Characterization of an ATP-Mg2+-dependent guanine nucleotide-stimulated adenylate cyclase from Neurospora crassa

A novel adenylate cyclase activity was found in crude homogenates of Neurospora crassa. The adenylate cyclase had substantial activity with ATP-Mg2+ as substrate differing significantly from the strictly ATP-Mn2+-dependent enzyme characterized previously. Additionally, the ATP-Mg2+-dependent activity was stimulated two- to fourfold by GTP or guanyl-5'-yl-imido-diphosphate (Gpp(NH)p). We propose that the ATP-Mg2+-dependent, guanine nucleotide-stimulated activity is due to a labile regulatory component (G component) of the adenylate cyclase which was present in carefully prepared extracts. The adenylate cyclase had a pH optimum of 5.8 and both the catalytic and G component were particulate. The Km for ATP-Mg2+ was 2.2 mM in the presence of 4.5 mM excess Mg2+. Low Mn2+ concentrations had no effect on adenylate cyclase activity whereas high concentrations of Mn2+ or Mg2+ stimulated the enzyme. Maximal Gpp(NH)p stimulation required preincubation of the enzyme in the presence of the guanine nucleotide and the K1/2 for Gpp(NH)p stimulation was 110 nM. Neither fluoride nor any of a variety of glycolytic intermediates or hormones, including glucagon, epinephrine, and dopamine, had an effect on ATP-Mg2+-dependent adenylate cyclase activity. However, the enzymatic activity was stimulated not only by GTP but also by 5'-AMP and was inhibited by NADH.     

Ca2+/Calmodulin Distinguishes Between Guanyl-5''-yl-Imidodiphosphate-and Opiate-Mediated Inhibition of Rat Striatal Adenylate Cyclase

The inhibition of adenylate cyclase from rat striatal plasma membranes by guanyl-5'-yl-imidodiphosphate [Gpp(NH)p] and morphine was compared to determine whether Gpp(NH)p-mediated inhibition accurately reflected hormone-mediated inhibition in this system. Inhibition of adenylate cyclase activity by Gpp(NH)p and morphine was examined with respect to temperature, divalent cation concentration, and the presence of Ca2+/calmodulin (Ca2+/CaM). Gpp(NH)p-mediated inhibition was dependent on the presence of Ca2+/CaM at 24 degrees C; the inhibition was independent of Ca2+/CaM at 18 degrees C; and inhibition could not be detected in the presence, or absence, of Ca2+/CaM at 30 degrees C. In contrast, naloxone-reversible, morphine-induced inhibition of adenylate cyclase was independent of both temperature and the presence of Ca2+/CaM. Mg2+ dose-response curves also reinforced the differences in the Ca2+/CaM requirement for Gpp(NH)p- and morphine-induced inhibition. Because Gpp(NH)p-mediated inhibition was independent of Ca2+/CaM at low basal activities (i.e., 18 degrees C, or below 1 mM Mg2+) and dependent on the presence of Ca2+/CaM at higher basal activities (24 degrees C, or above 1 mM Mg2+), the inhibitory effects of Gpp(NH)p were examined at 1 mM Mg2+ in the presence of 100 nM forskolin. Under these conditions, both Gpp(NH)p- and morphine-induced inhibition of adenylate cyclase were independent of Ca2+/CaM. The results demonstrate that the requirement for Ca2+/CaM to observe Gpp(NH)p-mediated inhibition depends on the basal activity of adenylate cyclase, whereas hormone-mediated inhibition is Ca2+/CaM independent under all conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of regulation of adenylate cyclase activity in human polymorphonuclear leukocytes by calcium, guanosyl nucleotides, and positive effectors.

