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1.
The inhibitors histidine and AMP cause the enzyme ATP phosphoribosyltransferase of E. coli to associate into a hexamer from its initial dimeric form. The behaviour of these inhibitors has been studied by three different methods. I) Equilibrium dialysis studies have shown that one mole of dimeric enzyme (67,000 g) binds one mole of histidine. II) By kinetic inhibition of the reaction studied at 21, 25 and 38 degrees C the enthalpy changes in the process of histidine and of AMP inhibition have been deduced. The inhibition has also been studied in function of enzyme concentration and temperature. The inhibition appears to be slightly negatively cooperative for histidine and positively cooperative for AMP. In neither case is it possible to obtain 100% maximal inhibition. III) By microcalorimetric analysis the values obtained for the enthalpies of histidine and of AMP interaction with the enzyme are similar.  相似文献   

2.
Culture of a wild-type strain of Escherichia coli in the presence of cyclic AMP leads to an impairment of uracil uptake. Half maximum inhibition of uracil uptake was observed at 1.5 mM cyclic AMP. The effect seems to be specific since no inhibition was found in cultures supplemented with ATP, ADP or 5'-AMP. Similarly the inhibition was not observed in cultures of a mutant deficient in the cyclic AMP receptor protein. The inhibition in uracil uptake, found in bacteria cultured in the presence of cyclic AMP, is not a consequence of a reduction in the growth rate. On the other hand, this inhibition was observed only in cultures containing glucose or pyruvate as carbon source.  相似文献   

3.
Phosphatidate bilayers composed of dilauroylphosphatidate, dimyristoylphosphatidate, dipalmitoylphosphatidate and dioleoylphosphatidate were prepared. Their interaction with AMP deaminase isolated from pig heart was investigated. Dioleoylphosphatidate bilayers were found to exert non-competitive inhibition on the AMP deaminase with a Ki of 15 x 10(-6) M. This inhibition is three orders of magnitude stronger than that exerted by orthophosphate. The phosphatidate species containing saturated fatty acids were either non-inhibitory or inhibited enzyme activity rather poorly. However, alkalinization of the medium from pH 6.5 to pH 7.9 led to the inhibition of pig heart AMP deaminase by dilauroylphosphatidate bilayers. This was accompanied by the fluidization of the saturated phosphatidate species, i.e. the lowering of their phase transition temperature in alkaline pH, as measured by light-scattering and fluorescence scans. The possible significance of these findings for the regulation of AMP deaminase activity in vivo by natural membranes is discussed.  相似文献   

4.
AMP deaminase [EC 3.5.6.4] purified from chicken erythrocytes was inhibited by phytic acid (inositol hexaphosphate), which is the principal organic phosphate in chicken red cells. Kinetic analysis has indicated that this inhibition is of an allosteric type. The estimated Ki value was within the normal range of phytic acid concentration, suggesting that this compound acts as a physiological effector. Divalent cations such as Ca2+ and Mg2+ were shown to affect AMP deaminase by potentiating inhibition by lower concentrations of phytic acid, and by relieving the inhibition at higher concentrations of phytic acid. These results suggests that Ca2+ and Mg2+ can modify the inhibition of AMP deaminase by phytic acid in chicken red cells.  相似文献   

