Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:4.6.1.2 (guanylate cyclase)
 8,497 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A 37,000 X g supernatant fraction prepared from fat lung homogenate demonstrated a 2- to 3-fold increase in guanylate cyclase activity after incubation at 30 degrees for 30 min (preincubation). Treatment of the supernatant fraction with Triton X-100 increased activity to approximately the same extent as preincubation, but would not increase the activity after preincubation. By chromatography on Sepharose 2B, before and after preincubation, it was demonstrated that the increase in activity was only associated with the soluble guanylate cyclase, and not the particulate enzyme. Activation by preincubation required O2. It was completely inhibited by thiols such as 2-mercaptoethanol, and by bovine serum albumin, KCN, and sodium diethyldithiocarbamate. These inhibitors suggested a copper requirement for activation, and this was confirmed by demonstrating that 20 to 60 muM CuCl2 could relieve the inhibition by 0.1 mM sodium diethyldithiocarbamate. 2-Mercaptoethanol inhibition could also be reversed by removal of the thiol on a Sephadex G-25 column, however, this treatment partially activated the enzyme. Addition of 2-mercaptoethanol to a preincubated preparation would not reverse the activation. H2O2 was found to activate guanylate cyclase, either by its generation in the lung supernatant with glucose oxidase and glucose, or by its addition to a preparation in which the catalase was inhibited with KCN. KCN or bovine serum albumin was able to partially inhibit activation by glucose oxidase plus glucose, however, larger amounts of glucose oxidase could overcome that inhibition, indicating a catalytic role for Cu2+ at low H2O2 concentrations. No direct evidence for H2O2 formation during preincubation could be found, however, indirect evidence was obtained by the spectrophotometric detection of choleglobin formation from hemoglobin present in the lung supernatant fluid. The H2O2 is believed to result from the reaction of oxyhemoglobin with ascorbate.
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PMID:Activation of soluble guanylate cyclase from rat lung by incubation or by hydrogen peroxide. 1 60

Native soluble and particulate guanylate cyclase from several rat tissues preferred Mn2+ to Mg2+ as the sole cation cofactor. Wtih 4mM cation, activities with Mg2+ were less than 25% of the activities with Mn2+. The 1 mM NaN3 markedly increased the activity of soluble and particulate preparations from rat liver. Wtih NaN3 activation guanylate cyclase activities wite similar with Mn2+ and Mg2+. Co2+ was partially effective as a cofactor in the presence of NaN3, while Ca2+ was a poor cation with or without NaN3. Activities with Ba, Cu2+, or Zn2+ were not detectable without or with 1 mM NaN3. With soluble liver enzyme both manganese and magnesium activities were dependent upon excess Mn2+ or Mg2+ at a fixed MnGTP or MgGTP concentration of 0.4 mm; apparent Km values for excess Mn2+ and Mg2+ were 0.3 and 0.24 mM, respectively. After NaN3 activation, the activity was less dependent upon free Mn2+ and retained its dependence for free Mg2+, at 0.4 mM MgGTP the apparent Km for excess Mg2+ was 0.3 mM. The activity of soluble liver guanylate cyclase assayed with Mn2+ or Mg2+ was increased with Ca2+. After NaN3 activiation, Ca2+ had no effect or was somewhat inhibitory with either Mn2+. After NaN activation, Ca2+ had no effect or was somewhat inhibitory with either Mn2+ or Mg2+. The stimulatory effect of NaN2 on Mn2+-and Mg2+-dependent guanylate cyclase activity from liver or cerebral cortex supernatant fractions required the presence of the sodium azide-activator factor. With partially purified soluble liver guanylate cyclase and azide-activator factor, the concentration (1 mjM) of NaN3 that gave half-maximal activation with Mn2+ or Mg2+ was imilar. Thus, under some conditions guanylate cyclase can effectively use Mg2+ as a sole cation cofactor.
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PMID:Appearance of magnesium guanylate cyclase activity in rat liver with sodium azide activation. 1 77

