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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)

The activity of calf uterus guanylate cyclase (EC 4.6.1.2) exists in at least two and most probably three distinct forms. The cytosolic enzyme exhibits hyperbolic substrate curves with respect to GTP and Mn2+, while the particulate cyclases (nuclear and microsomal)display sigmoidal (GTP) and hyperbolic (Mn2+) relationships. The Hill coefficient for the GTP dependence is 0.9 for the cytosolic, 1.5 for the nuclear, and 1.4 for the microsomal enzyme. The cytosolic enzyme has a Km for GTP of 70 muM while half maximal velocity occurs at 90 and 100 muM GTP for the nuclear and microsomal enzymes, respectively. The Ka for Mn2+ is 0.57, 0.71 or 0.75 mM for the cytosolic, nuclear, or microsomal enzyme, respectively.
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PMID:Guanylate cyclase. Existence of different forms and their regulation by nucleotides in calf uterus. 0 17

1. Guanylate cyclase of washed particles and plasma membranes showed S-shaped progress curves when titrated with either GTP or Mn2+ ions; similar results were obtained with Triton X-100-solubilized enzyme preparation from washed particles. Hill plots of these data revealed multiple metal-nucleotide and free-metal binding sites. 2. Guanylate cyclase of supernatant fractions displayed typical Michaelis-Menten properties when enzyme required excess of (free) Mn2+ (over GTP) for maximal activities; Ka (free Mn2+) was about 0.15-0.25 mM at subsaturating concentrations of GTP. 4 MnATP, MnADP, and MnGDP were found to increase the activities of both particulate and superantant enzyme, when MnGTP concentration was below saturation and free Mn2+ ion concentration was low (less than 100 muM); MnATP (50muM-1 mM) inhibited both these activities at high free Mn2+ concentration (1.5 mM) and inhibition of the particulate enzyme was greater than that of supernatant enzyme. 5. Ca2+ ions stimulated supernatant-enzyme activity; the stimulatory concentration of Ca2+ ions depended on the concentration of Mn2+ and GTP. 6. A modest stimulation of particulate guanylate cyclase by pyrophosphate (0.02-1 mM) was observed; the pyrophosphate effect appeared to be competitive with respect to GTP. At a higher concentration (2 mM), pyrophosphate produced a marked inhibition of particulate enzyme; the nature of inhibitory effect appeared complex. 7. Inorganic salts (e.g. NaCl, KCl, LiBr, NaF) produced inhibition of particulate enzyme; the degree of inhibition of Triton X-100-stimulated activity was less than that of unstimulated activity. 9. Treatment of sarcolemmal or microsomal membranes with either phospholipase C or trypsin decreased, whereas phospholipase A increased, the activity of guanylate cyclase.
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PMID:Properties of particulate, membrane-associated and soluble guanylate cyclase from cardiac muscle, skeletal muscle, cerebral cortex and liver. 1 Aug 91

In a submaximal concentration carbachol contracted the rabbit colon muscle and increased the cyclic GMP level. The cyclic AMP level was reduced. In a Ca++-depleted muscle carbachol reduced the cyclic GMP level while the effect on the cyclic AMP content of the muscle was unchanged. Carbachol had no effect on the guanylate cyclase activity of the "plasma membrane fraction" (the 35-45% fraction). In the homogenate and the microsomal fractions Ca++ had no effect on the guanylate activity while it stimulated the enzyme in a soluble fraction. In the "plasma membrane fraction" cyclic GMP released Ca++ from the preloaded fraction and inhibited the Ca++ accumulation. These effects were not found in the vesicular microsomal fraction (the 35% fraction). In both fractions, however, cyclic GMP counteracted the stimulating effect of cyclic AMP. These results indicate that cyclic AMP and GMP may have antagonistic roles on the Ca++ metabolism in the colon muscle. It is suggested that cyclic GMP may act as some kind of positive feedback mechanism which may have a modulating effect on the release of Ca++ from one pool to another in rabbit colon.
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PMID:Effects of carbachol and calcium on the cyclic guanosine-3',5'-monophosphate (cyclic GMP) metabolism in intestinal smooth muscle. 1 81

