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1.
We report that the adenylate cyclase system in human platelets is subject to multiple regulation by guanine nucleotides. Previously it has been reported that GTP is either required for or has little effect on the response of the enzyme to prostaglandin E1. We have found that when platelet lysates were prepared in the presence of 5 mM EDTA, GTP lowered the basal and prostaglandin E1-stimulated adenylate cyclase activity when the enzyme was assayed in the presence of Mg2+. The basal and prostaglandin E1-stimulated adenylate cyclase activities were also increased by washing, which presumably removes endogenous GTP. The analog, guanyl-5′-yl-imidodiphosphate mimics the inhibitory effect of GTP on prostaglandin E1-stimulated adenylate cyclase activity but it stimulates basal enzyme activity. The onset of the inhibitory effect of GTP on the adenylate cyclase system is rapid (1 min) and is maintained at a constant rate during incubation for 10 min. GTP and guanyl-5′-yl-imidodiphosphate were noncompetitive inhibitors of prostaglandin E1. An increase in the concentration of Mg2+ gradually reduces the effect of GTP while having little influence on the effect of guanyl-5′-yl-imidodiphosphate. Neither the substrate concentration nor the pH (7.2–8.5) is related to the inhibitory effect of guanine nucleotides. The inhibition by nucleotides was found to show a specificity for purine nucleotides with the order of potency being guanyl-5′-yl-imidodiphosphate > dGTP > GTP > ITP > XTP > CTP > TTP. The inhibitory effect of GTP is reversible while the effect of guanyl-5′-yl-imidodiphosphate is irreversible. The GTP inhibitory effect was abolished by preparing the lysates in the presence of Ca2+. However, the inhibitory effect of guanyl-5′-yl-imidodiphosphate persisted. Substitution of Mn2+ for Mg2+ in the assay medium resulted in a diminution of the inhibitory effect of GTP on basal activity and converted the inhibitory effect of GTP on prostaglandin E1-stimulated activity to a stimulatory effect. At a lower concentration of Mn2+ (less than 2 mM) guanyl-5′-yl-imidodiphosphate inhibited prostaglandin E1-stimulated adenylate cyclase activity, but at a higher concentration of Mn2+, it caused an increase in enzyme activity exceeding that occuring in the presence of prostaglandin E1. In the presence of Mn2+, dGTP mimics the effect of GTP and is 50% as effective as GTP. Our data suggest that the inhibitory effect of GTP on prostaglandin E1-stimulated adenylate cyclase is mainly due to its direct effect on the enzyme itself, whereas the stimulatory effect of GTP on prostaglandin E1-stimulated adenylate cyclase is due to enhancement of the coupling between the prostaglandin E1 receptor and adenylate cyclase. These studies also indicate that the method of preparation of platelet lysates can profoundly alter the nature of guanine nucleotide regulation of adenylate cyclase.  相似文献   

2.
Activation of adenylate cyclase by guanine nucleotide and catecholamines was examined in plasma membranes prepared from rabbit skeletal muscle. The GTP analog, 5′-guanylyl imidodiphosphate caused a time and temperature-dependent activation of the enzyme which was persistent, the Ka was 0.05 μM. 5′-Guanylyl imidodiphosphate binding to the membranes was time and temperature dependent, KD 0.07 μM. Beta adrenergic amines accelerated the rate of 5′-guanylyl imidodiphosphate activation of the enzyme with an order of potency isoproterenol ≈ soterenol ≈ salbutamol > epinephrine ? norepinephrine. Catecholamine activation was antagonized by propranolol and the β2 antagonist butoxamine; the β1 antagonist practolol was inactive. [3H]Dihydroalprenolol bound to the membranes and binding was antagonized by β adrenergic agonists with an order of potency similar to the activation of adenylate cyclase and was antagonized by butoxamine but not by practolol. The data are consistent with the idea that adenylate cyclase in skeletal muscle plasma membranes is coupled to adrenergic receptors of the β2 type.  相似文献   

3.
We found that adenylate cyclase activity of human erythrocytes is potentially labile during isolation of their plasmalemma. Addition of 1 mM EGTA to solution used to remove hemoglobin from lysed cells protected activity. Human erythrocyte adenylate cyclase is minimally activated by catecholamines, in the absence or presence of exogenous guanyl nucleotide, but substantially by 5′-guanylyl imidodiphosphate or sodium fluoride and concentration-dependently by Mg2+ or Mn2+. Basal catalytic activity is an age-dependent component of the human erythrocyte; 5′-guanylyl imidodiphosphate- or fluoride-activated activities decline with cellular maturation proportionally to the decrease in basal activity.  相似文献   