The effects of copper deficiency on smooth muscle relaxation were studied in the cremaster muscle microcirculation. Male Sprague-Dawley rats were fed either a copper-adequate diet (CuA, 5 micrograms copper/g diet) or copper-deficient diet (CuD, no added copper) for 17-27 d before experimentation. In vivo television microscopy was used to quantify agonist-induced diameter changes in third-order arterioles. Endothelium-dependent relaxation, which is hypothesized to be mediated by nitric oxide, was attenuated by copper deficiency. Both receptor (acetylcholine, 10(-7) to 10(-4) mol/L) and nonreceptor (calcium ionophore A23187, 10(-8) to 10(-7) mol/L) relaxation was decreased. Nitric oxide-mediated dilation, which was endothelium-independent (10(-7) to 10(-5) mol/L sodium nitroprusside), was also attenuated by copper deficiency. Maximal responses were as follows: for acetylcholine, 136 +/- 16% CuA vs. 45 +/- 15% CuD; for A23187, 104 +/- 16% CuA vs. 21 +/- 11% CuD; and for nitroprusside, 125 +/- 12% CuA vs. 46 +/- 13% CuD. There was no difference in microvascular dilation between groups treated with 10(-6) to 10(-4) mol/L of the phosphodiesterase inhibitor papaverine (e.g., CuA 109 +/- 11% vs. CuD 133 +/- 21% with 10(-4) mol/L). These results suggest that copper deficiency inhibits the nitric oxide-mediated mechanism of vascular smooth muscle relaxation without altering the capacity of the smooth muscle to relax. We suggest that copper deficiency either decreases nitric oxide radical availability or disrupts the nitric oxide-guanylate cyclase interaction.
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PMID:Copper deficiency alters vasodilation in the rat cremaster muscle microcirculation. 161 79

The influence of native (N-) and oxidized (Ox-) low density lipoproteins (LDLs) on endothelium-dependent vasomotion is still controversial. We investigated the short-term effects of N-LDL and Ox-LDL on the formation of endothelium-derived relaxing factor (EDRF) in native and cultured endothelial cells and on its inactivation after release from the cells. N-LDL was isolated from fresh human plasma via sequential ultracentrifugation and oxidized by incubation with Cu2+. EDRF released from cultured endothelial cells was inactivated by both N-LDL and Ox-LDL (1 mg/ml) as detected in a bioassay system. N-LDL reduced the EDRF-mediated vasodilations of the detector segments by 38.5 +/- 5.3%, and Ox-LDL, by 55.5 +/- 4.6%. The effects of lipoproteins on EDRF formation were studied in cultured endothelial cells preincubated with either N-LDL or Ox-LDL (1 mg/ml for 1 hour) and stimulated for EDRF release with bradykinin after washout of the lipoproteins. EDRF was assessed by measuring its stimulatory effect on the activity of a purified, soluble guanylate cyclase. Both N-LDL and Ox-LDL did not reduce the bradykinin-induced EDRF formation. Consistent with this finding, acetylcholine-induced, EDRF-mediated dilations of intact rabbit femoral artery segments were not impaired by luminal exposure to N-LDL or Ox-LDL (1 mg/ml for 1 hour). However, these relaxations were significantly reduced by preincubation of aortic ring preparations with the same concentrations of the same charges of N-LDL and Ox-LDL. In conclusion, neither N-LDL nor Ox-LDL acutely impairs the formation of EDRF but does inactivate EDRF after its release from endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of native and oxidized low density lipoproteins on formation and inactivation of endothelium-derived relaxing factor. 198 98