This study presents the results of a kinetic investigation of adenylate cyclase in human polymorphonuclear leukocytes. In the presence of a saturating concentration of substrate (1 mM), the basal activity was increased severalfold by increasing Mg2+ from 1 to 25 mM. A Hill coefficient of 1.9 was obtained for Mg2+ or ATP. The data suggest cooperative interactions between the substrate binding sites in the neutrophil adenylate cyclase complex. It has been observed that guanyl-5'-yl imidodiphosphate (Gpp(NH)p) (S0.5 = 10 MUM) significantly increased and Ca2+ (S0.5 = 0.5 MM) significantly decreased only the Vmax without affecting the Hill coefficient or S0.5 for ATP. The Hill coefficients for Ca2+ or Gpp(NH)p were 0.9 and 0.8, respectively. The Hill coefficient for Ca2+ was not changed by the increased Gpp(NH)p concentrations. It appears that neutrophil adenylate cyclase has distinct binding sites for Gpp(NH)p and Ca2+, one for each compond. The binding of ligands is not changed by the other effectors and the action is directed only toward the Vmax of the enzyme. The stimulatory action of positive effectors (prostaglandin E1, isoproterenol, histamine) was enhanced by Gpp(NH)p and depressed by Ca2+. No preferential stimulation by Gpp(NH)p nor inhibition by Ca2+ of the action of the positive effectors has been found. The data suggests that only one type of catalytic subunit responds to the action of several positive effectors. Extracellular Gpp(NH)p or Ca2+ do not affect the cyclic adenosine 3':5'-monophosphate (cAMP) level in whole neutrophils and the effect of positive effectors on cAMP production is also not significantly changed by 5 mM Ca2+ or 0.1 mM Gpp(NH)p. Ionophore A23187 in the presence of 5 mM Ca2+ enhances Ca2+ entry into cells and decreases the basal cAMP formation. It appears that Gpp(NH)p or Ca2+ act only at the intracellular site of the adenylate cyclase complex.     

Forskolin-activated adenylate cyclase. Inhibition by guanyl-5'-yl imidodiphosphate

Forskolin activated adenylate cyclase of purified rat adipocyte membranes in the absence of exogenous guanine nucleotides. Guanyl-5'-yl imidodiphosphate (Gpp(NH)p) inhibited the forskolin-activated cyclase immediately upon addition of the nucleotide at concentrations too low to activate adenylate cyclase (10(-9) to 10(-7) M). Inhibition seen with a very high concentration of Gpp(NH)p (10(-4) M) lasted for 3-4 min and was followed by an increase in the synthetic rate which remained constant for at least 15 min. The length of the transient inhibition did not vary with forskolin concentrations above 0.05 microM but low Gpp(NH)p (10(-8) M) exhibited a lengthened (6-7 min) inhibitory phase. The transient inhibitory effects of Gpp(NH)p were eliminated by 10(-7) M isoproterenol, high (40 mM) Mg2+, or preincubation with Gpp(NH)p in the absence of forskolin. While forskolin stimulated fat cell cyclase in the presence of Mn2+, this ion blocked the inhibitory effects of Gpp(NH)p. The well documented inhibitory effects of GTP on the fat cell adenylate cyclase system were also observed in the presence of forskolin. However, the inhibition by GTP is not transitory. These findings indicate that Gpp(NH)p regulation of forskolin-stimulated cyclase has at least two components: 1) an inhibitory component which acts through an undetermined mechanism and which acts immediately to decrease cyclase activity; and 2) an activating component which modulates the inhibited cyclase activity through the guanine nucleotide regulatory protein.     

Inhibition of hepatic adenylate cyclase by NADH

Adenylate cyclase activity in isolated rat liver plasma membranes was inhibited by NADH in a concentration-dependent manner. Half-maximal inhibition of adenylate cyclase was observed at 120 microM concentration of NADH. The effect of NADH was specific since adenylate cyclase activity was not altered by NAD+, NADP+, NADPH, and nicotinic acid. The ability of NADH to inhibit adenylate cyclase was not altered when the enzyme was stimulated by activating the cyclase was not altered when the enzyme was stimulated by activating the Gs regulatory element with either glucagon or cholera toxin. Similarly, inhibition of Gi function by pertussis toxin treatment of membranes did not attenuate the ability of NADH to inhibit adenylate cyclase activity. Inhibition of adenylate cyclase activity to the same extent in the presence and absence of the Gpp (NH) p suggested that NADH directly affects the catalytic subunit. This notion was confirmed by the finding that NADH also inhibited solubilized adenylate cyclase in the absence of Gpp (NH)p. Kinetic analysis of the NADH-mediated inhibition suggested that NADH competes with ATP to inhibit adenylate cyclase; in the presence of NADH (1 mM) the Km for ATP was increased from 0.24 +/- 0.02 mM to 0.44 +/- 0.08 mM with no change in Vmax. This observation and the inability of high NADH concentrations to completely inhibit the enzyme suggest that NADH interacts at a site(s) on the enzyme to increase the Km for ATP by 2-fold and this inhibitory effect is overcome at high ATP concentrations.     

