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)

[3H]5-HT exhibited specific binding in membrane preparations of Hymenolepis diminuta. The specific binding was saturable, reversible and temperature dependent. A non-linear Scatchard plot was obtained in a concentration range of 11 nM - 1000 nM [3H]5-HT, which could be resolved into sites having apparent dissociation constants (KD) of 0.10 microM and 6.25 microM for the high-affinity and low-affinity components, respectively. The latter could be selectively eliminated by binding [3H]5-HT to H. diminuta membranes in the presence of 10(-3) M nitroimipramine. Drug displacement studies, using 0.20 microM and 2.0 microM [3H]5-HT, revealed that while low-affinity [3H]5-HT binding was displaced by unlabelled 5-HT and inhibitors of 5-HT uptake, high affinity [3H]5-HT binding was affected only by tryptamine derivatives and, to a lesser extent, methysergide. In addition, high-affinity binding was stimulated by MgCl2 while low-affinity binding showed sodium-dependency. The data implicate the low-affinity site as a putative 5-HT transporter and the high-affinity site as a putative 5-HT 1 receptor. Exposure of H. diminuta membranes to 5-HT resulted in a 3-4 fold stimulation of cAMP levels. The EC 50 for the 5-HT-induced activation of adenylate cyclase (0.76 microM) was of the same order of magnitude as the apparent KD for high-affinity binding. Furthermore, the order of drug potency for the elevation of cAMP levels by 5-HT agonists and reversal by 5-HT antagonists was identical to the order of drug potency for the inhibition of high-affinity binding, suggesting linkage of the putative 5-HT 1 receptor to adenylate cyclase in H. diminuta.
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PMID:Characterization of a serotonin transporter and an adenylate cyclase-linked serotonin receptor in the cestode Hymenolepis diminuta. 302 90

Vasoactive intestinal peptide (VIP)-responsive adenylate cyclase and VIP binding sites were investigated in membranes prepared from ciliary processes dissected from albino rabbit eyes. High-affinity binding sites for VIP (Kd, 0.95 nM; 607 fmol/mg of protein), in addition to beta adrenergic sites labeled by dihydroalprenolol (Kd, 0.48 nM; 123 fmol/mg of protein), were present. Activation of adenylate cyclase by VIP had a Ka of 65 nM, and the maximal response was 3.3-fold greater than that for I-isoproterenol (Ka, 102 nM). A peptide fragment of VIP (sequence 10-28) was inactive in all assays and did not inhibit VIP-stimulated adenylate cyclase at 10 microM. Responses to VIP and isoproterenol in combination were additive at lower doses but less than additive at maximal doses. Responses to VIP in combination with a low dose of forskolin (0.1 microM) were potentiated at all dose levels, whether assays were done in presence of MgCl2 or MnCl2. VIP- and forskolin-activated adenylate cyclase was associated with the nonpigmented epithelial cell fraction and not with pigmented epithelial cells separated on Percoll density gradients after dissociation of cells from processes by collagenase digestion. Intravitreous injection of 10 nmol of VIP into the rabbit eye caused a maximal reduction in intraocular pressure at 40 to 50 hr lasting beyond 72 hr. VIP-responsive and beta adrenergic-responsive adenylate cyclase are present on the same cell type (nonpigmented epithelial cells) and appear to share components of the adenylate cyclase system in the same membrane. VIP may participate in the physiologic regulation of aqueous humor secretion at the level of the epithelial cell membrane.
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PMID:Vasoactive intestinal peptide and intraocular pressure: adenylate cyclase activation and binding sites for vasoactive intestinal peptide in membranes of ocular ciliary processes. 303 1

