding the receptor. The interaction of the receptor with the guanine nucleotide-binding protein to form the high-affinity component of agonist binding was thermodynamically described by larger negative changes in enthalpy and entropy than the values for formation of the low-affinity component of agonist binding. There was a correlation between the efficacies of ligands in activating adenylate cyclase and the delta H degree and delta S degree values for high-affinity binding of agonists. Thus, the extent or nature of the interaction between the guanine nucleotide-binding protein and the receptor may determine the efficacies of ligands.
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PMID:Thermodynamic properties of agonist interactions with the beta adrenergic receptor-coupled adenylate cyclase system. I. High- and low-affinity states of agonist binding to membrane-bound beta adrenergic receptors. 287 Jan 74

The thermodynamic parameters associated with the interactions of agonists and antagonists with digitonin-solubilized beta adrenergic receptors were determined. A rapid method for measuring the binding of [125I]iodopindolol to soluble receptors using glass-fiber filters was developed. The binding of [125I]iodopindolol, an antagonist with intrinsic sympathomimetic activity, to soluble receptors was temperature-sensitive as is the binding of the ligand to membrane-bound receptors. The interactions of propranolol and timolol with soluble receptors were independent of temperature. In contrast, the binding of agonists to soluble receptors was sensitive to temperature, although insensitive to GTP. Thermodynamically, the interactions of the antagonists timolol and propranolol with soluble beta adrenergic receptors were entropy-driven, with little contribution from changes in enthalpy. This is consistent with a hydrophobic interaction between the receptor and the antagonist. The binding of [125I]iodopindolol was enthalpy-driven. The binding of full agonists with soluble receptors was described thermodynamically by changes in enthalpy and entropy that were negative relative to the values for propranolol and timolol, suggesting that the guanine nucleotide-binding protein required for stimulation of adenylate cyclase activity and an intact lipid environment are not involved in the thermodynamics of formation of the low-affinity component of agonist binding. These results are consistent with an agonist-induced change in the conformation of the receptor.
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PMID:Thermodynamic properties of agonist interactions with the beta adrenergic receptor-coupled adenylate cyclase system. II. Agonist binding to soluble beta adrenergic receptors. 287 Jan 75

Low concentrations of ethanol (10-100 mM), added to assays in vitro, altered agonist (isoproterenol) binding to mouse cerebral cortical beta-adrenergic receptors in a reversible manner. Ethanol decreased the affinity of the high affinity form of the receptor for isoproterenol but had no effect on the affinity of the low affinity form of the receptor, the proportion of high and low affinity forms of the receptor, the total number of agonist-binding sites, or antagonist binding. The selective effect of ethanol on the properties of the high affinity agonist-binding site suggested that ethanol alters the characteristics of the complex of the receptor and Gs, the guanine nucleotide-binding protein. In cerebral cortical membranes of mice that had ingested ethanol chronically, isoproterenol binding data were best fit by a one-site model, even in the absence of guanine nucleotides. This change, when considered together with previously reported changes in adenylate cyclase activity, is reminiscent of heterologous desensitization of the beta-adrenergic receptor. Thus, both acute and chronic ethanol administration may produce changes in adrenergic function in brain.
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PMID:Effect of ethanol on mouse cerebral cortical beta-adrenergic receptors. 288 6

Somatostatin receptors from a rat pancreatic acinar cell line, AR4-2J, were characterized biochemically, structurally, and functionally. Binding of 125I-[Tyr11]somatostatin to AR4-2J cells was saturable, exhibiting a single class of high-affinity binding sites (Kd = 0.55 +/- 0.06 nM) with a maximal binding capacity of 258 +/- 20 fmol/10(6) cells. Somatostatin receptor structure was analyzed by covalently cross-linking 125I-[Tyr11]somatostatin to its plasma membrane receptors. Gel electrophoresis and autoradiography of cross-linked proteins revealed a peptide (Mr 80,000) containing the somatostatin receptor. Somatostatin inhibited vasoactive intestinal peptide (VIP)-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) formation in a dose-dependent manner. The concentration of somatostatin that caused half-maximal inhibition of cAMP formation (IC50 = 0.4 nM) was close to the receptor affinity for somatostatin. Pertussis toxin pretreatment of AR4-2J cells prevented somatostatin inhibition of VIP-stimulated cAMP formation as well as somatostatin binding. We conclude that AR4-2J cells exhibit functional somatostatin receptors that retain both specificity and affinity of the pancreatic acinar cell somatostatin receptors and act via the pertussis toxin-sensitive guanine nucleotide-binding protein Ni to inhibit adenylate cyclase.
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PMID:Functional somatostatin receptors on a rat pancreatic acinar cell line. 289 95

