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1.
Highly purified rat lung soluble guanylate cyclase was activated with nitric oxide or sodium nitroprusside and the degree of activation varied with incubation conditions. With Mg2+ as the action cofactor, about 2- to 8-fold activation was observed with nitric oxide or sodium nitroprusside alone. Markedly enhanced activation (20-40 fold) was observed when 1 muM hemin added to the enzyme prior to exposure to the activating agent. The activation with hemin and sodium nitroprusside was prevented in a dose-dependent manner by sodium cyanide. The level activation was also increased by the addition of 1 mM dithiothreitol, but unlike hemin which had no effect on basal enzyme activity, dithiothreitol led to a considerable increase in basal activity. Activated guanylate cyclase decayed to basal activity within one hour at 2 degrees C and the enzyme could be reactivated upon re-exposure to nitroprusside or nitric oxide. Under basal conditions, Michaelis-Menten kinetics were observed, with a Km for GTP of 140 muM with Mg2+ cofactor. Following activation with nitroprusside or nitric oxide, curvilinear Eadie-Hofstee transformations of kinetic data were observed, with Km's of 22 MuM and 100 MuM for Mg-GTP. When optimal activation (15-40 fold) was induced by the addition of hemin and nitroprusside, multiple Km's were also seen with Mg-GTP and the high affinity form was predominant (22 MuM). Similar curvilinear Eadie-Hofstee transformations were observed with Mn2+ as the cation cofactor. These data suggest that multiple GTP catalytic sites are present in activated guanylate cyclase, or alternatively, multiple populations of enzyme exist.  相似文献   

2.
Activation of cerebral guanylate cyclase by nitric oxide.   总被引:15,自引:0,他引:15  
Mouse cerebral guanylate cyclase was activated by catalase in the presence of sodium azide (NaN3), which is known to form catalase-NO complex, while nitrosamines and nitric oxide (NO gas) were capable of activating cerebral guanylate cyclase in the absence of catalase. The activation of guanylate cyclase by NaN3-catalase or nitrosamines was markedly inhibited by ferrohemoglobin which has a high affinity for NO, but not by ferrihemoglobin. These data suggest that NO or NO containing compounds may activate guanylate cyclase, whereas ferrohemoglobin may exhibit an inhibitory effect on the activation of guanylate cyclase, possibly by interacting with NO or NO containing compounds.  相似文献   

3.
4.
Analysis of purified soybean and rabbit reticulocyte 15-lipoxygenase (15-LOX) and PA317 cells transfected with human 15-LOX revealed a rapid rate of linoleate-dependent nitric oxide (.NO) uptake that coincided with reversible inhibition of product ((13S)-hydroperoxyoctadecadienoic acid, or (13S)-HPODE) formation. No reaction of .NO (up to 2 microM) with either native (Ered) or ferric LOXs (0.2 microM) metal centers to form nitrosyl complexes occurred at these .NO concentrations. During HPODE-dependent activation of 15-LOX, there was consumption of 2 mol of .NO/mol of 15-LOX. Stopped flow fluorescence spectroscopy showed that.NO (2.2 microM) did not alter the rate or extent of (13S)-HPODE-induced tryptophan fluorescence quenching associated with 15-LOX activation. Additionally, .NO does not inhibit the anaerobic peroxidase activity of 15-LOX, inferring that the inhibitory actions of .NO are due to reaction with the enzyme-bound lipid peroxyl radical, rather than impairment of (13S)-HPODE-dependent enzyme activation. From this, a mechanism of 15-LOX inhibition by .NO is proposed whereby reaction of .NO with EredLOO. generates Ered and LOONO, which hydrolyzes to (13S)-HPODE and nitrite (NO2-). Reactivation of Ered, considerably slower than dioxygenase activity, is then required to complete the catalytic cycle and leads to a net inhibition of rates of (13S)-HPODE formation. This reaction of .NO with 15-LOX inhibited. NO-dependent activation of soluble guanylate cyclase and consequent cGMP production. Since accelerated .NO production, enhanced 15-LOX gene expression, and 15-LOX product formation occurs in diverse inflammatory conditions, these observations indicate that reactions of .NO with lipoxygenase peroxyl radical intermediates will result in modulation of both .NO bioavailability and rates of production of lipid signaling mediators.  相似文献   

