Showing papers on "Cyclase published in 1982"
TL;DR: It is shown here that EF is an adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1] produced by Bacillus anthracis in an inactive form and nearly equals that of the most active known cyclase.
Abstract: Anthrax toxin is composed of three proteins: protective antigen (PA), lethal factor (LF), and edema factor (EF). These proteins individually cause no known physiological effects in animals but in pairs produce two toxic actions. Injection of PA with LF causes death of rats in 60 min, whereas PA with EF causes edema in the skin of rabbits and guinea pigs. The mechanisms of action of these proteins have not been determined. It is shown here that EF is an adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] produced by Bacillus anthracis in an inactive form. Activation occurs upon contact with a heat-stable eukaryotic cell material. The specific activity of the resulting adenylate cyclase nearly equals that of the most active known cyclase. In Chinese hamster ovary cells exposed to PA and EF, cAMP concentrations increase without a lag to values about 200-fold above normal, remain high in the continued presence of toxin, and decrease rapidly after its removal. The increase in cAMP is completely blocked by excess LF. It is suggested that PA interacts with cells to form a receptor system by which EF and perhaps LF gain access to the cytoplasm.
926 citations
TL;DR: It is concluded that islet-activating protein enhances receptor-mediated GTP-induced activation of membrane adenylate cyclase as a result of ADP-ribosylation of a membrane protein, probably one of the components of the receptor-adenylatecyclase system.
Abstract: GTP and isoproterenol activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in washed membranes prepared from C6 gliomas cells was enhanced by incubation with islet-activating protein, one of the pertussis toxins, if the incubation mixture was supplemented with NAD and ATP. The action of the protein was observed immediately after its addition and increased progressively in magnitude as the protein concentration or the incubation time increased. There was simultaneous incorporation of radioactivity from the ADP-ribose moiety of variously labeled NAD into the membrane protein with a molecular weight of 41,000. We conclude that islet-activating protein enhances receptor-mediated GTP-induced activation of membrane adenylate cyclase as a result of ADP-ribosylation of a membrane protein, probably one of the components of the receptor-adenylate cyclase system.
770 citations
TL;DR: The requirement for Al3+ is highly specific; of 28 other metals tested, only Be2+ promoted activation of G/F by F-.
Abstract: Activation of the purified guanine nucleotide-binding regulatory component (G/F) of adenylate cyclase by F- requires the presence of Mg2+ and another factor. This factor, which contaminates commercial preparations of various nucleotides and disposable glass test tubes, has been identified as Al3+. In the presence of 10 mM Mg2+ and 5 mM F-, AlCl3 causes activation of G/F with an apparent activation constant of approximately 1-5 muM. The requirement for Al3+ is highly specific; of 28 other metals tested, only Be2+ promoted activation of G/F by F-.
591 citations
TL;DR: It is concluded that IAP, added to intact cells or isolated membranes, causes unique modification of the receptor-adenylate cyclase coupling mechanism as a result of ADP ribosylation of the Mr = 41,000 protein which is presumably one of the subunits, other than the cholera toxin substrates, of the guanine nucleotide regulatory component of the cyclase system.
561 citations
TL;DR: There is no reversal of activation or binding under activating conditions and the rate constant for activation is unchanged from 10 mi to 100 PM GTPyS, so reversal of binding and activation, which occurs only in the absence of divalent cation, is not a first order process.
486 citations
TL;DR: This work has shown that Bordetella species elaborate a soluble, heat-stable, and highly active adenylate cyclase that is internalized by phagocytic cells and catalyzes the unregulated formation of adenosine 3',5'-monophosphate (cyclic AMP), thereby disrupting normal cellular function.
Abstract: For unknown reasons, humans infected with the bacterium Bordetella pertussis are exceptionally vulnerable to secondary infections. Bordetella species elaborate a soluble, heat-stable, and highly active adenylate cyclase. This enzyme is internalized by phagocytic cells and catalyzes the unregulated formation of adenosine 3',5'-monophosphate (cyclic AMP), thereby disrupting normal cellular function. This unusual phenomenon may explain Bordetella-induced aphylaxis and may prove to be useful for investigating a variety of cyclic AMP-governed processes.
434 citations
TL;DR: It is reported here that the toxin also catalyzes ADP-ribosylation of the alpha-subunit of transducin in ROS membranes, which indicates close functional homology between trans Ducin of ROS and the N protein of adenylate cyclase.
201 citations
01 Jan 1982
TL;DR: In this paper, it was shown that although forskolin can stimulate C in intact cells, full expression of maximal response to for-skolin and potentia- tion of hormonal response by the diterpene require the presence of functional N.membranes and intact cells.