Isoproterenol increased the activity of the adenylate cyclase of rabbit heart sarcolemmal membranes in the absence of added GTP. ATP, the ATP-regenerating system, and the sarcolemmal membrane preparation were eliminated as possible sources of contaminating GTP. Isoproterenol-stimulation increased as ATP was raised. At 0.5 mM ATP, isoproterenol increased activity by 19% whereas at 5 mM ATP isoproterenol increased activity by 121%. There was no change in basal activity between 0.5 and 5 mM ATP. Stimulation by Gpp(NH)p and NaF increased slightly between 0.5 and 5 mM ATP; stimulation by KCl was unaffected. GTP does not activate cyclase d. GTP does not activate cyclase to the same extent as Gpp(NH)p even though the two act at the same site on Ns (the stimulatory guanine nucleotide-binding protein). GTP decreased cyclase activation by Gpp(NH)p in a concentration-dependent fashion when the two were added to the assay simultaneously. Increasing ATP from 0.5 to 5 mM did not reduce activation by Gpp(NH)p when both were added simultaneously to the assay. This suggests that ATP does not interact with the same site as Gpp(NH)p. ATP gamma S, an analogue of ATP which irreversibly thiophosphorylates proteins, did not irreversibly support activation by isoproterenol. The effect of ATP in supporting isoproterenol stimulation is not, therefore, thought to be due to phosphorylation of a protein.
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PMID:GTP-independent stimulation of rabbit heart adenylate cyclase by isoproterenol at physiological ATP concentrations. 292 4

Using modifications of the methods of Bokoch et al. (Bokoch, G.M., Katada, T., Northup, J. K., Ui, M., and Gilman, A. G. (1984) J. Biol. Chem. 259, 3560-3567) and Codina et al. (Codina, J., Hildebrandt, J. D., Sekura, R. D., Birnbaumer, M., Bryan, J., Manclark, C. R., Iyengar, R., and Birnbaumer, L. (1984) J. Biol. Chem. 259, 5871-5886), we have purified a pertussis toxin substrate with the expected characteristics of the inhibitory guanine nucleotide-binding protein (Ni) essentially to homogeneity. The purified protein consists of 3 subunits of Mr 40,000, 35,000, and less than 10,000. The Mr 40,000 band is found, upon close examination, to consist of a poorly resolved doublet. Starting with the membranes from 1,320 g of bovine forebrain we purified the protein some 100-fold with approximately 20% yield to obtain 13 mg of a greater than 95% pure protein. Chromatography on octyl-Sepharose provided efficient separation of Ni from Ns (the stimulatory guanine nucleotide-binding protein). Analytical ultracentrifugation indicates an Mr of 82,000 and a sedimentation coefficient S20,w of 5.1. The protein is able to restore opiate-mediated inhibition of adenylate cyclase to membranes prepared from NG 108-15 cells which had been treated with pertussis toxin. Bovine brain Ni has the enzymatic properties of a low Km GTPase with a turnover number of 0.3 and affinities for nucleotides in the order GppNHp greater than or equal to GTP greater than or equal to GDP much greater than ATP, CTP, UTP, and GMP. Na+ specifically stimulates the GTPase and low concentrations of Mg2+ (less than 50 microM) are inhibitory. Some Mg2+ is apparently necessary because EDTA, but not EGTA, abolishes the GTPase activity.
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PMID:The inhibitory guanine nucleotide-binding protein (Ni) purified from bovine brain is a high affinity GTPase. 298 5

Rat reticulocytes contain a cytosol activator protein (RCAP) that augments catecholamine-sensitive adenylate cyclase activity in reticulocyte membranes. A highly purified preparation of RCAP, obtained by Sephacryl S-200 chromatography, was used to elucidate further its mechanism of action. The specific activity of the S-200 fraction to augment isoproterenol responsiveness was increased approximately 1,100-fold over the starting material, from 1.2 to 1,300 nmol cAMP formed per mg RCAP. The mol wt of RCAP is approximately 20,000. The effect of RCAP to enhance isoproterenol responsiveness was apparent within 20 sec, virtually abolishing the normal lag time of hormone-activated adenylate cyclase. In addition to its effects on catecholamine-responsive adenylate cyclase, RCAP significantly increased basal [21 +/- 3 (+/- SEM) to 41 +/- 4 pmol/mg protein X 30 min; P less than 0.02], guanyl-5'-yl-imidodiphosphate-associated (3173 +/- 213 to 4339 +/- 365 pmol/mg X 30 min; P less than 0.03), and fluoride-associated (5152 +/- 64 to 5807 +/- 58 pmol/mg X 30 min; P less than 0.05) adenylate cyclase activities. RCAP also altered the characteristics of agonist binding to the beta-adrenergic receptor of reticulocyte membranes, causing an increase in the apparent IC50 for isoproterenol from 0.7 +/- 0.2 to 7.9 +/- 1.6 microM (P less than 0.001). Similar to its effects on reticulocytes, RCAP enhanced isoproterenol- and prostaglandin E2-sensitive adenylate cyclase activity in the wild-type S49 lymphoma cell and shifted the binding isotherm for isoproterenol rightward. In cyc-, the mutant that lacks the stimulatory guanine nucleotide-binding protein (Ns) and in UNC, the mutant in which receptors are uncoupled from N, RCAP was ineffective. Moreover, RCAP decreased agonist affinity for the beta-adrenergic receptor in wild-type S49 cells, but not in cyc- or UNC cells. These observations suggest that RCAP requires a functional Ns unit for its effects on hormone-sensitive adenylate cyclase activity.
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PMID:Cytosol activator protein from rat reticulocytes requires the stimulatory guanine nucleotide-binding protein for its actions on adenylate cyclase. 298 18

