Absence Of Gpp Research Articles

Stimulatory and inhibitory effects of guanine nucleotides on arginine-vasotocin-sensitive adenylate cyclase in the epithelial cell membranes of the bullfrog bladder.

The effects of arginine-vasotocin and nucleotides on the steady-state kinetics of the adenylate cyclase activity in the epithelial cell membranes of the bullfrog (Rana catesbiana) bladder were studied. Arginine-vasotocin stimulated adenylate cyclase more effectively than oxytocin or arginine-vasopressin, with respect to both the maximal hormonal activation ratio relative to basal, and the hormone concentration yielding a half-maximal response (apparent Km). Arginine-vasotocin, GTP and its analogue guanyl-5'-yl imidodiphosphate (Gpp(NH)p) increased the Vmax of the basal adenylate cyclase activity, but showed no effect of the apparent Km of the system for ATP. In addition, Gpp(NH)p enhanced the arginine-vasotocin-stimulated adenylate cyclase activity, further increasing the Vmax, while GTP showed no statistically significant effect. Dual effects of GDP were apparent: it was stimulatory at 1 x 10(-5) mol/l and inhibitory at 1 x 10(-3) mol/l, on both the basal and the arginine-vasotocin-stimulated adenylate cyclase activity. Guanosine 5'-monophosphate, CTP, UTP and ITP showed no apparent effect on the enzyme activity. Sodium fluoride acted in the same manner as GTP on the adenylate cyclase system, increasing only basal activity. Adenylate cyclase activities exhibited pH optima that were less distinct in the presence than in the absence of Gpp(NH)p. The Arrhenius plot of the temperature experiment showed that a high-energy step was involved for activation by Gpp(NH)p or arginine-vasotocin. When the relative activation ratios by arginine-vasotocin at different ATP concentrations were studied, a distinct activation optimum was shown at 2.5 x 10(-4) mol ATP/l, either in the absence or presence of Gpp(NH)p. The possibility that GTP, GDP nd ATP play a regulatory role in the epithelial cells of the bullfrog bladder by adjusting the responsiveness of the system to a natural hormone, arginine-vasotocin, is discussed.

Lymphocyte adenylate cyclase and human aging.

Adenylate cyclase activity was determined by enzymatic conversion of [32P]ATP to [32P]cAMP using peripheral lymphocytes freshly isolated from human subjects. The lymphocyte enzyme was stimulated by the potent beta-adrenergic catecholamine agonist isoproterenol and by the nonhydrolyzable GTP-analog Gpp[NH]p. The two activators had a synergistic effect, and agonist-dependent enzyme activity followed simple Michaelis-Menten kinetics with respect to isoproterenol in the presence but not in the absence of Gpp[NH]p. Cyclic AMP production by intact lymphocytes, determined by protein binding assay, also followed simple Michaelis-Menten kinetics with respect to isoproterenol. Kact of isoproterenol was the same in intact cells and the broken cell assay in the presence of Gpp[NH]p, suggesting the indispensable role the GTP-binding coupling factors play in the intact lymphocyte. In 31 human subjects between the age of 21 and 103, adenylate cyclase activity in the presence of isoproterenol, Gpp[NH]p, or isoproterenol in the presence of Gpp[NH]p decreased with the increasing age of the subject. The sensitivity of the enzyme to stimulation by isoproterenol, defined as the Kact and determined in the presence of Gpp[NH]p, was the same in lymphocytes from young (less than 45 years) or elderly (greater than 75 years) subjects. These results suggest a deficiency in the lymphocyte adenylate cyclase system distal to the beta-adrenergic catecholamine receptor could account for deterioration of cAMP-mediated components of the immune response which occur with age.

A sensitive method for assaying thyroid stimulating immunoglobulins of Graves' disease: use of the guanyl nucleotide-amplified thyroid adenylate cyclase assay.