Abstract: membranes and intact cells. We find that although forskolin can stimulate C in intact cells, full expression of maximal response to forskolin and potentia- tion of hormonal response by the diterpene require the pres- ence of functional N. Potentiation of hormonal response by forskolin can occur even in cells that are desensitized to isoproterenol. In addition, we show that forskolin may alter N
200 citations
TL;DR: The activation of cyclic AMP systems by forskolin is rapid and reversible, and appears to involve both direct activation of adenylate cyclase and facilitation and/or enhancement of receptor‐mediated activation of the enzyme.
Abstract: The diterpene forskolin markedly activates adenylate cyclase in membranes from various rat brain regions and elicits marked accumulations of radioactive cyclic AMP in adenine-labeled slices from cerebral cortex, cerebellum, hippocampus, striatum, superior colliculi, hypothalamus, thalamus, and medulla-pons. In cerebral cortical slices, forskolin has half-maximal effects at 20-30 microM on cyclic AMP levels, both alone and in the presence of the phosphodiesterase inhibitor ZK 62771. The presence of a very low dose of forskolin (1 microM) can augment the response of brain cyclic AMP-generating systems to norepinephrine, isoproterenol, histamine, serotonin, dopamine, adenosine, prostaglandin E2, and vasoactive intestinal peptide. Forskolin does not augment responses to combinations of histamine-norepinephrine adenosine-norepinephrine, or histamine-adenosine. For norepinephrine and isoproterenol in rat cerebral cortical slices and for histamine in guinea pig cerebral cortical slices, the presence of 1 microM-forskolin augments the apparent efficacy of the amine, whereas for adenosine, prostaglandin E2, and vasoactive intestinal peptide, the major effect of 1 microM-forskolin is to increase the apparent potency of the stimulatory agent. In rat striatal slices, forskolin reveals a significant response of cyclic AMP systems to dopamine and augments the dopamine-elicited activation of adenylate cyclase in rat striatal membranes. The activation of cyclic AMP systems by forskolin is rapid and reversible, and appears to involve both direct activation of adenylate cyclase and facilitation and/or enhancement of receptor-mediated activation of the enzyme.
198 citations
TL;DR: A functional linkage between serotonergic and noradrenergic systems in the rat cortex is demonstrated, with beta- adrenergic receptors and neurohormonal sensitivity of the adenosine 3',5'-monophosphate-generating system being under separate regulatory control.
Abstract: The action of desipramine on the norepinephrine-sensitive adenylate cyclase system and the density of beta-adrenergic receptors in rat cortex was studied after selective lesioning of serotonergic neurons with 5,7-dihydroxytryptamine. In animals with lesions desipramine failed to reduce the density of beta-adrenoceptors but decreased the response of adenosine 3',5'-monophosphate to isoproterenol and norepinephrine to the same degree as in animals without lesions. The results demonstrate a functional linkage between serotonergic and noradrenergic systems in the rat cortex, with beta-adrenergic receptors and neurohormonal sensitivity of the adenosine 3',5'-monophosphate-generating system being under separate regulatory control.
174 citations
TL;DR: The initial rate of activation of the regulatory component of adenylate cyclase by toxin was found to be linearly related to the amount of factor present in the reaction, which has allowed the quantitation and partial purification of the factor from turkey erythrocyte membranes.
TL;DR: IAP would appear to enhance beta-receptor-coupled stimulation of adenylate cyclase, in a manner distinct from cholera toxin, by rendering more GTP available to the GTP sites on the regulatory subunit of the receptor-enzyme system.
TL;DR: The molecular basis of the unresponsiveness of the cells from the rachitic child appears to be due to defective or absent 1,25(OH)2D3 receptors.
Abstract: Lymphocyte adenylate cylase activity is reduced in aging man, despite unaltered beta-adrenergic receptor characteristics. To define the component of human lymphocyte adenylate cyclase which is reduced in aging, we assessed nucleotide-regulatory protein (N-protein) function by complementation of eye- S49 mouse lymphoma cells and catalytic unit activity by forskolin stimulation. Within the enzyme complex, N-protein function is unchanged, while catalytic unit activity is 3.6 fold greater in young than elderly. The decreased responsiveness to catecholamines seen in aging man may be due, in some tissues, to reduced adenylate cyclase activity, specifically, the catalytic unit of the enzyme complex.
TL;DR: A general hypothesis is proposed, which proposes that hormone binding to receptors leads to the release of previously bound GDP (an ineffective stimulator), which allows occupancy by GTP and thus attainment of a more active R s • N s • C complex.