Manganous chloride was compared with magnesium chloride in supporting maximal stimulations of the adenylate cyclase system in ocular ciliary process membranes by isoproterenol, vasoactive intestinal peptide, sodium fluoride, guanosine 5'-(beta, gamma-imino) triphosphate (GppNHp), or forskolin, and in supporting synergism between isoproterenol and forskolin. The primary effect of Mn2+ (2 mM) was due to an interaction at the catalytic unit. Mn2+ had no significant effect on the function of the GTP-binding stimulatory G-protein (Gs) which couples beta-adrenergic receptors to the catalytic unit of adenylate cyclase. However, Gs-protein function was impaired by Mn2+ relative to Mg2+ when GppNHp was used instead of GTP as the ligand for the Gs-protein. Compared with Mg2+, Mn2+ caused a 4-5.5-fold increase in adenylate cyclase responsiveness to all the activators tested (except GppNHp, where the increase was 2.5-3.5-fold). Thus Mn2+ ions appeared to be intrinsically more effective at divalent cation binding sites on the catalytic unit that control its enzymatic activity. Ciliary process membranes differ from erythrocyte and S49 lymphoma cell membranes where 2 mM Mn2+ strongly inhibits hormone-Gs-protein-mediated stimulations of adenylate cyclase. Divalent cations bound to the catalytic unit also affected the degree of synergism between hormone-activated Gs and forskolin to stimulate adenylate cyclase activity. In the presence of MgCl2 all effective doses of isoproterenol and forskolin in combination showed marked synergism. In contrast, with MnCl2 there was no synergism with high-dose isoproterenol-forskolin combinations, which gave only additive responses.
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PMID:Manganous chloride stimulation of adenylate cyclase responsiveness in ocular ciliary process membranes. 314 94

[3H]Forskolin binds to human platelet membranes in the presence of 5 mM MgCl2 with a Bmax of 125 fmol/mg of protein and a Kd of 20 nM. The Bmax for [3H]forskolin binding is increased to 455 and 425 fmol/mg of protein in the presence of 100 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p) and 10 mM NaF, respectively. The increase in the Bmax for [3H]forskolin in the presence of Gpp(NH)p or NaF is not observed in the absence of MgCl2. The EC50 values for the increase in the number of binding sites for [3H]forskolin by Gpp(NH)p and NaF are 600 nM and 4 mM, respectively. The EC50 value for Gpp(NH)p to increase the number of [3H]forskolin binding sites is reduced to 35 mM and 150 nM in the presence of 50 microM PGE1 or PGD2, respectively. The increase in the number of [3H]forskolin binding sites observed in the presence of NaF is unaffected by prostaglandins. The binding of [3H]forskolin to membranes that are preincubated with Gpp(NH)p for 120 min or assayed in the presence of PGE1 reaches equilibrium within 15 min. In contrast, a slow linear increase in [3H]forskolin binding is observed over a period of 60 min when Gpp(NH)p and [3H]forskolin are added simultaneously to membranes. A slow linear increase in adenylate cyclase activity is also observed as a result of preincubating membranes with Gpp(NH)p. In human platelet membranes, agents that activate adenylate cyclase via the guanine nucleotide stimulatory protein (Ns) increase the number of binding sites for [3H]forskolin in a magnesium-dependent manner. This is consistent with the high affinity binding sites for [3H]forskolin being associated with the formation of an activated complex of the Ns protein and adenylate cyclase. This state of the adenylate cyclase may be representative of that formed by a synergistic combination of hormones and forskolin.
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PMID:Binding of [3H]forskolin to human platelet membranes. Regulation by guanyl-5'-yl imidodiphosphate, NaF, and prostaglandins E1 and D2. 346 64

Stimulation and inhibition of adenylate cyclase activity are mediated by the guanine nucleotide regulatory proteins Gs and Gi, respectively. Two general mechanisms have been proposed for the inhibition of activated adenylate cyclase: direct inhibition of the catalyst by Gi, and indirect inhibition of the activated catalyst mediated by Gi inhibition of Gs. We have assessed direct inhibition of adenylate cyclase by evaluating the ability of Gpp(NH)p to inhibit the forskolin-stimulated enzyme in the presence of various concentrations of magnesium ions and the guanine nucleotide. Gpp(NH)p inhibition of adenylate cyclase activity was only observed in the presence of forskolin and low concentrations of MgCl2. Muscarinic agonists did not increase Gpp(NH)p inhibition of the forskolin-stimulated enzyme, even in the presence of low concentrations of MgCl2 and guanine nucleotide (near the respective Kact or Ki). Whether in the absence or presence of muscarinic agonists, no concentration of Gpp(NH)p was found to inhibit basal adenylate cyclase activity in the absence of forskolin. In addition, muscarinic agonists had no effect on the rate constant (kon) for Gpp(NH)p activation of the enzyme. In contrast to these data, the muscarinic agonist methacholine stimulated the inactivation rate constant (koff) for isoproterenol plus GTP-activated adenylate cyclase activity 15-fold, and the increase in koff was blocked by atropine. Moreover, the sarcolemma displayed specific, high affinity GTP hydrolytic activity which was stimulated by methacholine activation of muscarinic receptors. These data further support our original hypothesis, indicating that although direct inhibition of the catalyst by Gi may occur in cardiac sarcolemma, physiologically relevant attenuation of adenylate cyclase activity by muscarinic agonists occurs by a mechanism linked to GTP hydrolysis.
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PMID:Muscarinic receptor regulation of cardiac adenylate cyclase activity. 356 Feb 38