The purpose of this study was to develop and validate a sensitive method for evaluating adenylate cyclase stimulation by thyroid-stimulating antibodies (TSAb), based on the measurement of thyroid membrane adenylate cyclase activity in the presence of a non-hydrolyzable GTP analogue, guanyl-5'-yl imidodiphosphate (Gpp(NH)p). The addition of Gpp(NH)p (10(-5)M) produced a 10-fold increase of the sensitivity of the system for both TSH and TSAb. Immunoglobulin G preparations from sera of 30 patients with Graves' disease were tested for the adenylate cyclase stimulation either in the presence or in the absence of Gpp(NH)p: a significant stimulation was observed in 27/30 patients when the GTP analogue was added to the system, while only 20/30 patients were positive in the absence of the nucleotide. The advantage of Gpp(NH)p addition was also evident in a large series which included 57 patients with Graves' disease, 15 with Hashimoto's thyroiditis or primary myxoedema and 22 normal subjects. In fact, 88% of patients with Graves' disease resulted positive, while no significant stimulation was elicited by Hashimoto's thyroiditis, primary myxoedema and by normal immunoglobulins. The sensitivity achieved in our system which employs thyroid plasma membranes was similar to that obtained by other investigators with the use of thyroid slices or thyroid cells in primary culture. Furthermore, methods based on thyroid plasma membranes are supposed to have a better reproducibility, since the same tissue preparation, if appropriately stored, may be used in several different tests.

Specific uncoupling by islet-activating protein, pertussis toxin, of negative signal transduction via alpha-adrenergic, cholinergic, and opiate receptors in neuroblastoma x glioma hybrid cells.

Exposure of NG108-15 hybrid cells to islet-activating protein (IAP), pertussis toxin, caused strong ADP-ribosylation of one of the membrane proteins with a molecular weight of 41,000. This ADP-ribosylation was paralleled by decreases in the inhibition of cAMP accumulation in intact cells or associated with reversal of the inhibition of GTP-dependent membrane adenylate cyclase, via alpha-adrenergic, cholinergic muscarinic, or opiate receptors. The affinity of these receptors for agonists was lowered by guanyl-5'-yl beta-gamma-imidodiphosphate (Gpp(NH)p) reflecting their coupling to the guanine nucleotide regulatory protein in this cell line. This effect of Gpp(NH)p was lost in membranes of IAP-treated cells; in the absence of Gpp(NH)p, the affinity for agonist was lower in treated than in nontreated cells. In contrast, the function of these receptors to bind antagonists remained unaltered in IAP-treated cells. Thus, IAP treatment of NG108-15 cells caused specific uncoupling of negative signal transduction from inhibitory receptors to the adenylate cyclase catalytic unit via the guanine nucleotide regulatory protein, as a result of ADP-ribosylation of one of the subunits of the regulatory protein.

High and low (Gpp(NH)p-sensitive) affinity sites for β 2-adrenergic blockers as antagonists of isoproterenol in the field-stimulated rat vas deferens

Schild plots for (-)-propranolol, timolol and IPS 339, but not penbutolol or carazolol, as antagonists of isoproterenol in the isolated, field-stimulated rat vas deferens exhibited biphasic curves indicating the presence of both high and low affinity sites for these agents in this preparation. Schild plots determined for (-)-propranolol in the presence of prazosin (0.3 μM) or using the prostatic end of the rat vas deferens yielded two similar affinity sites suggesting the lack of involvement of postsynaptic α 1-adrenergic mechanisms in the observed response. In the presence of Gpp(NH)p(4μM), (-)-propranolol, timolol and IPS 339 exhibited only a single affinity site which corresponded to their high affinity site in antagonizing isoproterenol in the absence of Gpp(NH)p. Similarly, only a single high affinity site was observed for (-)-propranolol when salbutamol, rather than isoproterenol, was used as the agonist. The data suggest the two affinity sites observed with some β-adrenergic blockers in this preparation reflects a possible action of isoproterenol and the antagonists upon two subpopulations of β 2-adrenergic receptors, one of which is sensitive to Gpp(NH)p.

Guanyl nucleotide potentiation of parathyroid hormone-stimulated adenylate cyclase in chicken renal plasma membranes: a receptor-independent effect.

We investigated the interaction of guanyl nucleotides with the parathyroid hormone (PTH) receptor-adenylate cyclase system in chicken renal plasma membranes. Micromolar concentrations of guanosine triphosphate and its hydrolysis-resistant analog 5'-guanylimidodiphosphate [Gpp(NH)p] increased both basal and PTH-stimulated adenylate cyclase activity. The enzyme activation produced by the amino-terminal 1--34 peptide of bovine PTH [bPTH-(1--34)] was potentiated by both guanyl nucleotides, although quantitatively greater effects were seen with Gpp(NH)p. The apparent activation constant for bPTH-(1--34) stimulation of adenylate cyclase, 16 nM, was reduced to 3.7 nM in the presence of 1.0 microM Gpp(NH)p. The interaction of guanyl nucleotides with the PTH receptor was evaluated by measurement of specific 125I-labeled bPTH-(1--34) binding to chicken renal plasma membranes in the presence and absence of Gpp(NH)p. There was no effect of the guanyl nucleotide on the rate of binding or dissociation of 125I-labeled bPTH-(1--34) from its renal receptor. Scatchard analysis of steady state PTH binding revealed that 1.0 microM Gpp(NH)p had minimal effect on either the affinity of PTH receptors (Kd increased from 25 nM to 30 nM) or their number (total number of binding sites increased from 8.6 to 9.4 pmol/mg protein). Separate experiments demonstrated a concentration-dependent effect on Gpp(NH)p to decrease the apparent activation constant for bPTH-(1--34) stimulation of adenylate cyclase, with no detectable guanyl nucleotide effect on the affinity of PTH receptors. The results suggest that guanyl nucleotides may enhance the coupling of occupied PTH receptors to adenylate cyclase independent of direct nucleotide effects on renal PTH receptors.