4.
Adenylate cyclase activities in cell-free preparations of isolated transitional epithelium from rabbit urinary bladders were shown to be stimulated by epinephrine, prostaglandin E1 (PGE1), 5-guanylyl imidodiphosphate (GMP-PNP), and NaF. ACTH, aldosterone, insulin, glucagon, oxytocin, parathyroid hormone and vasopressin were without effect at the concentrations tested. The effects of epinephrine, PGE1, and GMP-PNP appeared to be additive. Essentially all of the adenylate cyclase activity was particulate, while approximately 70% of the cyclic nucleotide 3':5'-phosphodiesterase activity was soluble. Single reciprocal plots of the phosphodiesterase data revealed non-linear kinetics.  相似文献   

5.
Synthetic substance P stimulated adenylate cyclase activity in particulate preparations from rat and human brain.The concentration of substance P for half maximal stimulation in rat brain was 1.8 · 10−7 M.The stimulatory effect of substance P on the rat brain adenylate cyclase activity was 88% compared with 48% by noradrenalin, 163% by prostaglandin E1 and 184% by prostaglandin E2.Both the basal and substance P-stimulated adenylate cyclase activity in rat brain were inhibited by concentration of Ca2+ above 10−6 M.The chelating agent ethyleneglycol-bis-(β-aminoethylether)-N,N′-tetraacetic acid at a concentration of 0.1 mM reduced the basal adenylate cyclase activity by 64% and eliminated the substance P-stimulated activity.The inhibition by ethyleneglycol-bis-(β-aminoethylether)-N,N′-tetraacetic acid was completely reversed by increasing concentrations of Ca2+.  相似文献   

6.
7.
The results reported here show some characteristics of adenylate cyclase (EC 4.6.1.1) derived from homogenates of rat spleen, and describe the in vitro stimulation of this enzyme by prostaglandins, nucleotides, and F under conditions where cyclic nucleotide degradative pathways are effectively inhibited.Particulate fractions from rat spleen homogenates contain high adenylate cyclase activities, and the highest specific activity is recovered in a particulate fraction prepared by low speed (1200 × g) centrifugation. Activity found in all particulate fractions is stimulated by fluoride, prostaglandins E1 and E2, catecholamines, and purine nucleotides. No stimulation is caused by prostaglandins F and F. Stimulation by prostaglandin E1 or E2 is augmented by GTP and other purine nucleotides, and stimulation by the combination of GTP and prostaglandin E1 is equal to that caused by optimal fluoride concentrations. Stimulation c caused by L-isoproterenol is additive to that caused by GTP but is not increased by GTP.  相似文献   

8.
GTP potentiated the stimulation by parathyroid hormone and prostaglandin E1 of adenylate cyclase in a renal cortex preparation enriched in proximal tubule basal-lateral plasma membranes. Adenylate cyclase in these membranes did not respond to epinephrine nor glucagon, in the absence or presence of GTP. Activation of basal activity by GMP-PNP was strongly inhibited by GTP. GTP also increased the sensitivity of renal adenylate cyclase to parathyroid hormone and prostaglandin E1. The synergistic effect of GTP was not inhibited by chelating nor thiol-reducing reagents.  相似文献   

9.
Thyroid homogenates and thyroid plasma membranes were prepared from human thyroid and the effects of thyroid-stimulating hormone (thyrotropin), NaF, and prostaglandins E1 and E2 on adenyl cyclase activity in these preparations were studied. The basal level of adenyl cyclase activity in plasma membranes was 5–8 times greater than that of the original homogenates. Adenyl cyclase activity in plasma membranes was stimulated 4.7-fold by 100 munits/ml of thyrotropin and 5-fold by 10 mM of NaF, but the activity in the homogenates was only stimulated 2-fold by either thyrotropin or NaF. Prostaglandin E1 (10?6?10?3 M) and prostaglandin E2 (10?7?10?4 M) failed to stimulate adenyl cyclase activity in plasma membranes, but they did stimulate adenyl cyclase activity in the homogenates. A marked stimulatory effect of prostaglandin E2 (10?5 M) on adenyl cyclase activity in plasma membranes resumed in the presence of GTP (10?7?10?4 M), although GTP itself only slightly stimulated enzyme activity. GDP and GMP were also effective in this respect, although their potencies varied from compound to compound. GTP potentiated slightly the action of thyrotropin on adenyl cyclase in plasma membranes, but it significantly depressed an increase of enzyme activity produced by NaF. Since GTP did not affect the ATP-regenerating system, it seems that GTP, GDP or GMP was required for the manifestation of prostaglandin E2 action on adenyl cyclases of human thyroid plasma membranes.  相似文献   