The purpose of this study was to elucidate the mechanisms by which arachidonic acid activates guanylate cyclase from guinea pig lung. Guanylate cyclase activities in both homogenate and soluble fractions of lung were examined. Guanylate cyclase activity was determined by measuring formtion of [32-P] cyclic GMP from alpha-[32-P] GTP in the presence of Mn2+, a phosphodiesterase inhibitor and a suitable GTP regenerating system. Arachidonic acid, and to a slight extent dihomo-gamma-linolenic acid, activated guanylate cyclase in homogenate but not soluble fractions. Similarly, phospholipase A2 activated homogenate but not soluble guanylate cyclase. Methyl arachidonate, linolenic, linoleic and oleic acids did not activate guanylate cyclase in either fraction. High concentrations of indomethacin, meclofenamate and aspirin inhibited activation of homogenate guanylate cyclase by arachidonic acid and phospholipase A2, without altering basal enzyme activity. These data suggested that a product of cyclooxygenase activity, present in the microsomal fraction, may have accounted for the capacity of arachidonic acid to activate homogenate guanylate cyclase. This view was supported by the findings that addition of the microsomal fraction to be soluble fraction enabled arachidonic acid to activate soluble guanylate cyclase, an effect which was reduced with cycloooxygenase inhibitors. Lipoxygenase activated guanylate cyclase in homogenate and soluble fractions. Arachidonic acid potentiated the activation of soluble guanylate cyclase by lipoxygenase, and this effect was inhibited with nordihydroguairetic acid, 1-phenyl-3-pyrazolidone and hydroquinone, but not with high concentrations of indomethacin, meclofenamate or aspirin. These data suggest that arachidonic acid activates guinea pig lung guanylate cyclase indirectly, via two independent mechanisms, one involving the microsomal fraction and the other involving lipoxygenase.
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PMID:Arachidonic acid activation of guinea pig lung guanylate cyclase by two independent mechanisms. 4 57

The subcellular localization of guanylate cyclase was examined in rat liver. About 80% of the enzyme activity of homogenates was found in the soluble fraction. Particulate guanylate cyclase was localized in plasma membranes and microsomes. Crude nuclear and microsomal fractions were applied to discontinuous sucrose gradients, and the resulting fractions were examined for guanylate cyclase, various enzyme markers of cell components, and electron microscopy. Purified plasma membrane fractions obtained from either preparation had the highest specific activity of guanylate cyclase, 30 to 80 pmol/min/mg of protein, and the recovery and relative specific activity of guanylate cyclase paralleled that of 5'-nucleotidase and adenylate cyclase in these fractions. Significant amounts of guanylate cyclase, adenylate cyclase, 5'-nucleotidase, and glucose-6-phosphatase were recovered in purified preparation of microsomes. We cannot exclude the presence of guanylate cyclase in other cell components such as Golgi. The electron microscopic studies of fractions supported the biochemical studies with enzyme markers. Soluble guanylate cyclase had typical Michaelis-Menten kinetics with respect to GTP and had an apparent Km for GTP of 35 muM. Ca-2+ stimulated the soluble activity in the presence of low concentrations of Mn-2+. The properties of guanylate cyclase in plasma membranes and microsomes were similar except that Ca-2+ inhibited the activity associated with plasma membranes and had no effect on that of microsomes. Both particulate enzymes were allosteric in nature; double reciprocal plots of velocity versus GTP were not linear, and Hill coefficients for preparations of plasma membranes and microsomes were calculated to be 1.60 and 1.58, respectively. The soluble and particulate enzymes were inhibited by ATP, and inhibition of the soluble enzyme was slightly greater. While Mg-2+ was less effective than Mn-2+ as a sole cation, all enzyme fractions were markedly stimulated with Mg-2+ in the presence of a low concentration of Mn-2+. Triton X-100 increased the activity of particulate fractions about 3- to 10-fold and increased the soluble activity 50 to 100%.
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PMID:Localization of particulate guanylate cyclase in plasma membranes and microsomes of rat liver. 23 12