Binding of [3H]forskolin to human platelet membranes. Regulation by guanyl-5'-yl imidodiphosphate, NaF, and prostaglandins E1 and D2

[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.     

Guanine Nucleotides Modulate Cortical S2 Serotonin Receptors

Abstract

Many radiolabelled receptors coupled to intracellular adenylate cyclase activity have been found to be modulated by physiological modulators such as GTP (guanosine triphosphate) and Gpp(NH)p (guanosine-imido-diphosphate). In particular, the apparent affinity of agonists competing for the binding of ³H-antagonist-labelled receptors is reduced in the presence of GTP and Gpp(NH)p. We report herein the agonist-specific effects of GTP and Gpp(NH)p on rat brain cortical S² serotonin receptors. The agonists serotonin, 5-methoxytryptamine, bufotenine, and tryptamine display threefold lower affinities for S₂ serotonin receptors in the presence of 10^-4M GTP or Gpp(NH)p than in the absence of the nucleotides. The antagonists spiperone, cinanserin, cyproheptadine and methysergide are unaffected by the guanine nucleotides. The Hill coefficients of the agonists increase from between 0.70–0.80 to 0.90–1.00 due to guanine nucleotides. ATP, ADP, and GDP have little or no effect. This pattern of guanine nucleotide effects has been found with receptors which are modulated by a guanine nucleotide regulatory protein and may indicate that the S₂ serotonin receptor may be coupled to intracellular adenylate cyclase activity.     

Adenosine and gpp(NH)p modulate mouse sperm adenylate cyclase

The possible roles of adenosine and the GTP analogue Gpp(NH)p in regulating mouse sperm adenylate cyclase activity were investigated during incubation in vitro under conditions in which after 30 min the spermatozoa are essentially uncapacitated and poorly fertile, whereas after 120 min they are capacitated and highly fertile. Adenylate cyclase activity, assayed in the presence of 1 mM ATP and 2 mM Mn²⁺, was determined by monitoring cAMP production. When adenosine deaminase (1 U/ml) was included in the assay to deplete endogenous adenosine, enzyme activity was decreased in the 30-min suspensions but increased in the 120-min samples (P < 0.02). This suggests that endogenous adenosine has a stimulatory effect on adenylate cyclase in uncapacitated spermatozoa but is inhibitory in capacitated cells. Since the expression of adenosine effects at low nucleoside concentrations usually requires guanine nucleotides, the effect of adding adenosine in the presence of 5 x 10^–5 M Gpp(NH)p was examined. While either endogenous adenosine or adenosine deaminase may have masked low concentration (10^?9?10^?7 M) effects of exogenous adenosine, a marked inhibition (P < 0.001) of adenylate cyclase activity in both uncapacitated and capacitated suspensions was observed with higher concentrations (>10^?5 M) of adenosine. Similar inhibition was also observed in the absence of Gpp(NH)p, suggesting the presence of an inhibitory P site on the enzyme. In further experiments, the effects of Gpp(NH)p in the presence and absence of adenosine deaminase were examined. Activity in 30-min suspensions was stimulated by the guanine nucleotide and in the presence of adenosine deaminase this stimulation was marked, reversing the inhibition seen with adenosine deaminase alone. In capacitated suspensions the opposite profile was observed, with Gpp(NH)p plus adenosine deaminase being inhibitory; again, this was a reversal of the effects obtained in the presence of adenosine deaminase alone, which had stimulated enzyme activity. These results suggest the existence of a stimulatory adenosine receptor site (R_a) on mouse sperm adenylate cyclase that is expressed in uncapacitated spermatozoa and an inhibitory receptor site (R_i) that is expressed in capacitated cells, with guanine nucleotides modifying the final response to adenosine. It is concluded that adenosine and guanine nucleotides may regulate mouse sperm adenylate cyclase activity during capacitation.     