5.
Thrombin-induced release of arachidonic acid from human platelet phosphatidylcholine is found to be more than 90% impaired by incubation of platelets with 1 mM dibutyryl cyclic adenosine monophosphate (Bt2 cyclic AMP) or with 0.6 mM 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), an intracellular calcium antagonist. Incorporation of arachidonic acid into platelet phospholipids is not enhanced by Bt2 cyclic AMP. The addition of external Ca2+ to thrombin-treated platelets incubated with Bt2 cyclic AMP or TMB-8 does not counteract the observed inhibition. However, when divalent cation ionophore A23187 is employed as an activating agent, much less inhibition is produced by Bt2 cyclic AMP or TMB-8. The inhibition which does result can be overcome by added Ca2+. Inhibition of arachidonic acid liberation by Bt2 cyclic AMP, but not by TMB-8, can be overcome by high concentrations of A23187. When Mg2+ is substituted for Ca2+, ionophore-induced release of arachidonic acid from phosphatidylcholine of inhibitor-free controls is depressed and inhibition by Bt2 cyclic AMP is slightly enhanced. The phospholipase A2 activity of platelet lysates is increased by the presence of added Ca2+, however, the addition of either A23187 or Bt2 cyclic AMP is without effect on this activity. We suggest that Bt2 cyclic AMP may promote a compartmentalization of Ca2+, thereby inhibiting phospholipase A activity. The compartmentalization may be overcome by ionophore. By contrast, TMB-8 may immobilize platelet Ca2+ stores in situ or restrict access of Ca2+ to phospholipase A in a manner not susceptible to reversal by high concentrations of ionophore.  相似文献   

6.
  • 1.1. The native rat-kidney cortex Fructose-1,6-bisphosphatase is differentially regulated by adenine nucleotides in the presence of divalent cations.
  • 2.2. Binding of AMP and ADP to the enzyme is co-operative. The inhibition by both nucleotides show an uncompetitive mechanism AMP being the most efficient inhibitor.
  • 3.3. Mg2+ decreases the inhibition produced by AMP and ADP by enhancing their I0.5 and completely annulates the inhibitory effect of ATP.
  • 4.4. In the presence of Mn2+ ADP behaves as an inhibitor but no inhibition is evident with AMP, suggesting the existence of different allosteric sites for each nucleotide.
  相似文献   

7.
Purified rat muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) binds tightly to rat myosin. The binding is abolished in the presence of low concentrations of various ligands. Pyrophosphate and GTP at concentrations as low as 0.1 micrometer were effective in abolishing the interaction between two proteins. Other nucleoside triphosphates were less effective than GTP and the concentrations required for 50% inhibition were approximately 0.3 to 0.7 micrometer. ADP and AMP are effective in inhibiting the interaction between two proteins, but they are less effective than the nucleoside triphosphates; 50% inhibition occurred at 34 micrometer with ADP and at 1 mM with AMP. Creatine phosphate and inorganic phosphate showed 50% inhibition at 5 to 6 mM. All of the compounds, which affected AMP deaminase activity, were effective in abolishing the interaction of the enzyme with myosin; however, the interaction-abolishing effects of the compounds are not parallel with their inhibitory effects on the deaminase activity. Although there exist three parental isozymes of AMP deaminase in the rat, all three enzymes interacted with myosin.  相似文献   

8.
To understand the mechanism of signal propagation involved in the cooperative AMP inhibition of the homotetrameric enzyme pig-kidney fructose-1,6-bisphosphatase, Arg49 and Lys50 residues located at the C1-C2 interface of this enzyme were replaced using site-directed mutagenesis. The mutant enzymes Lys50Ala, Lys50Gln, Arg49Ala and Arg49Gln were expressed in Escherichia coli, purified to homogeneity and the initial rate kinetics were compared with the wild-type recombinant enzyme. The mutants exhibited kcat, Km and I50 values for fructose-2,6-bisphosphate that were similar to those of the wild-type enzyme. The kinetic mechanism of AMP inhibition with respect to Mg2+ was changed from competitive (wild-type) to noncompetitive in the mutant enzymes. The Lys50Ala and Lys50Gln mutants showed a biphasic behavior towards AMP, with total loss of cooperativity. In addition, in these mutants the mechanism of AMP inhibition with respect to fructose-1,6-bisphosphate changed from noncompetitive (wild-type) to uncompetitive. In contrast, AMP inhibition was strongly altered in Arg49Ala and Arg49Gln enzymes; the mutants had > 1000-fold lower AMP affinity relative to the wild-type enzyme and exhibited no AMP cooperativity. These studies strongly indicate that the C1-C2 interface is critical for propagation of the cooperative signal between the AMP sites on the different subunits and also in the mechanism of allosteric inhibition of the enzyme by AMP.  相似文献   