Basal and stimulated adenylate cyclase specific activity was characterized in gill plasma membrane of freshwater-adapted trout by measuring the conversion of [alpha-32P]ATP into [alpha-32P]cyclic AMP. Both basal and isoproterenol- or sodium fluoride-stimulated enzyme activities were linear with time and protein concentration. The optimum activities were obtained using a pH buffer of 7.5 and a temperature of 20 degrees. The Km for ATP was 0.5 mM in the presence or absence of the stimulators. The presence of 10(-5) M guanosine-5'-triphosphate and 4 X 10(-3) M MgCl2 (2.41 X 10(-3) M free Mg2+) was required to optimize not only the basal activity but also the stimulation ratio (test/control) produced by these agents. On the contrary, Ca2+ was inhibitory. IC50 for CaCl2 was 5 X 10(-4) M (10(-7) M free Ca2+) in the presence or absence of the stimulators. Under these conditions, the basal adenylate cyclase specific activity was 400-450 pmol/mg protein/10 min. A maximal stimulation was produced by isoproterenol or PGE1 10(-5) M (50% increase over basal activity) or by glucagon 5.7 X 10(-10) M (30%). In addition, this enzyme displayed high sensitivity to sodium fluoride which induced a particularly large maximal effect (370%) at a concentration of 10(-2) M.
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PMID:Basal and stimulated adenylate cyclase activity in the gill epithelium of the rainbow trout. 362 74

We have established previously that the regulation of adenylate cyclase is abnormal in adipose tissue membranes of ob/ob mice. To help establish the nature of the defect, we studied the time course of guanine nucleotide activation and inhibition of adenylate cyclase. The activation of adenylate cyclase by Gpp(NH)p in adipocyte membranes of normal (+/+) and ob/ob mice proceeds with a lag phase. In +/+ membranes, this lag could be shortened by increasing the concentration of Mg2+ in the incubation medium or by pretreatment of the membranes with cholera toxin, and it could be abolished by isoproterenol in combination with 4 mM MgCl2. In contrast, in the ob/ob membranes, only pretreatment with cholera toxin was effective in shortening the lag phase. These results indicate an impediment in the activation of adenylate cyclase in ob/ob membranes. In the +/+ membranes, Gpp(NH)p inhibited foreskolin-stimulated adenylate cyclase, following a short lag phase, producing lower steady-state velocities than those seen with forskolin alone. The inhibitory effect of Gpp(NH)p on forskolin-stimulated activity was abolished by pertussis but not by cholera toxin treatment. In the ob/ob membranes, neither Gpp(NH)p nor pertussis treatment had any effect on the steady-state velocity of the forskolin-stimulated activity. These data have been interpreted as meaning that an anomaly in Ni rather than in Ns is likely to be responsible for the impairment of adenylate cyclase activity in the membranes of the ob/ob mouse.
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PMID:The hysteretic effect of Gpp(NH)p on adenylate cyclase is not altered by Mg2+ in adipocyte membranes of ob/ob mice. 377 59

The adenylate cyclase coupled inhibitory nucleotide regulatory protein (Ni) and the bovine retinal nucleotide regulatory protein transducin (T) appear to share some common functional properties since their GTPase activity is stimulated to similar extents by the retinal photoreceptor rhodopsin. In the present work, we sought to assess whether these functional similarities might extend to their interaction with adenylate cyclase. This necessitated the development of reconstitution systems in which guanine nucleotide regulatory protein mediated inhibition of adenylate cyclase activity could be demonstrated and characterized in a lipid milieu. In the absence of the pure human erythrocyte stimulatory nucleotide regulatory protein (Ns), the insertion into phospholipid vesicles of either pure Ni from human erythrocytes or pure bovine T with the resolved catalytic moiety of bovine caudate adenylate cyclase (C) does not establish GppNHp inhibition of either Mg2+- or forskolin-stimulated adenylate cyclase. However, the coinsertion into lipid vesicles of either Ni or T with Ns and resolved C results in an inhibition of Ns(GppNHp) stimulatable C activity. As is the case in intact membranes, the reconstituted inhibition of the Ns-stimulated C activity extends into the steady-state phase of time courses of activity. This inhibition is highly sensitive to the MgCl2 concentration. At 2 mM MgCl2, the inhibition is greater than 80% while at 50 mM MgCl2 it is only approximately 20%.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Transducin and the inhibitory nucleotide regulatory protein inhibit the stimulatory nucleotide regulatory protein mediated stimulation of adenylate cyclase in phospholipid vesicle systems. 393 56