Modulation of Follicle-Stimulating Hormone-Sensitive Rat Testicular Adenylate Cyclase Activity by Guanyl Nucleotides*

We have studied modulation of FSH-sensitive adenylate cyclase activity in testes of immature rats by guanyl nucleotides. Highly purified hFSH alone stimulated adenylate cyclase activity 2.2-fold over basal levels. Addition of the GTP analog, 5'-guanylyl imidodiphosphate [Gpp(NH)p], caused an additional 2.8-fold augmentation of adenylate cyclase activity to 6 times over basal levels and 3.7 times greater than that seen in the presence of Gpp(NH)p alone. GTP did not significantly stimulate basal levels of adenylate cyclase and augmented FSH stimulated activity by 1.4-fold; other nucleotides were without effect. Half-maximum activation of adenylate cyclase in each instance was produced by approximately similar concentrations of either guanyl nucleotide (about 10 microM). The Km for hormone activation of adenylate cyclase was nearly the same in the presence and absence of Gpp(NH)p. Maximum adenylate cyclase stimulation in the presence of nucleotide and/or hRSH was always less than obtained by fluoride alone. Of all nucleotides tested, only GTP and its analog, Gpp(NH)p, significantly augmented FSH stimulation of testicular adenylate cyclase activity. Gpp(NH)p also markedly inhibited binding of radiolabeled hFSH to testicular receptor, but at a concentration 15-fold greater than that required for significant stimulation of testicular adenylate cyclase activity. The results suggest a specific role for guanyl nucleoside triphosphate in regulation of FSH effects on testicular adenylate cyclase activity.

Properties of detergent-dispersed myocardial adenylate cyclase

Adenylate cyclase from rabbit ventricle was solubilized in 30 to 50% yield by the nonionic detergent Lubrol PX. The detergent, when present in the assay at concentrations above 0.05%, rapidly inactivated the enzyme in assays conducted above 26 °C; assays were valid only when conducted below this temperature. The solubilized enzyme was eluted from diethylaminoethyl (DEAE)-Bio-Gel A (DEAE-agarose) with 100 m m NaCl in a yield of 25% and was free of detergent. Several properties of the solubilized detergent-free enzyme were similar to properties of the native membrane-bound species. The K m for substrate was 0.1 m m, the K a for Mg 2+ was 2.5 m m, and ATP in excess of Mg 2+ was inhibitory. The enzyme was activated by F − and guanyl-5′-yl imidodiphosphate [Gpp(NH)p] in a time- and temperature-dependent manner, and activation by the latter was persistent. Activation by F − and Gpp(NH)p reduced the K a for Mg 2+. Activation by Gpp(NH)p was increased by Mg 2+; the apparent K a for activation was 0.1 μ m. Multiple binding sites for Gpp(NH)p were present: one class with a K d value of 0.11 μ m was probably associated with activation of the enzyme. The soluble enzyme was insensitive to catecholamines, in both the presence and the absence of Gpp(NH)p. Sensitivity to catecholamines was not restored by the addition of phospholipids, particularly phosphatidyl inositol, in either the presence or the absence of Gpp(NH)p, and this phospholipid did not increase the sensitivity of the membrane-bound enzyme to epinephrine. Catecholamine binding sites were present, and their association with adenylate cyclase was seemingly not affected by phospholipids.