Regulation of human platelet adenylate cyclase by epinephrine, prostaglandin E1, and guanine nucleotides. Evidence for separate guanine nucleotide sites mediating stimulation and inhibition.

A method for preparing human platelet membranes with high adenylate cyclase activity is described. Using these membranes, epinephrine and GTP individually are noted to inhibit adenylate cyclase slightly. When present together, epinephrine and GTP act synergistically to cause a 50% inhibition of basal activity. The epinephrine effect is an alpha-adrenergic process as it is reversed by phentolamine but not propranolol. The quasi-irreversible activation of adenylate cyclase by Gpp(NH)p is time, concentration, and Mg2+-dependent but is not altered by the presence of epinephrine. Adenylate cyclase activated by Gpp(NH)p, and extensively washed to remove unbound Gpp(NH)p, is inhibited by the subsequent addition of Gpp(NH)p, GTP, and epinephrine. This effect of epinephrine is also an alpha-adrenergic phenomenon. In contrast to epinephrine which inhibits the cyclase, PGE1 addition results in enzyme stimulation. PGE1 stimulation does not require GTP addition. PGE1 accelerates the rate of Gpp(NH)p-induced activation. Low GTP concentrations (less than 1 x 10(-6) M) enhance PGE1 stimulation while higher GTP concentrations cause inhibition. These observations suggest that human platelet adenylate cyclase possesses at least two guanine nucleotide sites, one which interacts with the alpha-receptor to result in enzyme inhibition and a second guanine nucleotide site which interacts with the PGE1 receptor and causes enzyme stimulation.     

Mechanism of adenylate cyclase activation by the rat lung cytoplasmic factors

Epinephrine, histamine and prostaglandin E₁ stimulated adenylate cyclase activity in lung membranes and their stimulation of the enzyme activity was completely blocked by propranolol, metiamide and indomethacin, respectively. A partially-purified activator from the adult rat lung also enhanced adenylate cyclase activity in membranes. However, stimulation of adenylate cyclase by the rat lung activator was not abolished by the above receptor antagonists. Further, epinephrine, NaF and Gpp(NH)p stimulated adenylate cyclase activity rather readily, whereas stimulation of the enzyme activity by the lung activator was evident after an initial lag phase of 10 min. Also, the lung activator produced additive activation of adenylate cyclase with epinephrine, NaF and Gpp(NH)p. These results indicate that the lung activator potentiates adenylate cyclase activity in membranes by a mechanism independent from those known for epinephrine, NaF and Gpp(NH)p. Incubation of lung membranes for 30 min at 40°C resulted in a loss of adenylate cyclase activation by NaF and Gpp(NH)p. Addition of the released proteins to the heat-treated membranes did not restore the enzyme response to these agonists. However, heat treatment of lung membranes in the presence of 2-mercaptoethanol or dithiothreitol prevented the loss of adenylate cyclase response to NaF and Gpp (NH)p. N-ethylmaleimide abolished adenylate cyclase activation by epinephrine, NaF, Gpp(NH)p and the lung activator. These results indicate that the sulfhydryl groups are important for adenylate cyclase function in rat lung membranes.Abbreviations Gpp(NH)p 5-Guanylimidodiphosphate     

The mode of action of chloride on rabbit heart adenylate cyclase

The mechanism by which chloride stimulates adenylate cyclase was investigated. Depletion of GDP increased basal adenylate cyclase activity and reduced the stimulation by isoprenaline. Restoration of bound GDP partially reversed these effects. Chloride stimulated cyclase activity by the same proportion in control, GDP-depleted and GDP-restored preparations, as did Gpp(NH)p. Fluoride increased adenylate cyclase activity to the same final level in both GDP-depleted and GDP-restored membranes; addition of Gpp(NH)p as well as fluoride had no further effect. Solubilisation of adenylate cyclase reduced the stimulatory effect of Gpp(NH)p only slightly, but greatly attenuated the activation by chloride. We conclude that chloride does not stimulate cyclase activity by an action on GDP exchange. Activation by chloride may be due to a disrupting or chaotropic effect on membrane/protein interactions.     