10.
11.
Adenylate cyclase activity associated with particulate preparations from rat, mouse, rabbit, and dog liver is stimulated 2-to 5-fold by prostaglandin E1 (PGE1). This stimulation is dependent upon the presence of guanosine-5′-triphosphate (GTP). Prostaglandins F1a and F2a do not alter the enzymatic activity under these same conditions. Optimal concentrations of PGE1 + GTP stimulate rat liver adenylate cyclase more than glucagon alone, but less than glucagon + GTP. Activity measured with glucagon + GTP is not affected by addition of PGE1. Stimulation from PGE1 + GTP is increased by glucagon to the same level measured with glucagon + GTP.  相似文献   

12.
We have investigated the effects of NaCl and GTP on the inhibition of platelet adenylate cyclase by 1-O-octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (1-octadecyl-2-acetyl-G-3-PC), using particulate fractions from human and rabbit platelets that had been frozen and thawed in the presence of ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetate to prevent Ca2+-dependent proteolysis. When 10 μM GTP was present, 100 mM NaCl stimulated the activity of the rabbit enzyme 5.6-fold and that of the human enzyme 2.2-fold. Under these conditions, maximum inhibitions of 90% and 64% were obtained on addition of 100 nM 1-octadecyl-2-acetyl-G-3-PC to rabbit and human preparations, respectively. These inhibitions resulted partly from an NaCl-independent inhibition of basal enzyme activity and partly from reversal of the stimulatory effect of NaCl. The relative abilities of the chlorides of different monovalent cations to enhance inhibition of rabbit platelet adenylate cyclase were: NaCl >LiCl >KCl >choline chloride. NaCl also increased the concentrations of 1-octadecyl-2-acetyl-G-3-PC required for half-maximal inhibition of adenylate cyclase but this action of NaCl did not correlate with its stimulatory effect on enzyme activity. After particulate fractions from platelets of either species were washed, 10 μM GTP inhibited basal adenylate cyclase activity in the absence of NaCl but stimulated the enzyme in the presence of NaCl. Inhibition of adenylate cyclase by 1-octadecyl-2-acetyl-G-3-PC was then either enhanced by GTP (rabbit material) or completely dependent on added GTP (human material). Stimulation of the activity of the washed human preparations by NaCl required GTP, but concentrations lower than required for potentiation of the inhibitory effect of 1-octadecyl-2-acetyl-G-3-PC by NaCl were effective.  相似文献   

13.
The binding of [3H]prostaglandin E1 to membranes of clones of normal rat kidney fibroblasts (NRK cells) has been measured. Cell lines that responded to prostaglandin E1, such as NRK and NRK transformed with Schmitt-Ruppin strain of Rous sarcoma virus (SR-NRK cells), have a high affinity prostaglandin E1 binding site. Murine-sarcoma-virus-transformed lines of NRK cells are unresponsive to prostaglandin E1 and have reduced prostaglandin E1 binding. Exposure of cells to prostaglandin E1 results both in decreases prostaglandin E1 responsiveness and reduced prostaglandin E1 binding.Activation of adenylate cyclase is correlated to binding of prostaglandin E1 to receptors in both NRK and SR-NRK cell membranes. Mathematical models suggest that GTP decreases the affinity of hormone for its receptor while increasing the catalytic efficiency of adenylate cyclase, and that aggregates of occupied receptors may play an important role in the activation of adenylate cyclase.  相似文献   

14.
The effects of guanine nucleotides were tested on basal and agonist-modulated adenylate cyclase in guinea-pig superior cervical ganglion crude membrane preparations. GTPS and Gpp(NH)p dose-dependently stimulate, while GDPS inhibits, both the basal and the prostaglandin E2-stimulated enzyme activity. Low GTP doses, up to 10–5M, stimulate, while higher doses inhibit, the ganglionic adenylate cyclase. The GTP-induced diphasic pattern is maintained also in the presence of prostaglandin E2,d-Ala2-Met-enkephalinamide, or a combination of the two drugs. However, the opioid inhibits the enzyme activity, but only at high GTP doses, while the prostaglandin stimulates the enzyme at all GTP concentrations. The effect is potentiated by a combination of prostaglandin and enkephalin. The enhancing effect of the prostaglandin and of the combination with enkephalin is maximally expressed at high, almost physiological, GTP doses.  相似文献   