We compared the effects of guanine nucleotides and Mg2+ on ADP-ribosylation of rat brain and liver membrane proteins catalysed by Bordetella pertussis toxin (IAP) and cholera toxin (CT). Labelling of proteins in the presence of [alpha-32P]NAD+, ATP and CT required GTP or guanosine 5'-[gamma-thio]triphosphate (GTP [S]). In contrast, labelling of one (liver) or two (brain) polypeptides by IAP was enhanced by guanosine 5'-[beta-thio]diphosphate (GDP[S]) or GTP, but was blocked by GTP[S] or guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG). The order of labelling intensity was GDP[S] greater than GTP greater than no addition greater than GTP[S] = p [NH]ppG. Mg2+ increased labelling by CT, but decreased labelling by IAP. In addition, Mg2+ potentiated the effects of the guanine nucleotides, increasing the inhibitory effects of GTP[S] and the activatory effects of GDP[S] or GTP. Preincubating liver membranes at 30 degrees C in the presence of 10 mm-MgCl2 inhibited labelling by IAP irreversibly. Pretreatment of liver membranes with 4.95 mM-N-ethylmaleimide decreased labelling by CT by approximately 15%, but almost completely blocked labelling by IAP. These results suggest that the undissociated, GDP-bound, conformation of Ni, the inhibitory GTP-binding protein of adenylate cyclase, is the preferred substrate for ADP-ribosylation by IAP. This conformation, which is prevalent in native membranes, is sensitive to temperature, Mg2+ ions and alkylating agents such as N-ethylmaleimide. At 30 degrees C, Mg2+ may cause dissociation and denaturation of Ni in native membranes.
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PMID:Pertussis toxin substrate is a guanosine 5'-[beta-thio]diphosphate-, N-ethylmaleimide-, Mg2+- and temperature-sensitive GTP-binding protein. 393 83

The divalent cations Mg2+ and Mn2+ in excess of the concentrations required to complex with ATP (excess Mg2+ or Mn2+) modulate the activity of adenylate cyclase. As a substrate, Mn X ATP was at least as effective as Mg X ATP in supporting adenylate cyclase activity in white and brown adipose tissue membranes. Both excess Mg2+ and Mn2+ had quantitatively different effects on the enzyme of lean and ob/ob mice and qualitatively different effects in white and brown adipose tissue. In white adipocyte membranes excess Mg2+ increase basal activity, as well as activity owing to guanylylimidodiphosphate (Gpp(NH)p) (with or without isoproterenol) and NaF. Maximal activation by Gpp(NH)p + isoproterenol required a higher concentration of Mg2+ in tissue from ob/ob than lean mice. Excess Mn2+ prevented the activation of the enzyme by Gpp(NH)p or Gpp(NH)p + isoproterenol in a dose-dependent manner. Mn2+ inhibited even in the presence of maximally effective Mg2+ concentrations. The enzyme of the ob/ob mouse membrane required a significantly higher dose of Mn2+ to achieve 50% inhibition. In brown adipose tissue, specific activities of the isoproterenol + Gpp(NH)p and NaF stimulated enzyme were significantly lower in the obese mice under all conditions studied. Except that NaF-stimulated activity was increased significantly more in the membranes of lean mice by the combination of Mg2+ + Mn2+, these cations did not produce significantly different dose-dependent effects in membranes from lean and ob/ob mice. Maximal activation occurred at lower concentrations MgCl2 (3-5 versus 10-20 mM) and required higher concentrations of MnCl2 (3-5 versus 1 mM) in brown than in white adipose tissue membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The effect of the divalent cations Mg2+ and Mn2+ on adenylate cyclase activity in white and brown adipose tissue of lean and obese (ob/ob) mice. 398 64


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