Showing papers on "Cyclase published in 1974"
TL;DR: The high sensitivity of this method permits detection of the small amounts of cyclic AMP formed at low enzyme concentrations or at early time points in kinetic studies.
3,935 citations
TL;DR: The VIP stimulates adenylate cyclase and active ion secretion in both rabbit and human ileal mucosa, and may be related to the pathogenesis of diarrhea in patients with the pancreatic cholera syndrome.
Abstract: Vasoactive intestinal peptide (VIP), originally isolated from hog small intestinal mucosa, has been shown to cause small intestinal secretion. More recently, this peptide has been identified in the plasma and tumors of patients with the so-called "pancreatic cholera" syndrome. In order to explore the possible role of VIP in the pathogenesis of this syndrome, we examined the effects of this peptide and other hormones on the cyclic AMP levels, adenylate cyclase activity, and ion transport in in vitro preparations of ileal mucosa. In rabbit ileal mucosa, VIP (20 mug/ml) caused a prompt fivefold increase in cyclic AMP level, whereas nine other hormones, which have been postulated to cause intestinal secretion, failed to exert such an effect. Pentagastrin and glucagon also failed to increase cyclic AMP levels in canine ileal mucosa. An increase in mucosal cyclic AMP levels was observed at a VIP concentration of 0.1 mug/ml and appeared to be nearly maximal at 2.0 mug/ml. VIP (100 mug/ml) stimulated adenylate cyclase activity in a membrane preparation from rabbit ileal mucosa. Secretin (6.0 x 10(-5) M) failed to do so. When added to the serosal side of isolated rabbit ileal mucosa clamped in an Ussing chamber, VIP (2 mug/ml) increased short-circuit current (SCC) and caused net secretion of both Cl and Na. Net Cl secretion exceeded net Na secretion. These effects of VIP on mucosal cyclic AMP metabolism and ion transport are similar to those observed with cholera enterotoxin and certain prostaglandins. VIP was also tested with normal human ileal mucosa. At a concentration of 2 mug/ml it caused a fivefold increase in cyclic AMP level and an increase in SCC of the same magnitude as that caused by 5 mM theophylline. Addition of a second 2-mug/ml dose of VIP and addition of theophylline after VIP produced no further change in SCC. We conclude the VIP stimulates adenylate cyclase and active ion secretion in both rabbit and human ileal mucosa. This may be related to the pathogenesis of diarrhea in patients with the pancreatic cholera syndrome.
372 citations
TL;DR: It is apparent that two forms of guanylate cyclase activity are present in cell-free preparations of rat heart and other rat tissues.
268 citations
TL;DR: The near equity between the concentration of cyclic AMP binding sites and the ligand itself provides a potential mechanism whereby agents can affect the total cyclicAMP content without directly affecting adenylate cyclase, cycling AMP phosphodiesterase, or cyclic AmP transport.
Abstract: When determined under the usual conditions of an excess of ligand over protein, the concentration of cyclic AMP necessary to activate pure preparations of cyclic AMP-dependent protein kinase (EC 2.7.1.37; ATP:-protein protein phosphotransferase) half-maximally is in the range of 0.2-0.3 μM when casein or glycogen synthetase is used as the substrate, i.e., essentially the same as the concentration of the nucleotide that is found in resting skeletal muscle. The apparent dissociation constant for cyclic AMP bound to the protein kinase is also about 0.2-0.3 μM when measured under similar conditions. The concentration of the protein kinase in muscle is relatively high (0.23 μM), however, and under these conditions the apparent activation constant of the enzyme for cyclic AMP is raised so that an increase in cyclic AMP levels in the tissue would cause a concomitant increase in protein kinase activity over a wide range of nucleotide concentration. As a result, it is unnecessary to invoke compartmentalization of cyclic AMP to explain how it can control protein kinase activity in vivo. Another factor that may increase the effectiveness of changes in cyclic AMP concentration is the heat-stable protein inhibitor of protein kinase that may function to inhibit the activity of nearly all the protein kinase catalytic subunit dissociated by basal concentrations of cyclic AMP. Finally, the near equity between the concentration of cyclic AMP binding sites and the ligand itself provides a potential mechanism whereby agents can affect the total cyclic AMP content without directly affecting adenylate cyclase, cyclic AMP phosphodiesterase, or cyclic AMP transport.
241 citations
TL;DR: GTP, in addition to its effects on glucagon action, stimulates the rate of dissociation of glucagon from its receptor and, as a consequence, causes a decrease in the steady state levels of bound glucagon.