The metabolism of glyceryl trinitrate (GTN) to nitric oxide (NO) was studied in the mouse macrophage cell line J774 and in the human monocytic cell line U937 in the absence or presence of Escherichia coli lipopolysaccharide (LPS). Two bioassay systems were used: inhibition of platelet aggregation and measurement of cGMP after stimulation by NO of guanylate cyclase in J774 cells. In addition, NO produced from GTN by cells or by cellular fractions was measured as nitrite (NO2-) one of its breakdown products. J774 cells (1.25 x 10(5) cells) treated with indomethacin (10 microM) enhanced the platelet inhibitory activity of GTN (22-352 microM) but not that of sodium nitroprusside (4 microM). This effect was abrogated by co-incubation with oxyhaemoglobin (oxyHb, 10 microM) indicating release of NO from GTN. U937 cells (up to 60 x 10(5)) did not metabolize GTN to NO. LPS (0.5 micrograms/mL for 18 hr) enhanced at least 2-fold the capacity of J774 cells but not that of U937 cells to form NO from GTN and this enhancement was attenuated when cycloheximide (10 micrograms/mL) was incubated together with LPS. In the absence of LPS stimulation, cycloheximide had no effect. Furthermore, when incubated with GTN (200 microM), J774 cells treated with LPS released more NO from GTN as indicated by a 3-fold greater increase in their level of cGMP which was prevented by oxyHb (10 microM). Incubation of J774 cells with GTN (75-600 microM) for 30 min led to a concentration-dependent increase in NO2- which was substantially reduced when the cells were boiled. The microsomal fraction was more potent than the cytosol in producing NO2- from GTN (1.2-2.4 mM). Release of NO2- from GTN by J774 cells was not affected by treating the cells with the NO synthase inhibitor, NG-monomethyl-L-arginine (MeArg, 300 microM). In J774 cells made tolerant to GTN, potentiation of the anti-platelet effects of GTN (11-352 microM) and release of NO2- from GTN was reduced. Thus, J774 cells but not U937 cells convert GTN to NO. This enzymic pathway (present mainly in the microsomal fraction of the J774 cells) is induced by LPS and is not regulated by endogenous NO released from L-Arg by the enzyme NO synthase. Furthermore, when compared to normal cells, tolerant J774 cells metabolize GTN to NO less effectively as assessed by a reduced capacity to potentiate the anti-platelet effect of GTN and to release NO2-.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The metabolism of glyceryl trinitrate to nitric oxide in the macrophage cell line J774 and its induction by Escherichia coli lipopolysaccharide. 137 39

The author reviews the problem of the pattern of lipid peroxidation in cancer cells with special reference to a comparison between normal liver cells and hepatomas both transplanted and induced by diethylnitrosamine. It is stated that the loss of lipid peroxidation is proportional to the degree of de-differentiation of hepatoma cells. During carcinogenesis, however, the loss is already evident at the stage of preneoplastic nodules. A common feature of all tumors, independently of the extent of the loss of peroxidation in basal conditions, is the lack of further stimulation by ADP/iron or by ascorbate/iron. As regards the reasons for the decline in lipid peroxidation, they are certainly not unique. An important cause is the low activity of the enzymes of the monooxygenase microsomal chain. Another very important one is the change in lipid composition of membranes, with a marked decrease in polyunsaturated fatty acids, which are the main substrate for lipid peroxidation. It has been shown that enrichment of membranes of hepatomas with arachidonic acid results in restoration of stimulation of peroxidation by ascorbate/iron, but not with ADP/iron. The last type of stimulation mostly reflects the behaviour of the monooxygenase chain, whereas ascorbate/iron-induced stimulation does not require the presence of an efficient cytochrome P450-chain. Another cause for decreased lipid peroxidation in tumors is the increased rigidity of membranes, due to the large increase in cholesterol content: this prevents to some extent the influx of oxygen inside the membranes. Yet another cause is the presence of increased amounts of antioxidants in both cytosol and membranes. The main toxic product of lipid peroxidation, 4-hydroxynonenal, has been found to elicit several actions at extremely low concentrations. In fact, 4-hydroxynonenal stimulates chemotaxis of polymorphonuclear leukocytes, stimulates plasma membrane adenylate cyclase, stimulates plasma membrane guanylate cyclase, and stimulates phospholipase C. The last three enzymes involve the action of G-proteins. The effect of the aldehyde is present at less than micromolar concentrations, which may occur inside the cells in certain conditions. Moreover, at concentrations from 10(-6) to 10(-7) M, the aldehyde is able to block oncogene c-myc expression in the human erythroleukemic K562 cell line, which at the same time becomes able to express the gamma-globin gene. These facts are discussed with reference to a possible biological meaning of the loss of lipid peroxidation in tumors.
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PMID:Lipid peroxidation and cancer: a critical reconsideration. 251 Mar 83