Adenylate cyclase of platelets from spontaneously hypertensive rats: reduction of the inhibition by GTP

We have compared the effects of Gpp[NH]p on adenylate cyclase activity of platelet membranes in SHR and WKY rats. In the presence of 50 microM forskolin, low concentrations of Gpp[NH]p (0.01 to 0.3 microM) inhibited the enzyme activity in both strains, but the maximal level of inhibition was significantly lower in SHR (- 20%). In the absence of forskolin, 0.1 microM Gpp[NH]p was inhibitory only in WKY and the adenylate cyclase activity was greater in hypertensive rats at this nucleotide concentration. Increasing Gpp[NH]p from 0.1 to 3 microM induced the same increase of enzyme activity in both strains. In SHR, GTP itself induced a lower inhibition of the enzyme stimulated by 50 microM forskolin or 0.1 microM prostaglandin E1. These results suggest that the modulatory effect of the guanine nucleotide inhibitory protein on adenylate cyclase may be reduced in platelets from SHR.     

The hysteretic effect of Gpp(NH)p on adenylate cyclase is not altered by Mg2+ in adipocyte membranes of ob/ob mice

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.     

Studies on the mechanism of inhibition of amphibian oocyte adenylate cyclase by progesterone

Progesterone treatment induces the meiotic maturation of Xenopus laevis oocytes. Previous evidence indicates that this hormonal effect may be due to inhibition of oocyte adenylate cyclase. The present work studies several aspects of the mechanism of adenylate cyclase inhibition by this hormone. Forskolin greatly stimulates oocyte adenylate cyclase in the absence of guanine nucleotides and this activity is not sensitive to progesterone inhibition. In addition the forskolin-activated enzyme is not inhibited by a wide range of guanine nucleotide, in the presence or absence of hormone. The time course of cAMP synthesis catalyzed by oocyte adenylate cyclase in the presence of guanyl-5′_l-imidodiphosphate (Gpp(NH)p) shows an initial lag period that does not depend on the concentration of Gpp(NH)p. Progesterone causes a very significant increase in the hysteresis of the reaction, at least doubling the half-time of enzyme activation. The hormonal effect on the lag cannot be reversed by saturating concentrations of Gpp(NH)p. Progesterone also decreases the steady-state rates of the reaction. This effect, however, depends on the concentration of Gpp(NH)p. High concentrations of Gpp(NH)p almost completely reverse the inhibition of the steady-state rates. Progesterone does not inhibit if it is added to the reaction after the initial lag period. Guanosine-5′-O-(2-thiodiphosphate) (GDP-β-S) is an efficient competitive inhibitor of Gpp(NH)p activation of adenylate cyclase. Progesterone inhibition is observed at all concentrations of GDP-β-S and is potentiated at high ratios of GDP-β-S to Gpp(NH)p. These data indicate that progesterone inhibits by interfering with the activation of the N_s subunit of the enzyme by guanine nucleotides, rather than through a mechanism involving a separate N_i subunit.     

Exchange of Guanine Nucleotides Between Tubulin and GTP-Binding Proteins That Regulate Adenylate Cyclase: Cytoskeletal Modification of Neuronal Signal Transduction