9.
Carbachol antagonizes isoproterenol-stimulable cyclic AMP accumulation in mouse atria by direct activation of cardiac muscarinic receptors. Inhibition by carbachol occurs rapidly and is completely reversed when the drug is removed. Neither nitroprusside nor 8-bromo-cyclic GMP mimics the actions of carbachol and low concentrations of carbachol block cyclic AMP accumulation without increasing the intracellular cyclic GMP content. Carbachol does not block cyclic AMP accumulation by activating phosphodiesterase since it is fully effective in the face of marked phosphodiesterase inhibition, nor does it appear to inhibit the catalytic activity of adenylate cyclase since it does not decrease either basal or cholera toxin-stimulated cyclic AMP accumulation. The interaction between carbachol and isoproterenol is not competitive, since cholinergic inhibition cannot be surmounted by increasing concentrations of isoproterenol. The site of muscarinic action therefore appears to involve the mechanisms coupling the hormone-receptor complex to adenylate cyclase. This site is distinct from that of cholera toxin action since there is no antagonism between the effects of cholera toxin and carbachol on cyclic AMP metabolism in the atrium.  相似文献   

10.
L C Yip  M E Balis 《Biochemistry》1975,14(14):3204-3208
Preassay-incubation of the highly purified human erythrocyte adenine phosphoribosyltransferase (EC 2.4.2.7) (AMP pyrophosphorylase) with one of its substrates, 5-phosphoribosyl 1-pyrophosphate (PRibPP), changes the apparent V max value of the enzyme reaction. The extent of inhibition by preassay-incubation with an inhibitor, fructose 1,6-diphosphate (FDP), or a destabilizer, hypoxanthine (Hx), is found not to be proportional to the amount of the inhibitor present. The maximum inhibition achieved by preassay-incubation was about 40%. The PRibPP, FDP, and Hx induced changes in AMP pyrophosphorylase do not require the presence of divalent ions. The inhibtion of AMP pyrophosphorylase produced by preincubation with Hx was prevented when PRibPP was added to the preassay-incubation system. However, the preassay-incubation effect of FDP was only partially diminished under the same conditions. Contrary to the PRibPP-bound AMP pyrophosphorylase, the adenine-bound enzyme was found to be more heat labile than the unbound enzyme. Similar thermal instability was also observed with FDP- and Hx-bound enzyme. Our experimental results indicate that a conformational change of AMP pyrophosphorylase induced by the binding of metabolites is a slow process as compared to the overall catalytic reaction. This hysteretic characteristic of AMP pyrophosphorylase may be one of the regulatory mechanisms in purine intermediary metabolism.  相似文献   

11.
1. The present study extends the finding of Krebs & Woodford (1965) that muscle fructose diphosphatase is more sensitive to AMP inhibition than liver fructose diphosphatase. 2. Hen breast fructose diphosphatase has a K(i) for AMP of 0.1mum; the plot of percentage inhibition is non-sigmoid and the reciprocal plot of activity against AMP concentration is sometimes linear. 3. Percentage inhibition plots for other muscle fructose diphosphatases are sigmoid curves which exhibit different threshold responses to the AMP concentration. 4. The intracellular content of AMP in all muscles tested exceeds the inhibition concentration range of AMP. 5. The sensitivity of muscle fructose diphosphatase to AMP inhibition is decreased by the presence of Mg(2+) or Mn(2+) ions; in the presence of Mn(2+) the inhibition curve for hen breast fructose diphosphatase becomes sigmoid. 6. From the formation constants for the Mg(2+) and Mn(2+) chelates, the effect of these ions in chelation of AMP can be calculated. Although chelation of AMP can explain the Mg(2+) effect, it cannot explain the marked relief of AMP inhibition by Mn(2+). 7. It is suggested that Mn(2+) has a specific effect on this enzyme which reduces the sensitivity to AMP inhibition.  相似文献   