We used an apparatus in which pieces of dark-adapted amphibian retinas (Rana pipiens, Bufo marinus) obtained under infrared illumination were exposed to precise intervals of 500-nm illuminations, and then frozen by contact of their outer segment surface with a liquid helium-cooled copper mirror. Sections of the frozen outer segment layer were obtained in a cryostat and then assayed for total extractable cyclic 3',5'-guanosine monophosphate (cGMP). Significant losses of cGMP with respect to the dark level were evident as early as 60 ms after light onset. With dim subsecond illuminations these losses were surprisingly large, which suggests a previously underestimated magnification in the cGMP cascade, or a transient substantial inhibition of guanylate cyclase activity in combination with increased cyclic GMP phosphodiesterase activity. Within the subsecond period, significant losses that were proportional to light intensity (2-log-unit range) and duration (60-550 ms) were generally not evident. However, losses significantly proportional to these factors became evident with durations of 1 s or longer. When pieces of retina were first illuminated (10 or 60 ms), then held in darkness for increasing periods before freezing, we observed a continuous loss of cGMP during the early postillumination dark period, followed by a recovery of the total cGMP level. The times for recovery to the preillumination level appear to be significantly longer than times reported for the recovery of the photoreceptor membrane potential after similar light exposures.
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PMID:Light-induced losses and dark recovery rates of guanosine 3',5'-cyclic monophosphate in rod outer segments of intact amphibian photoreceptors. 285 Oct 28

The mechanism of nitroglycerin-induced vasodilation was examined in isolated arteries. Nitroglycerin relaxed the large coronary artery preferentially whereas nitroprusside and the so-called non-specific vasodilators showed the same activities on both the large and small coronary arteries. Nitroglycerin-induced vasodilation but not the vasodilation induced by the other agents was antagonized markedly by pretreatment with CuSO4. Other metal ions except Fe2+ had no antagonizing effect. The Cu2+-induced antagonism was restored by treatment with sulfhydryl reagents. Nitroglycerin formed inorganic nitrite by non-enzymatic reaction with the tissue sulfhydryl groups; the reaction was also inhibited by Cu2+ . Cu2+ suppressed the membrane stabilizing effect of Ca2+. It seems that nitroglycerin reacts with the sulfhydryl groups on the inner surface of the cell membrane, which may take part in the selectivity of this drug, and subsequently the intermediate(s) formed may activate guanylate cyclase.
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PMID:Inhibitory effect of Cu2+ on nitroglycerin-induced vasodilation. 632 72

1. The effect of copper on the activity of the S-nitrosothiol compounds S-nitrosocysteine (cysNO) and S-nitrosoglutathione (GSNO) was investigated, using the specific copper chelator bathocuproine sulphonate (BCS), and human washed platelets as target cells. 2. Chelation of trace copper with BCS (10 microM) in washed platelet suspensions reduced the inhibition of thrombin-induced platelet aggregation by GSNO; however, BCS had no significant effect on the anti-aggregatory action of cysNO. BCS inhibited cyclic GMP generation in response to both cysNO and GSNO. 3. The effect of BCS was rapid (within 30 s), and could be abolished by increasing the platelet concentration to 500 x 10(9) l-1. 4. In BCS-treated platelet suspensions, the addition of Cu2+ ions (0.37-2.37 microM) led to a restoration of both guanylate cyclase activation and platelet aggregation inhibition by GSNO. 5. The anti-aggregatory activity of GSNO was reduced in a concentration-dependent manner by the copper (I)-specific chelators BCS and neocuproine, and to a smaller extent by desferal. No effect was observed with the copper (II) specific chelator, cuprizone, the iron-specific chelator, bathophenanthroline sulphonate, or the broader-specificity copper chelator, D-penicillamine. 6. In both BCS-treated and -untreated platelet suspensions, cys NO was more potent than GSNO as a stimulator of guanylate cyclase. In BCS-treated platelet suspensions there was no significant difference between the anti-aggregatory potency of cysNO and GSNO; however, in untreated suspensions, GSNO was significantly more potent than cysNO. Thus, when copper was available, GSNO produced a greater inhibition of aggregation than cysNO, despite being a less potent activator of guanylate cyclase. 7. The breakdown of cysNO and GSNO was measured spectrophotometrically by decrease in absorbance at 334 nm. In Tyrode buffer, cysNO (10 microM) broke down at a rate of 3.3 microM min-1. BCS (10 microM)reduced this to 0.5 microM min-1. GSNO, however, was stable, showing no fall in absorbance over a period of 7 min even in the absence of BCS.8. We conclude that copper is required for the activity of both cysNO and GSNO, although its influence on anti-aggregatory activity is only evident with GSNO. The stimulatory effect of copper is unlikely to be explained solely by catalysis of S-nitrosothiol breakdown. The enhancement by copper of the anti-aggregatory activity of GSNO, relative to cysNO, suggests that copper may be required for biological activity of GSNO which is independent of guanylate cyclase stimulation.
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PMID:Copper chelation-induced reduction of the biological activity of S-nitrosothiols. 778 Jun 43