Characterization of the β-adrenergic receptor and adenylate cyclase in skeletal muscle plasma membranes

Subcellular fractions of rat skeletal muscle enriched in plasma membrane (sarcolemma) were used to characterize properties of the β-adrenergic receptor and adenylate cyclase. The equilibrium binding of (−)-[ 3 H]dihydroalprenolol to the sarcolemmal membrane was characterized by a high affinity, K =3.6×10 9 liters/mol and 1.2×10 −9 mol/g of membrane protein of homogenous noninteracting sites. The binding was reversible, saturable, and stereospecific. Displacement of labeled dihydroalprenolol by adrenergic ligands revealed an order of potency corresponding to the β 2 -type response: (−)-isoproterenol>(−)-epinephrine >norepinephrine. The (+)-stereoisomers and the α-adrenergic antagonist phentolamine showed little ability to compete with (−)-[ 3 H]dihydroalprenolol for occupancy of the binding sites. In the membrane fractions, a substantial basal activity of adenylate cyclase activity was demonstrated (35–50 pmol of cyclic AMP/mg/5 min). This activity was stimulated 40- to 50-fold by sodium fluoride, 7-to 8-fold by isoproterenol, and 5- to 6-fold by guanylyl 5′-imidodiphosphate [Gpp(NH)p]. Maximum activation depended upon optimal Mg 2+ ion concentration (10 m m ). In the presence of 0.1 m m Gpp (NH)p., the isoproterenol activation of adenylate cyclase was increased twofold. In addition, the concentration of hormone required for half-maximal stimulation of enzyme activity was shifted from 2.2×10 −7 m in the absence of Gpp(NH)p to 2.5×10 −8 m in the presence of Gpp(NH)p. Gpp(NH)p also stimulated epinephrine and norepinephrine activation without altering the β 2 -type potency series for the enzyme. Gpp(NH)p had no effect on either the number or the affinity of (−)-[ 3 H]dihydroalprenolol binding sites, but it did increase the dissociation constant for isoproterenol by approximately fivefold.

The adenylate cyclase system of planaria Polycelis tenuis: Activation by serotonin and guanine nucleotides

The particulate fraction prepared after homogenization of planaria Polycelis tenuis in a buffer containing 3 mM EDTA and 15 mM 2-mercaptoethanol possesses an adenylate cyclase activity which was enhanced two-fold by serotonin and 20-fold by the nucleotide analog guanosine 5′-(ß-γ-imino)triphosphate, Gpp(NH)p; when present together, the two activators exhibited a marked synergistic effect. The effect of serotonin was dose dependent, with a K A of 2 μM and a Hill coefficient of 0.4. In the presence of 10μM Gpp(NH)p, these values became 45 nM and 1.5 respectively. The effect of serotonin was due to an increase in the maximal velocity of the enzyme and was specifically inhibited by methiotepin. The effect of methiotepin was half-maximal at 0.2 μM in the absence of Gpp(NH)p and at 5.0 μM in its presence. Planaria thus appear to be the lowest organisms in which guanine nucleotides are active upon adenylate cyclase. As serotonin in normally present in planaria, it is postulated that a serotonin-dependent regulation of adenylated cyclase activity plays a physiological role in this species.

Irreversible activation of adenylate cyclase of toad erythrocyte plasma membrane by 5'-guanylylimidodiphosphate.

The irreversible activation of adenylate-cyclase by 5'guanylylimidodiphosphate, a phosphoramidate analog of 5'GTP, has been examined in toad (Bufus marinus) plasma membranes using the technique of preincubating the membranes with the nucleotide under various controlled conditions followed by washing and subsequent assay of enzyme activity. Activation of adenylate cyclase by Gpp(NH)p, but not GTP, is essentially permanent and persists following extensive washing, prolonged incubation at 30 degrees C in the absence of the nucleotide, and after dissolution of the membranes with Lubrol PX. (-)-Isoproterenol increases the activation observed with maximal concentrations of Gpp(NH)p from eight- to 10-fold (in the absence of hormone) to 50- to 100-fold; final activities as high as 10-15 nmoles of cyclic AMP per min per mg protein are achieved. The activated state obtained with isoproterenol and Gpp(NH)p is also permanent and is not inhibited by propranolol. The synergism between Gpp(NH)p and hormone requires the simultaneous presence of these compounds, and the time-dependent enhancement of activation with (-)-isoproterenol may be interrupted by addition of propranolol. The stimulation is slow, and may proceed for as long as 45 min at 30 degrees C in the presence of maximal concentrations of Gpp(NH)p and (-)-isoproterenol. Very little activation occurs at 0 degrees C. The time course of activation at 30 degrees C exhibits an accelerating phase lasting from 5 to 30 min when Gpp(NH)p is added directly during assay of cyclase activity or when the membranes are preincubated for various times and washed prior to assay for a fixed time. The lag period occurs in the presence and absence of (-)-isoproterenol, although the rate of increase in velocity is greater with hormone. The length of the accelerating phase decreases with increasing concentrations of Gpp(NH)p, although it is still evident with maximal levels of Gpp(NH)p and hormone. However, prewarming the membranes at 30 degrees C for 10 min in the absence of Gpp(NH)p or (-)-isoproterenol results in an immediate onset of linear activation at a rate which is achieved in untreated membranes only after about 10 min. The events occurring during prewarming at 30 degrees C are readily reversible since chilling the warmed membranes to 0 degrees C results in a time course of activation identical to that of membranes maintained at 0 degrees C until addition of Gpp(NH)p. Activation is proportional to the concentration of Gpp(NH)p within the range of 10(-8) to 10(-4) mM. The apparent affinity for Gpp(NH)p increases with increasing time of incubation. The primary effect of increasing the concentration of Gpp(NH)p is to decrease the time required to obtain a maximal rate of activation. The possible relevance of these findings to the mechanism of action of Gpp(NH)p, adenylate cyclase and hormones is discussed within the context of current views of biological membranes which recognize the lateral mobility of membrane molecules.