225 citations
TL;DR: The binding of [ 3 H]-alprenolol (a potent β-adrenergic antagonist) to sites in frog erythrocyte membranes has been studied by a centrifugal assay as discussed by the authors.
217 citations
TL;DR: Gpp(NH)p appears to be a useful probe for investigating the mechanism of hormone and nucleotide action on adenylate cyclase systems in eukaryotic cells.
Abstract: 5′-Guanylylimidodiphosphate (Gpp(NH)-p) stimulates adenylate cyclase [ATP-pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity in plasma membranes isolated from frog and salmon erythrocytes, from rat adrenal, hepatic, and fat cells, and from bovine thyroid cells. The nucleotide acts cooperatively with the various hormones (glucagon, secretin, ACTH, thyrotropin, and catecholamines) that stimulate these adenylate cyclase systems with resultant activities that equal or exceed those obtained with hormone plus GTP or with fluoride ion. In the absence of hormones, Gpp(NH)p is a considerably more effective activator than GTP, and, under certain conditions of incubation, stimulates rat fat cell adenylate cyclase to levels of activity (about 20 nmoles of 3′,5′-adenosine monophosphate mg protein per min) far higher than reported hitherto for any adenylate cyclase system examined. The nucleotide activates frog erythrocyte adenylate cyclase when the catecholamine receptor is blocked by the competitive antagonist, propranolol, and activates the enzyme from an adrenal tumor cell line which lacks functional ACTH receptors. In contrast, Gpp(NH)p does not stimulate adenylate cyclase in extracts from Escherichia coli B. Gpp(NH)p appears to be a useful probe for investigating the mechanism of hormone and nucleotide action on adenylate cyclase systems in eukaryotic cells.
215 citations
TL;DR: The results suggest that leukocyte cyclic AMP may act as a "second messenger" to suppress plaque formation in vitro, similar to their effects on in vitro models of immediate and cell-mediated hypersensitivity.
Abstract: Histamine, beta-adrenergic amines, and prostaglandins inhibited hemolytic plaque formation by splenic leukocytes from immunized mice. The same agents had previously been shown to prevent both the IgE-mediated release of histamine from human basophils and the immunologically specific cytolytic activity of murine lymphocytes, through stimulation of the production of cyclic AMP in leukocytes. We therefore tested the hypothesis that cyclic AMP might mediate an inhibitory effect of these drugs by comparing the ability of these agents to inhibit plaque formation with their effects on cyclic AMP accumulation in leukocytes. In splenic cells from three mouse strains, the dose-dependent effects of these agents of cyclic AMP correlated with their inhibition of plaque formation. Beta- but not alpha-adrenergic agonists were effective in both systems, and the effects of isoproterenol were inhibited by propranolol. Histamine was approximately equipotent with isoproterenol in both systems. Two prostaglandins (E(1) and E(2)) were effective in both systems, but prostaglandin F(2alpha) was not. Dibutyryl cyclic AMP, a lipid-soluble analog of the endogenous nucleotide, inhibited plaque formation by cells of all three strains. Theophylline, an inhibitor of cyclic AMP degradation, inhibited plaque formation slightly, but potentiated the effects of histamine, isoproterenol, and the prostaglandins on both cyclic AMP accumulation and plaque formation. Finally, cholera enterotoxin, a potent activator of adenyl cyclase, produced a delayed inhibition of plaque formation and a parallel increase in leukocyte cyclic AMP content; both effects of the toxin were blocked by canine antitoxin. These results suggest that leukocyte cyclic AMP may act as a "second messenger" to suppress plaque formation in vitro. The inhibitory effects of hormones and cyclic AMP on plaque formation are strikingly similar to their effects on in vitro models of immediate and cell-mediated hypersensitivity. The physiologic significance of these findings is not yet known.
214 citations
TL;DR: The stereospecificity of the interaction with the iodinated β-blocking agent and the correspondence between affinity for site and biological potency of analogs suggested that this interaction is involved in function of the β-adrenergic receptor.