12.
The ability of various adenosine analogs to inhibit cholera toxin activation of the intestinal epithelial cell adenylate cyclase-cyclic AMP system was investigated. After incubation of cells with cholera toxin for 6 hr, large increases in cellular cyclic AMP content were observed. Addition of 2', 5'-dideoxyadenosine during the last 30 min of this 6-hr incubation resulted in 70% reduction in elevated cyclic AMP content. Other analogs were not effective inhibitors. 2', 5'-Dideoxyadenosine was also a potent inhibitor of cholera toxin-activated intestinal cell adenylate cyclase activity with half-maximal inhibition occuring at 16 muM. NaF-stimulated cyclase was less susceptible to inhibition. The data suggest that inhibition by 2', 5'-dideoxyadenosine is due at least in part to direct inhibition of the cholera toxin-activated intestinal adenylate cyclase activity.  相似文献   

13.
F Marcus 《Biochemistry》1976,15(16):3505-3509
Modification of pig kidney fructose-1,6-bisphosphatase with 2,3-butanedione in borate buffer (pH 7.8) leads to the loss of the activation of the enzyme by monovalent cations, as well as to the loss of allosteric adenosine 5'-monophosphate (AMP) inhibition. In agreement with the results obtained for the butanedione modification of arginyl residues in other enzymes, the effects of modification can be reversed upon removal of excess butanedione and borate. Significant protection to the loss of K+ activation was afforded by the presence of the substrate fructose 1,6-bisphosphate, whereas AMP preferentially protected against the loss of AMP inhibition. The combination of both fructose 1,6-bisphosphate and AMP fully protected against the changes in enzyme properties on butanedione treatment. Under the latter conditions, one arginyl residue per mole of enzyme subunit was modified, whereas three arginyl residues were modified by butanedione under conditions leading to the loss of both potassium activation and AMP inhibition. Thus, the modification of two arginyl residues per subunit would appear to be responsible for the change in enzyme properties. The present results, as well as those of a previous report on the subject (Marcus, F. (1975), Biochemistry 14, 3916-3921) support the conclusion that one arginyl residue per subunit is essential for monovalent cation activation, and another arginyl residue is essential for AMP inhibition. A likely role of the latter residue could be its involvement in the binding of the phosphate group of AMP.  相似文献   

14.
The effects of ionic environment on both the intrinsic rate of differentiation and the response to exogenous cyclic AMP in Dictyostelium discoideum have been examined. K+ specifically inhibits the rate of early development when present at concentrations > 15 mM. Na+ does not inhibit at concentrations up to 25 mM, and can partially overcome K+ inhibition. The maximum rate of development also depends upon the presence of adequate levels of extracellular Ca++. The effect of exogenous cyclic AMP on the rate of development is inhibited by the absence of Ca++, and/or the presence of high concentrations of K+. Under optimal ionic conditions, the only effect of exogenous cyclic AMP on early developments of K+. Under optimal ionic conditions, the only effect of exogenous cyclic AMP on early development is a specific inhibition. The implications of these results for current models of early developmental regulation are discussed.  相似文献   