TL;DR: There are two at least partially distinct regulatory sites involved in opposing hormonal regulations of adenylate cyclase activity, with that involved in hormonal inhibition being highly susceptible to inactivation by NEM.
Journal Article•
TL;DR: The results indicate that forskolin increases cyclic AMP production in adipocytes through an activation of adenylate cyclase, and lipolysis is activated by forSkolin but at higher concentrations of total cyclicAMP than for catecholamines.
Abstract: Forskolin increased cyclic AMP accumulation in isolated adipocytes and markedly potentiated the elevation of cyclic AMP due to isoproterenol. In adipocyte membranes, forskolin stimulated adenylate cyclase activity at concentrations of 0.1 microM or greater. Forskolin did not affect the EC50 for activation of adenylate cyclase but did increase the maximal effect of isoproterenol. Neither the soluble nor particulate low-Km cyclic AMP phosphodiesterase activity was affected by forskolin. Low concentrations of forskolin (0.1-1.0 microM), which significantly elevated cyclic AMP levels, did not increase lipolysis, whereas similar increases in cyclic AMP levels due to isoproterenol elevated lipolysis. Forskolin did not inhibit the activation of triacylglycerol lipase by cyclic AMP-dependent protein kinase or the subsequent hydrolysis of triacylglycerol. Higher concentrations of forskolin (10-100 microM) did increase lipolysis. Both the increased cyclic AMP production and lipolysis due to forskolin were inhibited by the antilipolytic agents insulin and N6-(phenylisopropyl)adenosine. Hypothyroidism reduced the ability of forskolin to stimulate cyclic AMP production and lipolysis. These results indicate that forskolin increases cyclic AMP production in adipocytes through an activation of adenylate cyclase. Lipolysis is activated by forskolin but at higher concentrations of total cyclic AMP than for catecholamines.
TL;DR: Data suggest that MLTC-1 cells possess a gonadotropin- responsive adenylate cyclase system consisting of a specific hormone receptor, a regulatory component, and a catalytic subunit.
Abstract: Several clonal Leydig tumor cell lines have been established by adapting the transplantable Leydig tumor, M548OP, to culture. One of these cell line, MLTC-1, has been characterized with regard to the gonadotropin-responsive adenylate cyclase system. The binding of 125I-labeled human chorionic gonadotropin (hCG) was blocked by excess unlabeled hCG and lutropin (LH) but not by follitropin, thyrotropin, or insulin, indicating the presence of specific receptors for hCG and LH. Based on the specific binding of hCG to isolated MLTC-1 membranes, the calculated dissociation constant was 1.0 +/- 0.2 X 10(-10) M. The receptors appeared identical to those from normal murine Leydig cells when analyzed by SDS PAGE and sucrose density gradient centrifugation. The molecular weight and sedimentation coefficient were 95,000 daltons and 8.5 S, respectively. MLTC-1 cells responded to hCG by accumulating cyclic AMP and producing progesterone. Cyclic AMP accumulation was time- and dose-dependent with a maximal accumulation occurring at approximately 0.2 nM hCG. At saturating levels of hCG, cAMP levels reached a maximum by 30 min and then declined very slowly. Adenylate cyclase activity in membranes prepared from MLTC-1 cells was stimulated by hCG, LH, NaF, cholera toxin, and guanyl-59-ylimidodiphosphate, Additionally, choleragen was found to ADP-ribosylate a membrane protein of 54,000 daltons. This protein resembles the proposed guanine nucleotide regulatory component in both size and choleragen-dependent reactivity. These data suggest that MLTC-1 cells possess a gonadotropin-responsive adenylate cyclase system consisting of a specific hormone receptor, a regulatory component, and a catalytic subunit.
TL;DR: The results provide a mechanism for the dual requirement for Na+ and GTP in the inhibitory coupling of opiate receptors to the adenylate cyclase system in these cells and may be of general significance to the action of other inhibitory hormones.
TL;DR: Results indicate that the inhibition of CRF, (-)-isoproterenol, and VIP-stimulated ACTH secretion by SRIF may be regulated by its inhibitory action on adenylate cyclase.