15.
Adenylate cyclase in the membrane fractions of bovine and rat brains, but not in rat liver plasma membranes, was solubilized by treatment with Fe2+ (10 μM) plus dithiothreitol (5 mM). Solubilization of the enzyme by these agents was completely prevented by simultaneous addition of N,N′-diphenyl-p-phenylenediamine (DPPD), an inhibitor of lipid peroxidation. Ascorbic acid also solubilized the enzyme from the brain membranes. Lipid peroxidation of the brain membranes was characterized by a selective loss of phosphatidylethanolamine. Solubilization of membrane-bound enzymes by Fe2+ plus dithiothreitol was not specific for adenylate cyclase, because phosphodiesterase, thiaminediphosphatase and many other proteins were also solubilized. Solubilized adenylate cyclase had a high specific activity and was not activated by either NaF, 5′-guanylyl imidodiphosphate (Gpp[NH]p) or calmodulin. These results suggested that lipid peroxidation of the brain membranes significantly solubilized adenylate cyclase of high specific activity.  相似文献   

16.
We first identified GTP cyclohydrolase I activity (EC 3.5.4.16) in the ciliated protozoa, Tetrahymena pyriformis. The Vmax value of the enzyme in the cellular extract of T. pyriformis was 255 pmol mg−1 protein h−1. Michaelis–Menten kinetics indicated a positive cooperative binding of GTP to the enzyme. The GTP concentration producing half-maximal velocity was 0.8 mM. By high-performance liquid chromatography (HPLC) with fluorescence detection, a major peak corresponding to -monapterin (2-amino-4-hydroxy-6-[(1′R,2′R)-1′,2′,3′-trihydroxypropyl]pteridine, -threo-neopterin) and minor peaks of -erythro-neopterin and -erythro-biopterin were found to be present in the cellular extract of Tetrahymena. Thus, it is strongly suggested that Tetrahymena converts GTP into unconjugated pteridine derivatives. In this study, dopamine was detected as the major catecholamine, while neither epinephrine nor norepinephrine was identified. Indeed, this protozoa was shown to possess the activity of a dopamine synthesizing enzyme, aromatic -amino acid decarboxylase. On the other hand, activities of tyrosine hydroxylase or tyrosinase which converts tyrosine into dopa, the substrate of aromatic -amino acid decarboxylase, could not be detected in this protozoa. Furthermore, neither dopamine β-hydroxylase activity nor phenylethanolamine N-methyltransferase activity could be identified by the HPLC methods.  相似文献   

17.
In hamster adipocyte ghosts, ACTH and β-adrenergic agonists stimulate adenylate cyclase by a GTP-dependent process; in contrast, inhibition of the enzyme by hormonal factors requires both GTP and sodium ions. The interaction of various monovalent cations and guanine nucleotides was studied on basal, stimulated and inhibited adenylate cyclase activities. In the presence of GTP (0.03–10 μM), which reduced basal activity by up to 90%, monovalent cations (10–500 mM, added as chloride salts) increased the enzyme activity by up to about 8-fold. The potency order obtained was Na+>Li+>K+>choline. The stable GTP analogue, guanylyl-5′-imidodiphosphate, which like GTP was capable of decreasing basal activity, diminished the cation-induced activation. The stimulatory effects of ACTH and isoproterenol on adipocyte adenylate cyclase activity were impaired by the cations in the potency order, Na+>Li+>K+>choline. Additionally, NaCl shifted the concentration-response for ACTH to the right and caused an increase in the maximal activation by the hormone. Similar to basal activity, fluoride-stimulated activity was increased by NaCl, when GTP was present. The inhibitory effect of prostaglandin E1 on basal adipocyte adenylate cyclase activity was revealed by the cations in the above mentioned potency order by an apparent reversal of the cation-induced activation. In the presence of NaCl, the ACTH- or fluoride-stimulated activities were also reduced by prostaglandin E1, but the inhibitory hormonal factor did not reverse the NaCl-induced shift in the concentration-response curve for ACTH. Guanylyl-5′-imidodiphosphate completely prevented hormonal inhibition. The data suggest that monovalent cations interact with the guanine nucleotide-binding regulatory component of the adipocyte adenylate cylase system and that this interaction somehow changes the properties of this component, now revealing hormone-induced inhibition partially impairing hormone-induced stimulation.  相似文献   