5.
Purification of soluble guanylate cyclase from rat liver resulted in an apparent loss of enzyme activation by nitric oxide that could be restored by dithiothreitol. methemoglobin, bovine serum albumin, or sucrose. Although hemoglobin also permitted some activation with nitric oxide, the effect of other agents to restore enzyme activation was prevented with hemoglobin. As a result of enzyme purification, there is an alteration of the dose-response relationship for nitric oxide activation. After partial enzyme purification, relatively high concentrations of nitric oxide that were stimulatory in crude enzyme preparations had no effect on enzyme activity. However, partially purified or homogeneous enzyme was activated by lower concentrations of nitric oxide. The bell-shaped dose-response curve for nitric oxide was shifted to the left with guanylate cyclase purification. The addition of dithiothreitol, methemoglobin, bovine serum albumin, or sucrose to enzyme markedly broadens the dose-response curve for nitric oxide. Thus, the apparent loss of responsiveness to nitric oxide with purification is a function of increased sensitivity of guanylate cyclase to nitric oxide. Increased sensitivity to nitric oxide with enzyme purification probably results from the removal of heme, proteins, and small molecules that can serve as scavengers or sinks for nitric oxide and prevent excessive oxidation of the enzyme.  相似文献   

6.
This study examines in endothelium-denuded bovine pulmonary arteries the effects of increasing heme oxygenase-1 (HO-1) activity on relaxation and soluble guanylate cyclase (sGC) activation by nitric oxide (NO). A 24-h organ culture with 0.1 mM cobalt chloride (CoCl2) or 30 microM Co-protoporphyrin IX was developed as a method of increasing HO-1 expression. These treatments increased HO-1 expression and HO activity by approximately two- to fourfold and lowered heme levels by 40-45%. Induction of HO-1 was associated with an attenuation of pulmonary arterial relaxation to the NO-donor spermine-NONOate. The presence of a HO-1 inhibitor 30 microM chromium mesoporphyrin during the 24-h organ culture (but not acute treatment with this agent) reversed the attenuation of relaxation to NO seen in arteries co-cultured with agents that increased HO-1. Relaxation to isoproterenol, which is thought to be mediated through cAMP, was not altered in arteries with increased HO-1. Inducers of HO-1 did not appear to alter basal sGC activity in arterial homogenates or expression of the beta(1)-subunit of sGC. However, the increase in activity seen in the presence of 1 microM spermine-NONOate was attenuated in homogenates obtained from arteries with increased HO-1. Since arteries with increased HO-1 had decreased levels of superoxide detected by the chemiluminescence of 5 microM lucigenin, superoxide did not appear to be mediating the attenuation of relaxation to NO. These data suggest that increasing HO-1 activity depletes heme, and this is associated with an attenuation of pulmonary artery relaxation and sGC activation responses to NO.  相似文献   

7.
Free nitric oxide (NO) activates soluble guanylate cyclase (sGC), an enzyme, within both pulmonary and vascular smooth muscle. sGC catalyzes the cyclization of guanosine 5'-triphosphate to guanosine 3',5'-cyclic monophosphate (cGMP). Binding rates of NO to the ferrous heme(s) of sGC have been measured in vitro. However, a missing link in our understanding of the control mechanism of sGC by NO is a comprehensive in vivo kinetic analysis. Available literature data suggests that NO dissociation from the heme center of sGC is accelerated by its interaction with one or more cofactors in vivo. We present a working model for sGC activation and NO consumption in vivo. Our model predicts that NO influences the cGMP formation rate over a concentration range of approximately 5-100 nM (apparent Michaelis constant approximately 23 nM), with Hill coefficients between 1.1 and 1.5. The apparent reaction order for NO consumption by sGC is dependent on NO concentration, and varies between 0 and 1.5. Finally, the activation of sGC (half-life approximately 1-2 s) is much more rapid than deactivation (approximately 50 s). We conclude that control of sGC in vivo is most likely ultra-sensitive, and that activation in vivo occurs at lower NO concentrations than previously reported.  相似文献   