Abstract: An iodine-labeled beta-adrenergic inhibitor ((125)l-hydroxybenzylpindolol) binds specifically to a site on turkey erythrocyte membranes. A series of beta-adrenergic agonists and inhibitors compete for this binding site, with apparent affinities paralleling biological effectiveness as activators or inhibitors of catecholaminestimulated adenylate cyclase. The activity of d-(+) agonists or inhibitors was 1 percent (or less) than that of the corresponding l-(-) isomers in competing for binding of the iodinated blocker as well as in affecting catecholamine-stimulated adenylate cyclase. 1-(-)-Norepinephrine was about one-tenth as active as l-(-)-isoproterenol in competing for the beta-blocking agent site. The stereospecificity of the interaction with the iodinated beta-blocking agent and the correspondence between affinity for site and biological potency of analogs suggested that this interaction is involved in function of the beta-adrenergic receptor.
212 citations
TL;DR: It was concluded that these two classes of hormones interact with separate receptor sites, but it is still unclear whether they affect the same or different catalytic sites.
164 citations
TL;DR: A number of minor differences in the response of platelets to PGD 2 and PGE 1 suggest the existence of at least two prostaglandin receptors influencing a single adenylate cyclase.
TL;DR: It was of interest to determine whether cyclic AMP might also stimulate catecholamine turnover in the brain, especially in synaptosomes prepared from brain homogenates.
Abstract: EVIDENCE that catecholamines stimulate the production of adenosine cyclic-3′,5′-monophosphate (cyclic AMP) in the pineal gland1, superior cervical ganglia2 and both in brain slices3,4 and homogenates5 has suggested that cyclic AMP has an important postsynaptic function in both peripheral and central adrenergic neurotransmission. Evidence has also been presented in the hypogastric nerve–vas deferens preparation6 and adrenal medulla7 that dibutyryl cyclic AMP increases the release of noradrenaline, suggesting a presynaptic role for cyclic AMP in modulating catecholamine metabolism in adrenergic terminals. In the central nervous system, adenylate cyclase and phosphodiesterase are, to a considerable extent, associated with the isolated nerve-ending (synaptosomal) fraction8 in which putative neurotransmitters are primarily localised. Thus it was of interest to determine whether cyclic AMP might also stimulate catecholamine turnover in the brain, especially in synaptosomes prepared from brain homogenates.
TL;DR: CAMP Phosphodiesterase, Adenyl Cyclase, and Release of cAMP.PROKARYOTES .
Abstract: PROKARYOTES . Cellular Pools of cAMP and Release of cAMP. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362 Adenyl Cyclase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363 cAMP Phosphodiesterase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
TL;DR: The results suggest that endogenous prostaglandins do not provide an essential link in the activation of intestinal mucosal adenyl cyclase by cholera enterotoxin, and that the effects of the enteringotoxin on mucosal cyclic AMP levels were not influenced by indomethacin under conditions where mucosal prostag landins synthesis was inhibited.
Abstract: Both cholera enterotoxin and certain prostaglandins have been shown to stimulate intestinal fluid secretion in vivo, to cause ion flux changes in vitro similar to those caused by addition of cyclic 3',5'-adenosine monophosphate (cyclic AMP), and to activate intestinal mucosal adenyl cyclase. It has been suggested that the effects of the enterotoxin on intestinal cyclic AMP metabolism may be indirect, and that locally synthesized prostaglandins may serve as required intermediates for the effects of the enterotoxin in activating intestinal mucosal adenyl cyclase. In order to clarify certain aspects of the mechanisms by which these two agents alter intestinal mucosal cyclic AMP metabolism and ion transport, their effects on cyclic AMP accumulation in rabbit ileal mucosa were examined in vitro. Addition of 5 mug per ml (75 mug per 150 mg mucosa) of purified cholera enterotoxin produced a peak increase in cyclic AMP level in 3 h but there was a time delay of at least 30 min before any effect was observed. Inhibition of cyclic nucleotide phosphodiesterase with theophylline failed to reduce this time delay. In contrast, addition of prostaglandin E(1) (PGE(1)) increased the cyclic AMP level rapidly, a peak effect being observed in 2 min. The time of the peak prostaglandin-induced changes in cyclic AMP level and short-circuit current correlated closely. A maximal increment in cyclic AMP level was achieved with 5 x 10(-5) M PGE(1). When 10(-4) M PGE(1) was added to mucosa already maximally stimulated with cholera toxin, the resulting cyclic AMP level was equal to the sum of the levels reached when each agent was added alone. Furthermore, the effects of the enterotoxin on mucosal cyclic AMP levels were not influenced by indomethacin under conditions where mucosal prostaglandins synthesis was inhibited. The results suggest that endogenous prostaglandins do not provide an essential link in the activation of intestinal mucosal adenyl cyclase by cholera enterotoxin. The present study also indicates that the effect of cholera enterotoxin on intestinal mucosal cyclic AMP metabolism involves a definite time delay which is not due to cyclic nucleotide phosphodiesterase activity.