The aim of the present study was to investigate the effect of Cu(II) ions on soluble guanylyl cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2; sGC] and to test for a possible physiological role of this putative cofactor of the enzyme [Gerzer et al., FEBS Lett. 132: 71-74, 1981]. CuSO4 was found to inhibit NO-stimulated 5GC with an IC50 of 2.2 +/- 0.3 microM. Virtually complete inhibition of guanosine-3',5'-cyclic monophosphate (cGMP) formation was observed at 10 microM of the copper salt. Presence of CuSO4 (2 microM) did not significantly affect the potency of 2,2-diethyl-1-nitroso-oxyhydrazine (DEA/NO) but did markedly decrease maximal cyclase activity from 3.71 +/- 0.2 mumol cGMP x mg-1 x min-1 to 1.75 +/- 0.2 mumol cGMP x mg-1 x min-1. The nonstimulated enzyme was also sensitive to CuSO4 (IC50 of 6.2 +/- 1.2 microM). Addition of glutathione, which potently complexes Cu(I) ions, induced a pronounced rightward shift of the concentration-response curves for inhibition by CuSO4 of both DEA/NO-stimulated and nonstimulated guanylyl cyclase. The inhibitory effect of CuSO4 was completely antagonized by the specific Cu(I) chelator neocuproine, with a half-maximal effect at 5.9 +/- 0.2 microM. In contrast, the Cu(II) chelator cuprizone and several thiols, which do not form stable Cu(I) complexes, were far less protective. Our results suggest that inhibition of soluble guanylyl cyclase by CuSO4 is unrelated to heme-mediated enzyme stimulation and may arise from the reversible high affinity binding of Cu(I) ions to a site of the protein that is critically involved in enzyme catalysis.
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PMID:Inhibition of purified soluble guanylyl cyclase by copper ions. 883 23

1. The potential role of copper (Cu2+) in modulating the activity of nitric oxide synthase (NOS) and guanylyl cyclase (GC) was investigated by use of diethyldithiocarbamic acid (DEDCA), a high affinity Cu2+ chelator. 2. DEDCA 100 microM inhibited sodium nitroprusside (SNP; 0.005-10 microM)-evoked relaxation of rat isolated aortic rings precontracted with 3 microM phenylephrine (PE). A lower concentration of DEDCA (10 microM) did not significantly attenuate SNP-evoked responses but did inhibit relaxation to the endothelium-dependent dilator, A23187 (0.01-10 microM). 3. The presence of 100 microM Cu2+, but not 100 microM Fe2+, alone enhanced A23187- and SNP-evoked relaxation of aortae precontracted with PE. 4. The inhibitory effect of DEDCA on SNP- and A23187-induced relaxation was reversed by equimolar concentrations of Cu2+ but not Fe2+, indicating that DEDCA does not act via removal of haem-iron from the NOS and GC complexes. 5. Superoxide dismutase (30 mu ml-1) was without effect on the inhibition of DEDCA relaxation induced by either SNP or A23187 in aortae precontracted with PE. 6. When assessed by radioimmunoassay, DEDCA inhibited SNP- and A23187-stimulated cyclic GMP formation with IC50 values of 0.5 microM and 50 microM, respectively. 7. These data demonstrate that Cu2+ plays a role in controlling NOS and GC activity in the rat aorta.
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PMID:Effect of copper on nitric oxide synthase and guanylyl cyclase activity in the rat isolated aorta. 915 47