8.
Guanylate cyclase activity in the soluble extract of bovine pulmonary arteries is activated by hydrogen peroxide generated by glucose oxidase only in the presence of catalase. This mechanism of guanylate cyclase activation is not blocked by scavengers for superoxide anion or hydroxyl radical, but is selectively inhibited by methylene blue, inactivation of catalase and ethanol. The time dependency of increases in guanylate cyclase activity in the presence of peroxides that are substrates for catalase are associated with the spectral detection of compound I, a species of catalase formed during the metabolism of peroxide. Thus, activation of soluble guanylate cyclase appears to be elicited by compound I of catalase or by a mediator generated by this species.  相似文献   

9.
Soluble guanylate cyclase (GC) from bovine lung is activated 4-fold by carbon monoxide (CO) and 400-fold by nitric oxide (NO). Spectroscopic and kinetic data for ligation of CO and NO with GC are summarized and compared with similar data for myoglobin (Mb), hemoglobin (Hb), and heme model compounds. Kinetic, thermodynamic, and structural data form a basis on which to construct a model for the manner in which the two ligands affect protein structure near the heme for heme proteins in general and for GC in particular. The most significant datum is that although association rates of ligands with GC are similar to those with Mb and Hb, their dissociation rates are dramatically faster. This suggests a delicate balance between five- and six-coordinate heme iron in both NO and CO complexes. Based on these and other data, a model for GC activation is proposed: The first step is formation of a six-coordinate species concomitant with tertiary and quaternary structural changes in protein structure and about a 4-fold increase in enzyme activity. In the second step, applicable to NO, the bond from iron to the proximal histidine ruptures, leading to additional relaxation in the quaternary and tertiary structure and a further 100-fold increase in activity. This is the main event in activation, available to NO and possibly other activators or combinations of activators. It is proposed, finally, that the proximal base freed in step 2, or some other protein base suitably positioned as a result of structural changes following ligation, may provide a center for nucleophilic substitution catalyzing the reaction GTP --> cGMP. An example is provided for a similar reaction in a derivatized protoheme model compound. The reaction mechanism attempts to rationalize the relative enzymatic activities of GC, heme-deficient GC, GC-CO, and GC-NO on a common basis and makes predictions for new activators that may be discovered in the future.  相似文献   

10.
Sodium nitroprusside, nitroglycerin, sodium azide and hydroxylamine increased guanylate cyclase activity in particulate and/or soluble preparations from various tissues. While sodium nitroprusside increased guanylate cyclase activity in most of the preparations examined, the effects of sodium azide, hydroxylamine and nitroglycerin were tissue specific. Nitroglycerin and hydroxylamine were also less potent. Neither the protein activator factor nor catalase which is required for sodium azide effects altered the stimulatory effect of sodium nitroprusside. In the presence of sodium azide, sodium nitroprusside or hydroxylamine, magnesium ion was as effective as manganese ion as a sole cation cofactor for guanylate cyclase. With soluble guanylate cyclase from rat liver and bovine tracheal smooth muscle the concentrations of sodium nitroprusside that gave half-maximal stimulation with Mn2+ were 0.1 mM and 0.01 mM, respectively. Effective concentrations were slightly less with Mg2+ as a sole cation cofactor. The ability of these agents to increase cyclic GMP levels in intact tissues is probably due to their effects on guanylate cyclase activity. While the precise mechanism of guanylate cyclase activation by these agents is not known, activation may be due to the formation of nitric oxide or another reactive material since nitric oxide also increased guanylate cyclase activity.  相似文献   