TL;DR: Binding of propranolol to the beta-receptor was found to be stereospecific for the L stereoisomer, and the turnover number of this adenylate cyclase would be close to 100 min(-1).
Abstract: Turkey erythrocyte ghosts (empty membranes) possess a class of receptors that can bind both L-[3H]isoproterenol and DL-[3H]propranolol. The binding of [3H]isoproterenol to these receptors occurs with a dissociation constant of 0.15 μM and can be fully inhibited by 1 μM propranolol. The binding of [3H]propranolol occurs with a dissociation constant of 2.5 nM and can be fully inhibited by 0.2 mM DL-isoproterenol. Ligand binding is sensitive to sonication, boiling, and 8 M urea. The cells possess 500 to 1000 β-adrenergic receptors per cell. Binding of propranolol to the β-receptor was found to be stereospecific for the L stereoisomer. If one assumed a 1:1 relationship between β-adrenergic receptors and adenylate cyclase, the turnover number of this adenylate cyclase would be close to 100 min-1.
TL;DR: Low concentrations of glucagon did not increase tissue accumulation or release of cyclic AMP in livers from diabetic rats but were effective in normal livers, and higher concentrations of the hormone produced normal responses in diabetic livers.
TL;DR: A new rapid and reliable assay for guanylate cyclase was established, which employs a simple procedure to isolate guanosine 3' : 5'-monophosphate with neutral aluminum oxide and Dowex 1 formate columns.
TL;DR: GTP caused stimulation of adenylate cyclase in myocardial membranes, was virtually without effect in frog erythrocytes and resulted in inhibition in adipose membranes, and ability to competitively antagonize stimulation by Gpp(NH)p provided a convenient way of assessing the relative affinities of nucleotides for the regulatory sites.
TL;DR: The functional consequences of the cholesterol filipin reaction in erythrocyte membranes are heterogeneous and the relevant complex formation was shown not to involve lysis and disintegration and to occur in that part of the membrane which is probed by the hydrophobic dye perylene.
TL;DR: Alumina is a useful adsorbent for the anion exchange chromatography of 3', 5'-cyclic nucleotides because it effects a sharp discrimination on the basis of charge, and the general availability and cheapness of alumina is recommended.
Abstract: Publisher Summary Alumina is a useful adsorbent for the anion exchange chromatography of 3', 5'-cyclic nucleotides because it effects a sharp discrimination on the basis of charge. The univalent cyclic nucleotides are eluted from alumina in high yield by a neutral aqueous medium, while the other nucleotides of higher valence are adsorbed. This behavior, coupled with the general availability and cheapness of alumina, recommends its use in the purification of cyclic nucleotides from enzyme reaction mixtures or from tissue extracts. This chapter describes the use of alumina columns in the assay of adenylate and guanylate cyclase. Because the experience has been confined to the purification of radioactive cyclic nucleotides from cyclase reaction mixtures, the chapter discusses the purification of cyclic nucleotides from tissue extracts.
TL;DR: It is proposed that GTP enhances adenylate cyclase by altering the interaction between hormone and receptor, and the receptor site specific for the ethanolamine function of the catecholamine molecule is influenced by the nucleotides.
TL;DR: Adenylate cyclase from the rat renal medulla has been solubilized with the nonionic detergents Triton X-100 and Lubrol PX and findings suggest that no more than 5 % of the surface of adenylates cyclase is involved in hydrophobic interactions with other membrane components.
TL;DR: Selective inhibition of the glucagon-inactivating components of liver cell membranes allows more meaningful kinetic characterization of the hormone-receptor interaction in these membranes and may also aid in further studies designed to isolate and purify the receptors.
TL;DR: Prostaglandin E2 (PGE2) stimulates steroidogenesis in vitro in human corpus luteum slices, to a similar extent as hCG and hLH, and this stimulation appears to be specific and dose related for these substances.