PGE2 and PGA2 incubated for 30 min at 25 degrees C with microsomal membranes isolated from Walker-256 tumour, in the presence of 50 microM indomethacin increase the lipid fluidity estimated by steady-state fluorescence anisotropy [(r0/r)-1]-1, using 1,6-diphenyl-1,3,5-hexatriene (DPH) as probe. The microsomal preparations of Walker-256 tumour contained calcium-stimulated and magnesium-dependent ATPase as well as calmoduling-dependent guanylate cyclese activities. A considerable decrease (approx. 65%) in the activity of the Ca2+-stimulated ATPase was observed when preparations were treated with 10 microM PGE2 and PGA2. A dramatic gradual decrease of the calmodulin-dependent guanylate cyclase activity was also observed at different concentrations of PGE2 and PGA2 (0.25-10 microM). The ATP-dependent uptake of calcium was reduced by approximately 60% in microsomal membranes treated with PGE2 and PGA2. The allosteric properties of Ca2+-stimulated ATPase by Na+, and of guanylate cyclase by Mn.GTP (as reflected by changes in the Hill coefficients, h) were modulated by PGE2 and PGA2. The apparent cooperativity of the Ca2+-ATPase (h + 1.73 +/- 0.21) in control membranes was abolished (h + 1.1 +/- 0.11 and h = 0.9 +/- 0.09) in membranes treated by PGE2 and PGA2 (10 microM), while the allosteric stimulation of guanylate cyclase by Mn.GTP was reduced from h = 2.78 +/- 0.24 in control membranes to h = 1.92 +/- 0.16 and h = 1.73 +/- 0.15 in membranes treated by PGE2 and PGA2 (10 microM), respectively, suggesting that the physical state of Ca2+-stimulated ATPase and guanylate cyclase lipid microenvironments changed from a gel phase to a liquid-crystalline phase. In conclusion, it is suggested that PGE2 and PGA2 promote a phase separation in Walker-256 tumour microsomal membranes. This may be relevant to the Ca2+-calmodulin system and tumour growth inhibition.
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PMID:PGE2 and PGA2 affect the allosteric properties and the activities of calmodulin-dependent guanylate cyclase and Ca2+-stimulated ATPase of Walker-256 tumour microsomal membranes. 256 56

Two hours after administration of Soman (120 micrograms/kg, s.c.), Sarin (150 micrograms/kg, s.c.), or Tabun (240 micrograms/kg, s.c.), microsomes and cytosol were prepared from rat striata. Microsomal and cytosolic calmodulin (CaM) levels, microsomal adenylate and guanylate cyclase activities, protein kinase activities, and Ca2+ + Mg2+-ATPase activities were determined while cytosolic phosphodiesterase (PDE) activities were determined. CaM levels in both cell fractions were significantly increased by Soman and Sarin. Cyclic AMP-PDE and adenylate cyclase activities were decreased by Soman and Sarin. All three agents decreased activities of cyclic GMP-PDE and guanylate cyclase. Sarin and Tabun administration caused significant increases in microsomal protein kinase activity and none of the agents affected activity of divalent cation ATPases. The intensity of effects of the three organophosphates roughly paralleled their observed neurotoxic potencies. The results indicate that components of the CaM system are implicated as either causative or adaptive changes induced by these agents.
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PMID:Acute effects of soman, sarin, and tabun on microsomal and cytosolic components of the calmodulin system in rat striatum. 286 34

Activities of adenylate and guanylate cyclases and cAMP and cGMP phosphodiesterases (cAPDE, cGPDE) were assayed in cell homogenates and subcellular fractions of cultured rabbit corneal epithelium, and effects of carbamylcholine on enzyme activities in each fraction were evaluated. Activity of cyclases and phosphodiesterases was detectable in control incubations of homogenates, nuclei, the mitochondrial/lysosomal fraction, microsomes, and cytosol, although microsomal guanylate cyclase represented a very small proportion of the total cellular activity. In homogenates, carbamylcholine significantly elevated guanylate cyclase and cAPDE and reduced cGPDE activity. In mitochondria/lysosomes, guanylate cyclase was elevated and cGPDE reduced, but the drug did not alter cAPDE activity. In microsomes, carbamylcholine enhanced cAPDE but did not alter guanylate cyclase of cGPDE activity. In the soluble cytoplasmic fraction the drug reduced guanylate cyclase activity. The purified nuclear fraction exhibited substantial activity of cyclases and phosphodiesterases. Carbamylcholine significantly elevated activity of nuclear guanylate cyclase and cAPDE and significantly reduced nuclear cGPDE activity. The drug did not significantly alter adenylate cyclase in homogenates or in any cell fraction. The presence of activity of enzymes of cyclic nucleotide metabolism in the cell nucleus and the sensitivity of nuclear guanylate cyclase, cAPDE and cGPDE to carbamylcholine, which in the same concentration range enhances activity of DNA and RNA polymerases, suggested the hypothesis that effects on cyclic nucleotide-dependent phosphorylation of nuclear proteins might be among regulatory mechanisms by which the drug alters rates of replication and transcription in corneal epithelial cells.
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PMID:Subcellular localization of muscarinic effects on enzymes of cyclic nucleotide metabolism in cultured corneal epithelial cells of the rabbit. 290 7


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