The radiolabeled agonist [3H]hydroxybenzylisoproterenol ([3H]HBI) and antagonist [125I]iodopindolol ([125I]IPIN) were used to investigate the properties of beta-adrenergic receptors on membranes prepared from L6 myoblasts and S49 lymphoma cells. The high affinity binding of (-)-[3H]HBI to membranes prepared from L6 myoblasts was stereoselectively inhibited by the active isomers of isoproterenol and propranolol. The density of receptors determined with (-)-[3H]HBI was less than that determined with [125I]IPIN. The binding of (-)-[3H]HBI was inhibited by guanine nucleotides, suggesting an agonist-mediated association of the receptor with a guanine nucleotide-binding protein, presumably the stimulatory guanine nucleotide-binding protein (Ns) of adenylate cyclase. Results obtained in studies with membranes prepared from wild-type S49 lymphoma cells and the adenylate cyclase-deficient variant (cyc-) were similar to those obtained in experiments carried out with membranes prepared from L6 myoblasts. Thus, the high affinity binding of (-)-[3H]HBI to membranes prepared from wild-type and cyc- S49 lymphoma cells was stereoselectively inhibited by the active isomers of isoproterenol and propranolol, and was inhibited by GTP. Moreover, the density of sites determined with (-)-[3H]HBI was less than that determined with [125I]IPIN. These results suggest either that cyc- cells contain a partially functional Ns, or alternatively, that the inhibitory guanine nucleotide-binding protein (Ni) is capable of interacting with beta-adrenergic receptors.
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PMID:Properties of beta-adrenergic receptors of cultured mammalian cells. Interaction of receptors with a guanine nucleotide-binding protein in membranes prepared from L6 myoblasts and from wild-type and cyc- S49 lymphoma cells. 299 14

Chronic treatment of neuroblastoma X glioma NG108-15 hybrid cells with 10 nM [D-Ala2,D-Leu5] enkephalin (DADLE) results in a reduction of cell-surface opiate delta receptors. Whether opiate receptor internalization requires the activation of the guanine nucleotide-binding protein (Ni) is unclear. Hence, activation of Ni was attenuated by treating hybrid cells with 100 ng/ml pertussis toxin (PT) for 3 h, which resulted in a decrease in DADLE's ability to inhibit adenylate cyclase activity. Despite this prior treatment with PT, chronic exposure of these cells to 10 nM DADLE resulted in a time-dependent decrease in both [3H]diprenorphine and [3H]DADLE binding. This reduction in 3H-ligand binding in cells previously treated with PT represented internalization of the receptors because translocation was observed of bound [3H]DADLE from plasma membrane fractions to the lysosomal fractions in the Percoll gradients. Thus, opiate receptors internalize without activation of Ni. The internalization of opiate receptors was not accompanied by Ni. By measuring the amount of the 41-kDa alpha subunit being labeled by PT with [32P]NAD+, it was determined that plasma membrane preparations, of both the control cells and cells treated with 10 nM of DADLE for 4 h, contained equal concentrations of Ni, 2 pmol of Ni/mg of protein. Additionally, there was no measurable alteration in the amount of PT substrate in the lysosomal fractions of the DADLE-treated cells as compared to that of control cells. Chronic DADLE treatment resulted in a decrease in Km value of NAD+ in the ADP-ribosylation of 41-kDa subunit of Ni. In summary, opiate receptors internalize as agonist-receptor complexes without the guanine nucleotide-binding component.
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PMID:Effect of pertussis toxin treatment on the down-regulation of opiate receptors in neuroblastoma X glioma NG108-15 hybrid cells. 299 25

Alpha 2-Adrenergic receptor-mediated inhibition of adenylate cyclase requires the guanine nucleotide-binding protein, Ni. This protein may also be required for stabilization of high-affinity alpha 2-adrenergic agonist binding. Human platelet membranes treated under alkaline conditions (pH 11.5) exhibited a selective loss of high-affinity agonist binding as measured by p-[3H]aminoclonidine and [3H]UK 14,304. Binding of the antagonist [3H]yohimbine was largely unaffected with retention of greater than 60% of control binding sites. Ni, determined by pertussis toxin-catalyzed [32P]ADP-ribosylation of cholate extracts from alkaline-treated membranes, was also markedly reduced. The parallel loss of alpha 2-agonist binding and Ni provides additional evidence that Ni is required for alpha 2-adrenergic agonist binding.
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PMID:Parallel inactivation of alpha 2-adrenergic agonist binding and Ni by alkaline treatment. 299 75


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