Abstract: Prostaglandin E2 (PGE2) stimulates steroidogenesis in vitro in human corpus luteum slices, to a similar extent as hCG. This prostaglandin, as well as hCG and hLH also stimulates the incorporation of [8-3H] adenine into [8-3H] cyclic AMP in this in vitro system and this stimulation appears to be specific and dose related for these substances. The increase of [8-3H] cyclic AMP accumulation was found to be due to the stimulation of the synthetic adenyl cyclase enzyme system rather than the inhibition of the destructive phosphodiesterase enzyme system. Throughout the study, corpora lutea of the menstrual cycle were shown to be much more responsive to stimulation (hCG, LH, PGE2) in terms of steroid synthesis and cyclic AMP formation than corpora lutea of pregnancy. The reason for this difference between the two types of corpora lutea is not clear at this time but does not appear to be due to a deficiency in the adenyl cyclase system of pregnancy corpora lutea. The data of the present study brings our ...
TL;DR: A high affinity purine nucleotide site is involved in regulating the catalytic function of the adenylate cyclase complex, which indicates that, although ATP is clearly the substrate for the enzyme, it is GTP and not ATP that interacts at the regulatory site (identified as a Gpp(NH)p binding site).
TL;DR: The activation of this enzyme by serotonin was selectively inhibited by extremely low concentrations of D-lysergic acid diethylamide (LSD), 2-bromo-LSD, and cyproheptadine, agents which are known to block certain serotonin receptors in vivo.
Abstract: An adenylate cyclase (EC 4.6.1.1) that is activated specifically by low concentrations of serotonin has been identified in homogenates of the thoracic ganglia of an insect nervous system. The activation of this enzyme by serotonin was selectively inhibited by extremely low concentrations of D-lysergic acid diethylamide (LSD), 2-bromo-LSD, and cyproheptadine, agents which are known to block certain serotonin receptors in vivo. The inhibition was competitive with respect to serotonin, and the calculated inhibitory constant of LSD for this serotonin-sensitive adenylate cyclase was 5 nM. The data are consistent with a model in which the serotonin receptor of neural tissue is intimately associated with a serotonin-sensitive adenylate cyclase which mediates serotonergic neurotransmission. The results are also compatible with the possibility that some of the physiological effects of LSD may be mediated through interaction with serotonin-sensitive adenylate cyclase.
TL;DR: Examination of components of the cAMP system in primary cultures of differentiating chick myoblasts revealed a basal intracellular cAMP level of 50–100 pmole/mg of DNA, which increased ten to fifteen-fold for approximately 1 hr between 37.5 and 39.5 hr of culture, only 5–6 hr before the initiation of myoblast fusion.
TL;DR: Adenyl cyclase activity was normal in muscle from patients with Duchenne or facioscapulohumeral dystrophy but the response to epinephrine was about 30% of normal and theresponse to sodium fluoride about half normal, suggesting that the mechanisms of these agents differ.
Abstract: Adenyl cyclase activity in normal human muscle is stimulated four to seven times by epinephrine and about 18 times by sodium fluoride. The enzyme is present in all particulate fractions of muscle (myofibrils, mitochondria, sarcoplasmic reticulum) but not in the soluble phase. The highest specific activity is found in isolated plasma membrane. Basal enzyme activity was normal in muscle from patients with Duchenne or facioscapulohumeral dystrophy but the response to epinephrine was about 30% of normal and the response to sodium fluoride about half normal. In polymyositis, responses were normal, and intermediate values were found in myotonic dystrophy. In denervated muscle, responses were enhanced. In fetal muscle there was no response to epinephrine but sodium fluoride stimulated, suggesting that the mechanisms of these agents differ.
TL;DR: It was concluded that hormones as well as fluoride ion decrease the sensitivity of the enzyme toward inhibition by ATP not complexed to magnesium, thereby favoring the formation of the productive Michaelis complex.
TL;DR: Of 12 neuroleptic drugs with reported antipsychotic efficacy, all inhibit stimulation of adenylate cyclase by 40 muM dopamine at micromolar concentrations, and among 14 other structurally related drugs that are not clinically effective as antippsychotic agents, 12 were almost ineffective while two drugs were moderate inhibitors of dopamine-sensitive adenYLate cyclases.
Abstract: Antipsychotic drugs and their clinically impotent congeners were examined as inhibitors of dopamine-sensitive adenylate cyclase (EC 4.6.1.1) in cell-free membrane preparations of the caudate-putamen of rat brain. Of 12 neuroleptic drugs with reported antipsychotic efficacy, all inhibit stimulation of adenylate cyclase by 40 μM dopamine at micromolar concentrations. Among 14 other structurally related drugs that are not clinically effective as antipsychotic agents, 12 were almost ineffective while two drugs were moderate inhibitors of dopamine-sensitive adenylate cyclase.