Tubulin, the primary constituent of microtubules, is a GTP-binding proteins with structural similarities to other GTP-binding proteins. Whereas microtubules have been implicated as modulators of the adenylate cyclase system, the mechanism of this regulation has been elusive. Tubulin, polymerized with the hydrolysis-resistant GTP analog, 5'-guanylylimidodiphosphate [Gpp(NH)p], can promote inhibition of synaptic membrane adenylate cyclase which persists subsequent to washing. Tubulin with Gpp(NH)p bound was slightly less potent than free Gpp(NH)p in the inhibition of adenylate cyclase, but tubulin without nucleotide bound had no effect on the enzyme. A GTP-binding protein from the rod outer segment (transducin), with Gpp(NH)p bound, was also without effect on adenylate cyclase. Tubulin (regardless of the nucleotide bound to it) did not alter the activity of the adenylate cyclase catalytic unit directly. When tubulin was polymerized with the hydrolysis-resistant photoaffinity GTP analog, [32P]P3(4-azidoanilido)-P1-5'-GTP ([32P]AAGTP), and this protein was added to synaptic membranes, AAGTP was transferred from tubulin to the inhibitory GTP-binding protein, Gi. This transfer was blocked by prior incubation of the membranes with Gpp(NH)p or covalent binding of AAGTP to tubulin prior to exposure of that tubulin to membranes. Incubation of membranes with Gpp(NH)p subsequent to incubation with tubulin-AAGTP results in a decrease in AAGTP bound to Gi and a compensatory increase in AAGTP bound to the stimulatory GTP-binding protein, Gs. Likewise, persistent inhibition of adenylate cyclase by tubulin-Gpp(NH)p could be overridden by the inclusion of 100 microM Gpp(NH)p in the assay inhibition. Whereas Gpp(NH)p promotes persistent inhibition of synaptic membrane adenylate cyclase without incubation at elevated temperatures, tubulin [with AAGTP or Gpp(NH)p bound] requires 30 s incubation at 23 degrees C to effect adenylate cyclase inhibition. Photoaffinity experiments yield parallel results. These data are consistent with synaptic membrane tubulin regulating neuronal adenylate cyclase by transferring GTP to Gi and, subsequently, to Gs.     

Calcium-dependent adenylate cyclase from rat cerebral cortex: Activation by guanine nucleotides

The adenylate cyclase activity of a participate preparation of rat cerebral cortex is composed of at least two contributing components, one of which requires a Ca²⁺-dependent regulator protein (CDR) for activity (Brostrom, C. O., Brostrom, M. A., and Wolff, D. J. (1977) J. Biol. Chem.252, 5677–5685). Each of these components of the activity was activated by GTP and its synthetic analog, 5-guanylylimidodiphosphate (Gpp(NH)p). The component of the adenylate cyclase activity which did not respond to CDR (CDR-independent activity) was stimulated approximately 60% by 100 μm GTP and 3.5-fold by 100 μm Gpp(NH)p. Concentrations of GTP required for maximal activation of the CDR-dependent adenylate cyclase component decreased as CDR concentrations in the assay were increased. Similarly, GTP pr Gpp(NH)p lowered the concentration of CDR required to produce half-maximal activation of this enzyme form. At saturating CDR concentrations, however, increases in activity were not observed with the addition of these nucleotides. The CDR-dependent component responded biphasically (activation followed by inhibition) to increasing free Ca²⁺ concentrations; both phases of this response occurred at lower free Ca²⁺ concentrations with GTP present in the assay. The concentration of chlorpromazine which inhibited activation of adenylate cyclase by CDR was elevated when GTP was present. The CDR-dependent form of activity, which is stabilized by CDR to thermal inactivation, was also stabilized by Gpp(NH)p. The increase in stability produced by Gpp(NH)p did not require the presence of CDR, and stabilization with both Gpp(NH)p and CDR was greater than that obtained with either Gpp(NH)p or CDR alone.     

Purification and partial characterization of two forms of urinary trypsin inhibitor