1. We have measured the ability of a range of NO donor compounds to stimulate cyclic GMP accumulation and inhibit collagen-induced aggregation of human washed platelets. In addition, the rate of spontaneous release of NO from each donor has been measured spectrophotometrically by the oxidation of oxyhaemoglobin to methaemoglobin. The NO donors used were five s-nitrosothiol compounds: S-nitrosoglutathione (GSNO), S-nitrosocysteine (cysNO), S-nitroso-N-acetyl-DL-penicillamine (SNAP), S-nitroso-N-acetyl-cysteine (SNAC), S-nitrosohomocysteine (homocysNO), and two non-nitrosothiol compounds: diethylamine NONOate (DEANO) and sodium nitroprusside (SNP). 2. Using 10 microM of each donor compound, mean+/-s.e.mean rate of NO release ranged from 0.04+/-0.001 nmol min(-1) (for SNP) to 3.15+/-0.29 nmol min(-1) (for cysNO); cyclic GMP accumulation ranged from 0.43+/-0.05 pmol per 10(8) platelets (for SNP) to 2.67+/-0.31 pmol per 10(8) platelets (for cysNO), and inhibition of platelet aggregation ranged from 40+/-6.4% (for SNP) to 90+/-3.8% (for SNAC). 3. There was a significant positive correlation between the rate of NO release and the ability of the different NO donors to stimulate intra-platelet cyclic GMP accumulation (r = 0.83; P = 0.02). However, no significant correlation was observed between the rate of NO release and the inhibition of platelet aggregation by the different NO donors (r= -0.17), nor was there a significant correlation between cyclic GMP accumulation and inhibition of aggregation by the different NO donor compounds (r = 0.34). 4. Comparison of the dose-response curves obtained with GSNO, DEANO and 8-bromo cyclic GMP showed DEANO to be the most potent stimulator of intraplatelet cyclic GMP accumulation (P < 0.001 vs both GSNO and 8-bromo cyclic GMP), but GSNO to be the most potent inhibitor of platelet aggregation (P < 0.01 vs DEANO, and P < 0.001 vs 8-bromo cyclic GMP). 5. The rate of NO release from GSNO, and its ability both to stimulate intra-platelet cyclic GMP accumulation and to inhibit platelet aggregation, were all significantly diminished by the copper (I) (Cu+) chelating agent bathocuproine disulphonic acid (BCS). In contrast, BCS had no effect on either the rate of NO release, or the anti-platelet action of the non-nitrosothiol compound DEANO. 6. Cyclic GMP accumulation in response to GSNO (10(-9) 10(-5) M) was undetectable following treatment of platelets with ODQ (100 microM), a selective inhibitor of soluble guanylate cyclase. Despite this abolition of guanylate cyclase stimulation, GSNO retained some ability to inhibit aggregation, indicating the presence of a cyclic GMP-independent component in its anti-platelet action. However, this component was abolished following treatment of platelets with a combination of both ODQ and BCS, suggesting that Cu+ ions were required for the cyclic GMP-independent pathway to operate. 7. The cyclic GMP-independent action of GSNO, observed in ODQ-treated platelets, could not be explained by an increase in intra-platelet cyclic AMP. 8. The impermeable thiol modifying agent p-chloromercuriphenylsulphonic acid (CMPS) produced a concentration-dependent inhibition of aggregation of ODQ-treated platelets, accompanied by a progressive loss of detectable platelet surface thiol groups. Additional treatment with GSNO failed to increase the degree of aggregation inhibition, suggesting that a common pathway of thiol modification might be utilized by both GSNO and CMPS to elicit cyclic GMP-independent inhibition of platelet aggregation. 9. We conclude that NO donor compounds mediate inhibition of platelet aggregation by both cyclic GMP-dependent and -independent pathways. Cyclic GMP generation is related to the rate of spontaneous release of NO from the donor compound, but transfer of the NO signal to the cyclic GMP-independent pathway may depend upon a cellular system which involves both copper (I) (Cu+) ions and surface membrane thiol groups. The potent anti-platelet action of GSNO
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PMID:Evidence for a cyclic GMP-independent mechanism in the anti-platelet action of S-nitrosoglutathione. 963 Mar 53


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