Abstract: The AtT-20/D16-16 mouse pituitary tumor cell secretes corticotropin (ACTH) in response to corticotropin-releasing factor (CRF), (-)-isoproterenol, and vasoactive intestinal peptide (VIP). These responses are associated with a rapid increase in cyclic AMP formation. Somatostatin (SRIF) markedly decreases the stimulatory effect of CRF, (-)-isoproterenol, and VIP on both cyclic AMP formation and immunoreactive ACTH secretion. Forskolin and cholera toxin, adenylate cyclase activators, also stimulate cyclic AMP formation and ACTH secretion in AtT-20 cells and these responses are all inhibited by SRIF. The ACTH secretory responses to melittin and to the calcium ionophore A23187, neither of which increases cyclic AMP in AtT-20 cells, were not inhibited by SRIF. SRIF did not affect the binding of a tritiated beta-adrenergic receptor antagonist to AtT-20 membranes nor did it decrease basal cyclic AMP formation even in the presence of excess phosphodiesterase inhibitor, indicating that the reduction of cyclic AMP levels by SRIF did not involve either an interference with beta-adrenergic agonist binding to receptors or stimulation of cyclic AMP degradation. These results indicate that the inhibition of CRF-, (-)-isoproterenol-, and VIP-stimulated ACTH secretion by SRIF may be regulated by its inhibitory action on adenylate cyclase.
TL;DR: Plasma membranes from rat striatum contain adenylate cyclase activity that is subject to dual regulation by GTP; the potencies of the opiates in mediating these effects mirror their affinities for 8 opiate receptors in striatum.
Abstract: Plasma membranes from rat striatum contain adenylate cyclase activity that is subject to dual regulation by GTP. Low concentrations (up to 30 nM) of the nucleotide increase activity whereas higher concentrations evoke a steady decline in activity; such behavior characterizes dually regulated adenylate cyclase systems. The opiates, morphine sulfate and D-Ala-Met-enkephalin, produce naloxone-reversible inhibition of the enzyme that is dependent on "inhibitory concentrations" of GTP (above 50 nM). In the absence of GTP no inhibition is observed. Sodium ions decrease the inhibition of activity promoted by GTP alone, but amplify the degree of inhibition seen in the presence of the opiates and GTP. The potencies of the opiates in mediating these effects mirror their affinities for delta opiate receptors in striatum. It is suggested that this action of the opiates may represent their primary action in striatum.
TL;DR: It is suggested that a reduced beta-adrenergic function is not an intrinsic component of allergic bronchial asthma but is rather the consequence of an active disease state, and that alterations distal to the receptor may play an important role in the observed decrease in beta- adrenergic responsiveness.
Abstract: Beta-adrenergic receptor characteristics and adenylate cyclase responses to different stimuli were measured in lymphocyte membrane preparations of six normal control subjects and five allergic asthmatic patients with mild airways disease and increased bronchial reactivity to histamine and acetylcholine. The determinations were performed just before and 24 hr after inhalation challenge with house-dust mite antigen to investigate the influence of an allergen-induced asthmatic attack on the beta-adrenergic receptor system. Before the house-dust mite challenge, the lymphocyte membranes of the patients showed a normal receptor density, dissociation constant for (-)3H-dihydroalprenolol, and adenylate cyclase response to isoproterenol, guanyl-5'-yl-imidodiphosphate, (GppNHp) and NaF. After the allergen-induced asthmatic reaction, however, the beta-adrenergic receptor number in the patients was significantly reduced by 21%, while the basal adenylate cyclase activity and isoproterenol-, GppNHp-, and NaF-induced cyclic AMP responses were simultaneously reduced by about 40%. The allergen challenge had no significant effect on the lymphocyte membranes of the control subjects. These results suggest (1) that a reduced beta-adrenergic function is not an intrinsic component of allergic bronchial asthma but is rather the consequence of an active disease state, and (2) that next to changes in beta-adrenergic receptor number, alterations distal to the receptor may play an important role in the observed decrease in beta-adrenergic responsiveness.
TL;DR: The ability of angiotensin II to inhibit cyclic AMP synthesis may provide an explanation for the observed effects of guanine nucleotides on 125I-angiotens in II binding to plasma membranes.
TL;DR: Epinephrine induces human platelet aggregation by interacting with α-adrenergic receptors by demonstrating the specificity of α2-receptors with the affinity of yohimbine » prazosin by radioligand-binding techniques using the new antagonist ligand, [3H]yOHimbine.
Abstract: Epinephrine induces human platelet aggregation by interacting with alpha-adrenergic receptors. These sites were demonstrated by radioligand-binding techniques using the new antagonist ligand, [3H]yohimbine. The sites labeled by [3H]yohimbine had the specificity of alpha 2-receptors with the affinity of yohimbine much greater than prazosin. Epinephrine-mediated inhibition of prostaglandin E1-stimulated adenylate cyclase activity in human platelet lysates was also found to have an alpha 2-receptor specificity. Competition curves of antagonists with [3H]yohimbine indicated a homogeneous population of alpha 2-receptors. In contrast, competition curves of a series of full and partial agonists with [3H]yohimbine were resolved into two distinct affinity states; the ratio of the dissociation constants of agonists for the low and high affinity states was positively correlated with the agonist's intrinsic activity for inhibition of adenylate cyclase. Guanine nucleotides were found to destabilize the high affinity form of the alpha 2-receptors. At high nucleotide concentrations, all high affinity states of the receptor were converted to the low affinity form. The formation of the high affinity agonist-binding state may reflect an interaction between the agonist-receptor complex and an additional membrane component, and probably reflects events involved in alpha 2-receptor-adenylate cyclase coupling.