18.
Pretreatment of isolated rat liver plasma membranes by washing with NaHCO3 buffer or by exposure to the chelator ethyleneglycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) with or without the ionophore A23187, produced a decrease in the sensitivity of adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) to subsequent stimulation by NaF or guanosine 5′-(β-γ-imino)triphosphate (GPP(NH)P). Sensitivity to activation by the nucleotide could be restored by addition of the lyophilized and ashed wash or by addition of Ca2+, Mg2+ or Mn2+. The factor extracted from the membranes by these various treatments which was responsible for loss of stimulation was identified as Ca2+. Determination of the metal ion content of isolated membranes by atomic absorption spectrometry indicated that Ca2+ was the only divalent cation present in sufficient concentration to support persistent activation by either NaF or GPP(NH)P.Pretreatment of liver plasma membranes with trifluoperazine, which inhibits the action of Ca2+-dependent regulator protein in other enzyme systems, reduced GPP(NH)P activation of adenylate cyclase and caused marked depletion of membrane Ca2+. The effects of low concentrations (less than 100 μM) of the phenothiazine could be reversed totally by Ca2+ and partly by regulator protein. At higher concentrations of trifluoperazine, slight restoration of enzyme activation was seen with either agent. The hypothesis is presented that Ca+ interacts with the nucleotide (GTP or GDP) regulatory site(s) of the adenylate cyclase. This interaction may be regulator-protein-dependent and may be important in determining the sensitivity of the enzyme to nucleotide activation in vivo.  相似文献   

19.
In hamster adipocyte ghosts, ACTH stimulates adenylate cyclase by a GTP-dependent process, whereas prostaglandin E E1, α-adrenergic agonists and nicotinic acid inhibit the enzyme by a mechanism which is both GTP- and sodium-dependent. The influence of the divalent cations Mn2+ and Mg2+, was studied on these two different, apparently receptor-mediated effects on the adipocyte adenylate cyclase. At low Mn2+ concentrations, GTP (1 μM) decreased enzyme activity by about 80%. Under this condition, ACTH (0.1 μM) stimulated the cyclase by 6- to 8-fold, and NaCl (100 mM) caused a similar activation. In the presence of both GTP and NaCl, prostaglandin E1 (1 or 10 μM) and nicotinic acid (30 μM) inhibited the enzyme by about 70–80% and epinephrine (300 μM, added in combination with a β-adrenergic blocking agent) by 40–50%. With increasing concentrations of Mn2+, the GTP-induced decrease and the NaCl-induced increase in activity diminished, with a concomitant decrease in prostaglandin E1?, nicotinic acid- and epinephrine-induced inhibitions as well as in ACTH-induced stimulation. At 1 mM Mn2+, inhibition of the enzyme was almost abolished and stimulation by ACTH was largely reduced, whereas activation of the enzyme by KF (10 mM) was only partially impaired. The uncoupling action of Mn2+ on hormone-induced inhibition was half-maximal at 100–200 μM and appeared not to be due to increased formation of the enzyme substrate, Mn · ATP. It occurred without apparent lag phase and could not be overcome by increasing the concentration of GTP. Similar but not identical findings with regard to adenylate cyclase stimulation and inhibition by hormonal factors were obtained with Mg2+, although about 100-fold higher concentrations of Mg2+ than of Mn2+ were required. The data indicate that Mn2+at low concentrations functionally uncouples inhibitory and stimulatory hormone receptors from adenylate adenylate cyclase in membrane preparations of hamster adipocytes, and they suggest that the mechanism leading to uncoupling involves an action of Mn2+ on the functions of the guanine nucleotide site(s) in the system.  相似文献   

20.
The rate of cyclic AMP formation by rabbit heart membrane particles decreased at assay temperatures greater than 30 °C. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity (assayed at 24 °C) decreased exponentially with time of preincubation at 30 or 37 °C, providing evidence for the instability of this enzyme. The half-life, t1/2, of the enzyme at 37 °C was 9.9 min in the absence and 4.4 min in the presence of MgCl2. The activity was most labile in the presence of 50 m m Mg2+ and 1 m m ATP, having t1/2 = 1.3min. Prior incubation of membranes with the GTP analog, guanyl-5′-yl imidodiphosphate [Gpp(NH)p], 0.1 m m, for 30 min at 37 °C produced maximal activation of adenylate cyclase; the rate of activation was temperature dependent and was increased in the presence of isoproterenol. The Gpp(NH)p-activated enzyme had increased thermal stability, t1/2 = 170 min, and was also markedly more stable in the presence of Mg-ATP, t1/2 = 72min, than nonactivated enzyme. Preactivation with F? (30 min at 24 °C) also stabilized the activity; t1/2 > 70 min in the absence or presence of Mg-ATP. The Mg2+ concentration required for maximal activity was reduced from approximately 60 m m for nonactivated enzyme to 10 m m for the Gpp(NH)p- and F?activated enzyme.  相似文献   

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