11.
Soluble guanylate cyclase (sGC) is the target of nitric oxide (NO) released by nitric-oxide synthase in endothelial cells, inducing an increase of cGMP synthesis in response. This heterodimeric protein possesses a regulatory subunit carrying a heme where NO binding occurs, while the second subunit harbors the catalytic site. The binding of NO and the subsequent breaking of the bond between the proximal histidine and the heme-Fe(2+) are assumed to induce conformational changes, which are the origin of the catalytic activation. At the molecular level, the activation and deactivation mechanisms are unknown, as is the dynamics of NO once in the heme pocket. Using ultrafast time-resolved absorption spectroscopy, we measured the kinetics of NO rebinding to sGC after photodissociation. The main spectral transient in the Soret band does not match the equilibrium difference spectrum of NO-liganded minus unliganded sGC, and the geminate rebinding was found to be monoexponential and ultrafast (tau = 7.5 ps), with a relative amplitude close to unity (0.97). These characteristics, so far not observed in other hemoproteins, indicate that NO encounters a high energy barrier for escaping from the heme pocket once the His-Fe(2+) bond has been cleaved; this bond does not reform before NO recombination. The deactivation of isolated sGC cannot occur by only simple diffusion of NO from the heme; therefore, several allosteric states may be inferred, including a desensitized one, to induce NO release. Thus, besides the structural change leading to activation, a consequence of the decoupling of the proximal histidine may also be to induce a change of the heme pocket distal geometry, which raises the energy barrier for NO escape, optimizing the efficiency of NO trapping. The non-single exponential character of the NO picosecond rebinding coexists only with the presence of the protein structure surrounding the heme, and the single exponential rate observed in sGC is very likely to be due to a closed conformation of the heme pocket. Our results emphasize the physiological importance of NO geminate recombination in hemoproteins like nitric-oxide synthase and sGC and show that the protein structure controls NO dynamics in a manner adapted to their function. This control of ligand dynamics provides a regulation at molecular level in the function of these enzymes.  相似文献   

12.
Particulate guanylate cyclase from rat lung was stimulated less than 2-fold by agents capable of activating the soluble guanylate cyclase, including sodium nitroprusside, MNNG, azide and hydroxylamine. The action of the first two agents was potentiated by 10 mM 2-mercaptoethanol, and that of the last two by catalase. Pretreatment of the particulate enzyme with the polyene antibiotic, filipin, potentiated the stimulatory effects of the activators, activity with 1 mM nitroprusside in the presence of 2-mercaptoethanol being increased 10.4-fold over basal. The enzyme treated with filipin and nitroprusside showed less specificity for Mn2+, as it was able to use Mg2+ as sole cation more efficiently than the untreated enzyme. Since filipin is known to alter membrane fluidity by interacting with membrane cholesterol, it is proposed that the activity of membrane bound guanylate cylase may be regulated in part by the fluid state of the phospholipid matrix.  相似文献   

13.
Bovine lung soluble guanylate cyclase was purified to apparent homogeneity in a form that was deficient in heme. Heme-deficient guanylate cyclase was rapidly and easily reconstituted with heme by reacting enzyme with hematin in the presence of excess dithiothreitol, followed by removal of unbound heme by gel filtration. Bound heme was verified spectrally and NO shifted the absorbance maximum in a manner characteristic of other hemoproteins. Heme-deficient and heme-reconstituted guanylate cyclase were compared with enzyme that had completely retained heme during purification. NO and S-nitroso-N-acetylpenicillamine only marginally activated heme-deficient guanylate cyclase but markedly activated both heme-reconstituted and heme-containing forms of the enzyme. Restoration of marked activation of heme-deficient guanylate cyclase was accomplished by including 1 microM hematin in enzyme reaction mixtures containing dithiothreitol. Preformed NO-heme activated all forms of guanylate cyclase in the absence of additional heme. Guanylate cyclase activation was observed in the presence of either MgGTP or MnGTP, although the magnitude of enzyme activation was consistently greater with MgGTP. The apparent Km for GTP in the presence of excess Mn2+ or Mg2+ was 10 microM and 85-120 microM, respectively, for unactivated guanylate cyclase. The apparent Km for GTP in the presence of Mn2+ was not altered but the Km in the presence of Mg2+ was lowered to 58 microM with activated enzyme. Maximal velocities were increased by enzyme activators in the presence of either Mg2+ or Mn2+. The data reported in this study indicate that purified guanylate cyclase binds heme and the latter is required for enzyme activation by NO and nitroso compounds.  相似文献   