15.
J Puurunen  H Karppanen 《Life sciences》1975,16(10):1513-1520
The effect of ethanol on the secretion of gastric acid and the content of cyclic AMP of the gastric mucosa was studied in rats. Intravenously, ethanol (10 to 800 mg/kg) had no effect on the output of acid. Upon local application into the stomach, ethanol (1 to 10%) caused a concentration-dependent inhibition of the output of gastric acid. The effect was evident within 5 min. At the concentration of 1 %,ethanol decreased the rate of acid secretion maximally by about 30%. At the concentration of 3 %, the maximal inhibition was about 70 %. At the concentration of 10 %, ethanol caused a total cessation of the output of acid within 20 to 60 min.Five and 25 min after the administration of 10 % ethanol into the stomach, the gastric mucosal content of cyclic AMP was decreased by approximately 50 %. Also in vitro, the mucosal content of cyclic AMP was decreased by ethanol within 5 min. The decrease was about 30 % with 2.5 % ethanol, approximately 60 % with 10 % ethanol, and approximately 45 % with 20 % ethanol. Alcohol inhibited the activity of the cyclic AMP phosphodiesterase of the gastric mucosa in a competitive manner. The Ki-value was 0.16 M which would correspond to an alcohol concentration of 9.1 % (v/v). Ethanol caused a concentration-dependent inhibition of the activity of the gastric mucosal adenyl cyclase. By 0.166 M (9.4 %) alcohol the inhibition was nearly 100 %.It is concluded that the ethanol-induced decrease of cyclic AMP in the gastric mucosa is due to a decreased formation of the nucleotide. The accompanying inhibition of the output of acid by ethanol is consistent with the view that cyclic AMP is an intracellular regulator of the gastric acid secretion. In view of the role of cyclic AMP in the control of the integrity of the cells, it is suggested that the ethanol-induced damage of gastric mucosa might also be, at least partly, due to the decreased mucosal content of cyclic AMP.  相似文献   

16.
Platelet cyclic AMP phosphodiesterase (PDE) has been partially purified, its properties studied and inhibition by certain vasodilators observed. Quazodine, dipyridamole, papaverine, Paveril® and other agents inhibit platelet adenosine uptake, potentiate both the inhibition of aggregation and PGE1 stimulated cyclic AMP synthesis and inhibit PDE. The mechanism of action of vasodilators as inhibitors of aggregation is discussed.  相似文献   

17.
Addition of the cyclic AMP phosphodiesterase inhibitors theophylline (10- minus 2 M) or papaverine (10- minus 4 M) leads to a complete inhibition of lactose synthesis in incubated guinea pig mammary gland slices. Addition of 10- minus 5 M cyclic AMP or dibutyryl cyclic AMP results in 1 30-40% inhibition of the synthesis, which effect is not increased by applying higher concentrations of these compounds. A 30-40% inhibition can also be obtained with epinephrine (5 - 10- minus 5 M), or isoproterenol (10- minus 4 M), but the polypeptide hormones glucagon (10- minus 7 M), insulin (1 munit/ml) and relaxin (10 mug/ml) do not significantly affect lactose synthesis. Cytochalasin B (5 mug/ml) inhibits lactose production by 58and colchicine (10- minus 5 M) by 25%. These experiments suggest that an increase in the intracellular level of cyclic AMP either through its addition, through hormonal stimulation of its synthesis, or through inhibition of its intracellular breakdown, leads to an inhibition of lactose production in lactating mammary gland. This effect of cyclic AMP is similar to that of progesterone, which is known to inhibit lactation in vivo and the withdrawal of which at parturition has been postulated to initiate lactogenesis.  相似文献   