The activation of bovine thyroid adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) by Gpp(NH)p has been studied using steady-state kinetic methods. This activation is complex and may be characterized by two Gpp(NH)p binding sites of different affinities with measured constants: Ka1 = 0.1 micro M and Ka2 = 2.9 micro M. GDP beta S does not completely inhibit the Gpp(NH)p activation: analysis of the data is consistent with a single GDP beta S inhibitory site which is competitive with the weaker Gpp(NH)p site. Guanine nucleotide effects upon F- activation of adenylate cyclase have been studied. When App(NH)p is the substrate, 10 micro M GTP along with 10 mM NaF gives higher activity than NaF alone, while GDP together with NaF inhibits the activity by 50% relative to NaF. These features are not observed when the complex is assayed with ATP in the presence of a nucleotide regenerating system or when analogs Gpp)NH)p or GDP beta S are used along with NaF. These effects were studied in three other membrane systems using App(NH)p as substrate: rat liver, rat ovary and turkey erythrocyte. No consistent pattern of guanine nucleotide effects upon fluoride activation could be observed in the different membrane preparations. Previous experiments showed that the size of soluble thyroid adenylate cyclase changed whether membranes were preincubated with Gpp(NH)p or NaF. This size change roughly corresponded to the molecular weight of the nucleotide regulatory protein. This finding, coupled with the present data, suggests that two guanine nucleotide binding sites may be involved in regulating thyroid cyclase and that these sites may be on different protein chains.     

Interaction of the Inhibitory GTP Regulatory Component with Soluble Cerebral Cortical Adenylate Cyclase

Functional interaction of the inhibitory GTP regulatory component (Ni) with the adenylate cyclase catalytic subunit has not previously been demonstrated after detergent solubilization. The present report describes a sodium cholate-solubilized preparation of rat cerebral cortical membrane adenylate cyclase that retains guanine nucleotide-mediated inhibition of activity. Methods of membrane preparation, cholate extraction, and assay conditions were manipulated such that guanosine-5'-(beta-gamma-imido)triphosphate [Gpp(NH)p] inhibited basal activity 40-60%. The rank order of potency among various GTP analogs was similar in cholate extracts and in membranes: guanosine-5'-0-(3-thiotriphosphate) greater than Gpp(NH)p greater than GTP. Inclusion of 0.1 mM EGTA reduced basal activity 70-90% and abolished Gpp(NH)p inhibition of basal activity in both membranes and cholate extracts. Forskolin-stimulated activity was also inhibited by Gpp(NH)p. Treatment of either membranes or cholate extracts with N-ethylmaleimide abolished Gpp(NH)p inhibition. Gel filtration of the cholate extract over a Sepharose 6B column in 0.1% Lubrol PX partially resolved the adenylate cyclase components. However, Gpp(NH)p inhibition of basal activity (60% of the control) was maintained in select column fractions. Sucrose gradient centrifugation totally resolved the catalytic subunit from both functional Ni and stimulatory GTP regulatory component (Ns) activities. The sedimentation of functional Ni activity was detected by assaying the ability of sucrose gradient fractions to confer Gpp(NH)p inhibition of the resolved catalytic activity. Labeling of gradient or column fractions with pertussis toxin and [32P]NAD revealed that both the 39,000- and 41,000-dalton substrates comigrated with the functional Ni activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenylate cyclase from Hirudo medicinalis segmental ganglia: Modulation by physiological and non-physiological agents

1. In Hirudo medicinalis segmental ganglia GTP is essential for the full expression of the stimulatory action of serotonin on the adenylate cyclase activity. The amine, in turn, increases the overall affinity of the enzymatic system for GTP.2. GTPγS and Gpp(NH)p, non-hydrolysable analogues of GTP, dose-dependently enhance the basal enzyme activity, but impair the stimulatory effect of serotonin.3. Fluoride ions biphasically modulate the leech adenylate cyclase both in the absence and in the presence of GTP. The ion effect is also influenced by non-physiological guanine nucleotides     

Inhibition of forskolin-stimulated cardiac adenylate cyclase activity by short-chain alcohols

The diterpene forskolin stimulated rat cardiac adenylate cyclase activity at least 20-fold and potentiated the effect of NaF. The stimulatory effect of forskolin was reduced in the presence of Gpp(NH)p. Ethanol markedly reduced the stimulation of adenylate cyclase by forskolin while potentiating NaF and Gpp(NH)p stimulation. The inhibitory effect of ethanol on forskolin stimulation appeared to be of a mixed type with both a competitive and a non-competitive component. Three other short-chain linear alcohols (methanol, propanol, butanol) also inhibited forskolin-stimulation, this effect being proportional to the number of carbon atoms.