14.
Nitric oxide (NO) is a physiologically relevant activator of the hemoprotein soluble guanylate cyclase (sGC). In the presence of NO, sGC is activated several hundredfold above the basal level by a mechanism that remains to be elucidated. The heme ligand n-butyl isocyanide (BIC) was used to probe the mechanism of NO activation of sGC. Electronic absorption spectroscopy was used to show that BIC binds to the sGC heme, forming a 6-coordinate complex with an absorbance maximum at 429 nm. BIC activates sGC 2-5-fold, and synergizes with the allosteric activator YC-1, to activate the enzyme 15-25-fold. YC-1 activates the sGC-BIC complex, and leads to an increase in both the V(max) and K(m). BIC was also used to probe the mechanism of NO activation. The activity of the sGC-BIC complex increases 15-fold in the presence of NO, without displacing BIC at the heme, which is consistent with previous reports that proposed the involvement of a non-heme NO binding site in the activation process.  相似文献   

15.
Hyperammonemia is the main responsible for the neurological alterations in hepatic encephalopathy in patients with liver failure. We studied the function of the glutamate-nitric oxide (NO)-cGMP pathway in brain in animal models of hyperammonemia and liver failure and in patients died with liver cirrhosis. Activation of glutamate receptors increases intracellular calcium that binds to calmodulin and activates neuronal nitric oxide synthase, increasing nitric oxide, which activates soluble guanylate cyclase (sGC), increasing cGMP. This glutamate-NO-cGMP pathway modulates cerebral processes such as circadian rhythms, the sleep-waking cycle, and some forms of learning and memory. These processes are impaired in patients with hepatic encephalopathy. Activation of sGC by NO is significantly increased in cerebral cortex and significantly reduced in cerebellum from cirrhotic patients died in hepatic coma. Portacaval anastomosis in rats, an animal model of liver failure, reproduces the effects of liver failure on modulation of sGC by NO both in cerebral cortex and cerebellum. In vivo brain microdialisis studies showed that sGC activation by NO is also reduced in vivo in cerebellum in hyperammonemic rats with or without liver failure. The content of alpha but not beta subunits of sGC are increased both in frontal cortex and cerebellum from patients died due to liver disease and from rats with portacaval anastomosis. We assessed whether determination of activation of sGC by NO-generating agent SNAP in lymphocytes could serve as a peripheral marker for the impairment of sGC activation by NO in brain. Chronic hyperammonemia and liver failure also alter sGC activation by NO in lymphocytes from rats or patients. These findings show that the content and modulation by NO of sGC are strongly altered in brain of patients with liver disease. These alterations could be responsible for some of the neurological alterations in hepatic encephalopathy such as sleep disturbances and cognitive impairment.  相似文献   

16.
D L Vesely  G S Levey 《Enzyme》1978,23(2):140-143
A variety of nitroso chemical carcinogens increase the activity of guanylate cyclase (EC 4.6.1.2), the enzyme catalyzing the production of guanosine 3',5'-monophosphate. In the present report, the first non-nitroso chemical carcinogen, butadiene diepoxide, was shown to activate guanylate in a variety of tissues over the concentration range 1-100 mmol/l. At 20 mmol/l concentration, increases were 2- to 17-fold above control. These observations have potential importance since guanosine 3',5'-monophosphate may be involved in cell growth and malignant transformation.  相似文献   