TL;DR: Adenylate cyclase in Drosophila melanogaster heads is stimulated 5–6‐fold by low concentrations of octopamine, and prolonged incubation with dopamine in vitro abolishes adenylates cyclase basal activity as well as responsiveness to guanyl nucleotides, NaF, and putative neurotransmitters.
Abstract: Adenylate cyclase in Drosophila melanogaster heads is stimulated 5-6-fold by low concentrations of octopamine. The octopamine stimulation is inhibited by low concentrations of the alpha-adrenergic ligands phentolamine and dihydroergotamine and of chlorpromazine, but not by low concentrations of the beta-antagonist propranolol and by the alpha-antagonist yohimbine. d-Tubocurarine enhances the octopamine effect. Tyramine, norepinephrine, and epinephrine also stimulate the cyclase, probably via the octopamine receptor. Serotonin and dopamine stimulate Drosophila adenylate cyclase 1.3-1.4-fold; at least the latter putative neurotransmitter seems to interact with a receptor distinct from the octopamine receptor. Prolonged incubation with dopamine in vitro abolishes adenylate cyclase basal activity as well as responsiveness to guanyl nucleotides, NaF, and putative neurotransmitters.
TL;DR: The observation suggests that upon addition of glucose to yeast cells cyclic AMP functions as the mediating signal for the protein kinase catalyzed phosphorylation of fructose-1,6-bisphosphatase.
TL;DR: A procedure for the functional reconstitution of beta-adrenergic receptors and the stimulatory guanine nucleotide-binding protein (G/F) of adenylate cyclase in phospholipid vesicles is described, which restored the ability to bind [125I]iodohydroxybenzylpindolol and [3H]dihydroalprenolol.
Abstract: A procedure for the functional reconstitution of beta-adrenergic receptors and the stimulatory guanine nucleotide-binding protein (G/F) of adenylate cyclase in phospholipid vesicles is described. beta-Adrenergic receptors were solubilized from turkey erythrocyte plasma membranes and reconstituted into phospholipid vesicles by the addition of dimyristoyl phosphatidylcholine and removal of detergent by gel filtration. This procedure restored the ability to bind [125I]iodohydroxybenzylpindolol and [3H]dihydroalprenolol. Purified rabbit hepatic G/F that was added to the receptor vesicles could be stably activated by guanosine 5'-[3-thio]triphosphate at a low rate, and this activation was increased up to 4-fold in the presence of beta-adrenergic agonists. This stimulation of the activation of G/F was specific for beta-adrenergic agonists and could be specifically blocked by beta-adrenergic antagonists. Stimulation was proportional to the concentration of vesicles containing active beta-adrenergic receptor. Under optimal conditions, 5 to 6 molecules of G/F were activated per receptor, indicating that catalytic activation of G/F by receptor was reconstituted.
Journal Article•
TL;DR: The rat islet cell membrane is equipped with alpha 2-adrenoceptors which are linked to adenylate cyclase to cause diminution of the cellular content of cyclic AMP; insulin secretion may be inhibited consequently.
Abstract: Glucose-induced secretion and cyclic AMP accumulation in isolated rat pancreatic islets as well as GTP-activated adenylate cyclase of the membrane-rich preparation from the islets were strongly inhibited by some alpha-adrenergic agonists. The relative potencies of the agonists, estimated according to their dose-dependent actions, were in such an order that clonidine greater than epinephrine (congruent to norepinephrine) greater than phenylephrine congruent to methoxamine, regardless of which of the three parameters (i.e., insulin release, cyclic AMP accumulation, and adenylate cyclase activity) was used for estimation. There was a highly significant correlation between the amounts of cyclic AMP accumulation and the rate of insulin release that were changed in response to these agonists. The order of the potencies of alpha-adrenergic antagonists to reverse epinephrine inhibition of these parameters was invariably yohimbine congruent to dihydroergotamine congruent to phenylephrine greater than prazosin. In conclusion, the rat islet cell membrane is equipped with alpha 2-adrenoceptors which are linked to adenylate cyclase to cause diminution of the cellular content of cyclic AMP; insulin secretion may be inhibited consequently.