18.
Lysine 274 is conserved in all known fructose-1,6-bisphosphatase sequences. It has been implicated in substrate binding and/or catalysis on the basis of reactivity with pyridoxal phosphate as well as by x-ray crystallographic analysis. Lys274 of rat liver fructose-1,6-bisphosphatase was mutated to alanine by the polymerase chain reaction, and the T7-RNA polymerase-transcribed construct containing the mutant sequence was expressed in Escherichia coli. The mutant and wild-type forms of the enzyme were purified to homogeneity, and their specific activity, substrate dependence, and inhibition by fructose 2,6-bisphosphate and AMP were compared. While the mutant exhibited no change in maximal velocity, its Km for fructose 1,6-bisphosphate was 20-fold higher than that of the wild-type, and its Ki for fructose 2,6-bisphosphate was increased 1000-fold. Consistent with the unaltered maximal velocity, there were no apparent difference between the secondary structure of the wild-type and mutant enzyme forms, as measured by circular dichroism and ultraviolet difference spectroscopy. The Ki for the allosteric inhibitor AMP was only slightly increased, indicating that Lys274 is not directly involved in AMP inhibition. Fructose 2,6-bisphosphate potentiated AMP inhibition of both forms, but 500-fold higher concentrations of fructose 2,6-bisphosphate were needed to reduce the Ki for AMP for the mutant compared to the wild-type. However, potentiation of AMP inhibition of the Lys274----Ala mutant was evident at fructose 2,6-bisphosphate concentrations (approximately 100 microM) well below those that inhibited the enzyme, which suggests that fructose 2,6-bisphosphate interacts either with the AMP site directly or with other residues involved in the active site-AMP synergy. The results also demonstrate that although Lys274 is an important binding site determinant for sugar bisphosphates, it plays a more significant role in binding fructose 2,6-bisphosphate than fructose 1,6-bisphosphate, probably because it binds the 2-phospho group of the former while other residues bind the 1-phospho group of the substrate. It is concluded that the enzyme utilizes Lys274 to discriminate between its substrate and fructose 2,6-bisphosphate.  相似文献   

19.
Cardiac sarcoplasmic reticulum-glycogenolytic complex, isolated as a single peak on sucrose density gradient, may function as a "compartmented" effector site for cyclic AMP resulting in modulation of both glycogenolysis and calcium transport. The conversion of phosphorylase b to a is stimulated by ATP and inhibited by protein kinase inhibitor. Cyclic AMP alone stimulated neither phosphorylase b to a conversion nor calcium uptake. An inhibitor of adenylate cyclase depressed both calcium uptake and phosphorylase activation and both functions were subsequently stimulated by micromolar concentrations of cyclic AMP. Endogenous phosphorylation of sarcoplasmic reticulum was also inhibited by adenylate cyclase inhibitor and the inhibition was reversed by cyclic AMP. These results suggest that the sarcoplasmic reticulum of cardiac muscle is an internal effector site for cyclic AMP which may regulate both calcium and metabolism. It appears that cyclic AMP formation in vitro is not the rate-controlling step in the activation sequence.  相似文献   

20.
Agents known to affect intracellular levels of cyclic AMP in many diverse systems have been tested for their effect on the chemotaxis induced by Escherichia coli culture filtrates, spontaneous motility and cyclic AMP levels of rabbit peritoneal neutrophils. Prostaglandin E1 and A1 but not prostaglandin F2alpha increased neutrophil cyclic AMP levels and, correspondingly, only the former two prostaglandins inhibited chemotaxis. Nevertheless, a quantitative relationship between prostaglandin stimulation of cyclic AMP and inhibition of chemotaxis could not be found. Epinephrine, isoproterenol, and, to a much lesser extent, norepinephrine increased neutrophil cyclic AMP through beta adrenergic stimulation. Only epinephrine and isoproterenol inhibited chemotaxis, but the inhibition was variable and not related to the ability of these catecholamines to increase intracellular cyclic AMP. Cholera toxin increased neutrophil cyclic AMP after a 30-min lag period which paralled its inhibitory effect on chemotaxis and spontaneous motility. However, the effect on chemotaxis require 50 ng/ml of toxin whereas the effect on cyclic AMP was manifested at 2 ng/ml of toxin. Prior to 30-min preincubation there was no effect of even 1250 ng/ml of toxin on either cyclic AMP or chemotaxis. Choleragenoid prevented the effects of toxin on both cyclic AMP and chemotaxis. The bacterial chemotactic factor obtained from E. coli culture filtrates did not effect a measurable change in levels of neutrophil cyclic AMP. The data indicate that even though cyclic AMP is not, in the main sequence of events, triggering the chemotactic response, increases in neutrophil cyclic AMP may modulate the movement and thus the chemotactic responsiveness of the neutrophil.  相似文献   

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