17.
Two monoclonal antibodies (mAbs) against bovine lung soluble guanylate cyclase (sGC) were prepared and characterized. mAb 3221 recognized both the alpha- and beta-subunits of sGC and had greater binding affinity to the enzyme in the presence of NO. mAb 28131 recognized only the beta-subunit and its affinity did not change with NO. Neither mAb cross-reacted with particulate GC. Cultured Purkinje cells from rats were treated with S-nitroso-N-acetylpenicillamine, an NO donor, and examined by immunocytochemical methods. The immunoreactivity associated with mAb 3221 increased with the cGMP content in a crude extract of cerebellum and the NO2 generated in the culture medium increased.  相似文献   

18.
The influence of adrenochrome and YC-1 activation of human platelet soluble guanylate cyclase was investigated. Adrenochrome (0.1–10.0 μM) had no effect on the basal activity, but it potentiated in a concentration- dependent manner the spermine NONO-induced activation of this enzyme. Adrenochrome also sensitized guanylate towards nitric oxide (NO) and produced the leftward shift of the spermine NONO concentration response curve. Addition of adrenochrome decreased the YC-1-induced leftward shift of the spermine NONO concentration response curve. Adrenochrome also inhibited enzyme activation byYC-1. Thus, synergistic activation of NO-stimulated guanylate cyclase activity by adrenochrome represents a new biochemical effect of this compound and indicates that adrenochrome may act as an endogenous regulator of the NO-dependent stimulation of soluble guanylate cyclase. This new property of adrenochrome, similar to YC-1 but more effective, should be taken into consideration especially under conditions of adrenochrome overproduction in the body.  相似文献   

19.
Effect of carnosine on the activation of soluble guanylate cyclase by sodium nitroprusside and protoporphyrin IX was studied using human platelet 105000 g supernatants and partially purified heme-deficient guanylate cyclase preparations. In experiments with 105000 g supernatants, carnosine (1 mM) inhibited the enzyme activation by nitroprusside by about 70%. With the partially purified heme-deficient guanylate cyclase, the enzyme activation by nitroprusside was lowered by 86%, and the remaining insignificant stimulatory effect remained unchanged upon carnosine addition. The stimulatory effect of protoporphyrin IX on the partially purified heme-deficient enzyme preparation did not differ from that observed with the 105000 g supernatant; carnosine addition had no effect on activation of guanylate cyclase by protoporphyrin IX. It was concluded that the inhibitory effect of carnosine on the ability of the enzyme to be activated by nitroprusside is due to the interaction of carnosine with guanylate cyclase, and that it is heme directed.  相似文献   

20.
The mechanisms through which thiol oxidation and cellular redox influence the regulation of soluble guanylate cyclase (sGC) are poorly understood. This study investigated whether promoting thiol oxidation via inhibition of NADPH generation by the pentose phosphate pathway (PPP) with 1 mM 6-aminonicotinamide (6-AN) or the thiol oxidant diamide (1 mM) alters sGC activity and cGMP-associated relaxation to nitric oxide (NO) donors [S-nitroso-N-acetylpenicillamine (SNAP) and spermine-NONOate]. Diamide and 6-AN inhibited NO-elicited relaxation of endothelium-denuded bovine pulmonary arteries (BPA) and stimulation of sGC activity in BPA homogenates. Treatment of BPA with the thiol reductant DTT (1 mM) reversed inhibition of NO-mediated relaxation and sGC stimulation by 6-AN. The increase in cGMP protein kinase-associated phosphorylation of vasodilator-stimulated phosphoprotein on Ser239 elicited by 10 microM SNAP was also inhibited by diamide. Activation of sGC by SNAP was attenuated by low micromolar concentrations of GSSG in concentrated, but not dilute, homogenates of BPA, suggesting that an enzymatic process contributes to the actions of GSSG. Relaxation to agents that function through cAMP (forskolin and isoproterenol) was not altered by inhibition of the pentose phosphate pathway or diamide. Thus a thiol oxidation mechanism controlled by the regulation of thiol redox by NADPH generated via the pentose phosphate pathway appears to inhibit sGC activation and cGMP-mediated relaxation by NO in a manner consistent with its function as an important physiological redox-mediated regulator of vascular function.  相似文献   

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