Role of 5′-Guanylylimidodiphosphate in the Activation of Adenylyl Cyclase by Parathyroid Hormone

We have studied the effects of guanylylimidodiphosphate (Gpp(NH)p), an analogue of GTP, on the stimulation of renal cortical adenylyl cyclase by bovine parathyroid hormone (bPTH, or bPTH-(1-84)). Incubation of canine renal membranes with bPTH-(3-34), a specific antagonist of parathyroid hormone, in either the presence or absence of Gpp(NH)p, prevented subsequently added bPTH-(1-84) from stimulating adenylyl cyclase. The addition of the antagonist to a cyclase system previously activated by both bPTH-(1-84) and Gpp(NH)p, however, produced no inhibition of enzyme activity. Removal of bPTH by washing the membranes virtually abolished activity, but washing after addition of bPTH plus Gpp(NH)p did not prevent continued accumulation of cAMP. The persistence of the activity of the enzyme brought about by the addition of Gpp(NH)p plus bPTH, despite washing or addition of specific inhibitor of bPTH action, indicates that the activity of the hormone-specific adenylyl cyclase in membrane suspensions is independent of cintinuous occupancy of the peptide-hormone receptor by bPTH in the presence of the guanyl-nucleotide analogue.

The hepatic adenylate cyclase system. I. Evidence for transition states and structural requirements for guanine nucloetide activiation

Previous studies have shown that guanine nucleotides, acting at a site termed nucleotide regulatory site, are required for activation of hepatic adenylate cyclase and that glucagon facilitates this process. This study shows that only guanine nucleotides containing triphosphate groups at the 5' position of ribose (or 3'-deoxyribose) are capable of activating the enzyme. The terminal phosphate is not utilized in the activation process since 5'-guanylylimidodiphosphate (Gpp(NH)p and 5'-guanylyl methylenediphosphonate, analogues of GTP that are not utilized in transferase or hydrolase reactions, stimulate enzyme activity. The nucleotides bind in their free form at the regulatory site; chelation by magnesium ion shifts the apparent concentration dependence for activation by Gpp(nh)p. GDP inhibits competitively Gpp(NH)p-stimulated activity and inhibits basal activity and activities stimulated by glucagon. Activation of the enzyme by Gpp(NH)p is a slow process; the length of the lag time increases as an inverse function of nucleotide concentration and is as long as 4 min before onset of increased enzyme activity. Following pretreatment with Gpp(NH)p and extensive washing of hepatic membranes, the enzyme displays immediate increases in activity with rates that are a function of the nucleotide concentration during pretreatment; the rates remain constant for at least 6 min despite the absence of Gpp(NH)p in the medium. Studies with labeled Gpp(NH)p show that the intact nucleotide remains firmly bound to the membranes after extensive washing, suggesting that the persistence of adenylate cyclase activity may be related to slow dissociation of the nucleotide from the regulatory site. Addition of 1 nM glucagon, a submaximal concentration, does not abolish the lag phase of Gpp(NH)p activation even at saturating concentration of the nucleotide (1 muM or higher). The maximal steady state rate is achieved under these conditions. Addition of 2 muM glucagon, a saturating hormone concentration, does not alter the steady state rate but abolishes the lag phase of Gpp(NH)p activation. The transient kinetics of Gpp(NH)p activation and the effects of glucagon thereon are discussed in terms of a three state model in which the guanine nucleotide induces the formation of an intermediate transition state that displays no increase in enzyme activity over the basal state and which slowly isomerizes to a high activity state of the adenylate cyclase system; glucagon acts by accelerating the rate of isomerization.