Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Products of the reactions catalyzed by highly purified preparations of soluble guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] from rat liver were identified and quantified with 31P NMR spectroscopy. Utilization of this technique necessitated modification of the standard assay conditions; higher concentrations of enzyme and substrate (2 mM), Mg2+ instead of Mn2+, and longer incubation times (up to 46 hr) at 30 degrees C were used. Revision of our reported procedure for purificaton of guanylate cyclase [Tsai, S-C., Manganiello, V. C. & Vaughan, M. (1978) J. Biol. Chem. 253, 8452-8457] to include chromatography on Ultrogel AcA34 and agarose-hexane-GTP provided an enzyme with specific activity higher than in our earlier preparations. 31P NMR spectra obtained during incubation of this enzyme showed that the rates of GTP disappearance and cyclic GMP (cGMP) accumulation were constant for approximately 16 hr. They indicated, however, that the preparations were contaminated with inorganic pyrophosphatase. This was removed by preparative electrophoresis, yielding enzyme with specific activities (900-1300 nmol/min per mg of protein) higher than those reported for guanylate cyclases from rat liver or lung. With this preparation, cGMP and PPi were the only products of GTP detected, consistent with the assumption that the guanylate and adenylate cyclase reactions are analogous.
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PMID:Products of reaction catalyzed by purified rat liver guanylate cyclase determined by 31p NMR spectroscopy. 610 59

Guanylate cyclase in neuroblastoma N1E 115 cells was readily solubilized upon homogenization of the cells with hypotonic buffer. When the supernatant was passed through cation exchangers such as a Chelex 100 Na+ column, the guanylate cyclase activity in the effluent fraction decreased to 4-6% of the original supernatant. The addition of the acid extract of neuroblastoma cells or rat tissues to the effluent restored guanylate cyclase activity, indicating that the supernatant of neuroblastoma cells contained an acid-soluble endogenous activator for guanylate cyclase which was adsorbed on cation exchangers. The activator was purified from rat brain and identified as L-arginine by 13C- and 1H-NMR spectroscopy and paper partition chromatography. L-Arginine, at a concentration of 1-2 x 10(-5) M, stimulated guanylate cyclase activity in the effluent fraction 15-25-fold, whereas D-arginine and other basic L-amino acids were ineffective. Peptides that contained L-arginine at the NH2- or COOH-terminal also resulted in an activation of guanylate cyclase to the extent similar to that of L-arginine, while peptides that contained L-arginine inside the peptide chain failed to stimulate the activity. The activation of L-arginine seemed to operate by a mechanism similar to that induced by nitroso compounds.
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PMID:L-Arginine identified as an endogenous activator for soluble guanylate cyclase from neuroblastoma cells. 612 10

The stereochemical course of the reaction catalyzed by the soluble form of bovine lung guanylate cyclase has been investigated using [alpha-18O]guanosine 5'-triphosphate (Rp diastereomer) and guanosine 5'-O-(1-thiotriphosphate) (Sp diastereomer) as substrates. The product from the 3-thiomorpholino-1',1'-dioxide sydnonimine-stimulated enzymatic cyclization of [alpha-18O] guanosine 5'-triphosphate was esterified with diazomethane. 31P NMR analysis of the triesters indicated that all of the 18O label was present in the axial position. Guanosine 5'-O-(1-thiotriphosphate) (Sp diastereomer) was cyclized under stimulated and basal enzyme activities and, in both cases, the Rp diastereomer of guanosine 3',5'-cyclic phosphorothioate was formed. This was determined by direct comparison with material synthesized chemically from guanosine 5'-phosphorothioate. The results from these experiments show that the reaction catalyzed by guanylate cyclase proceeds with inversion of configuration at phosphorus and this indicates that the reaction proceeds by way of a single direct displacement reaction.
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PMID:The stereochemical course of the reaction catalyzed by soluble bovine lung guanylate cyclase. 613 67

This study was designed to determine if the antinociception produced by intrathecally (i.t.) administered muscarinic agonists in male Sprague-Dawley rats is mediated by an L-arginine/nitric oxide/cyclic GMP cascade. Seven days after implantation of intrathecal catheters, antinociception was produced with graded doses of (+)-cis-methyldioxolane (CD). The ED50 values for CD in the hot-plate and tail-flick tests were 2.6 and 2.0 nmol, respectively. The corneal and righting reflexes, as well as stepping and negative geotaxic responses, were not affected by CD. Six-minute pretreatment with 50 nmol of the nitric oxide synthase inhibitor L-NG-nitroarginine (NNR) significantly inhibited CD-produced hot-plate and tail-flick antinociception as evidenced by 6-fold shifts to the right of the CD dose-response curves. Coadministration of 150 nmol of L-arginine with NNR reversed the NNR-induced inhibition of the antinociception produced by CD. D-Arginine was without effect. Pretreatment with 500 nmol of the guanylyl cyclase inhibitor methylene blue also antagonized the antinociception produced by CD in both the hot-plate and tail-flick tests. In parallel with CD, coadministration of L-arginine with NMR reversed the NMR-induced inhibition of (+)-muscarine-produced antinociception in both nociceptive tests. Intrathecal administration of buffer, 50 nmol of NNR, 150 nmol of L- or D-arginine or 500 nmol of methylene blue, did not alter nociceptive responses when injected alone. In contrast, i.t. administration of the membrane-permeable cyclic GMP analogs, dibutyryl cyclic GMP and 8-bromo cyclic GMP, produced antinociception in both nociceptive tests when injected alone.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Pharmacologic evidence that spinal muscarinic analgesia is mediated by an L-arginine/nitric oxide/cyclic GMP cascade in rats. 796 74

24 new thiazole-2-nitrosimines were prepared and described by means of spectroscopical methods (NMR, IR, MS, UV). At pH 7 in cell free systems as well as in platelet rich plasma the compounds are stable against hydrolysis and do not react with the platelet glutathione. The chemical stability is underlined by the mass spectra: M+. is of high intensity and sometimes even forms the base peak (e.g. 8a). Thermal elimination of N2 is of minor importance. The =N-NO bond in solution is susceptible to cleavage by visible light. The metabolite so formed is able to inhibit the platelet aggregation induced by collagen (Born-test). Five compounds exhibit this activity in concentrations below 10 mumol/L (IC50). This is due to the release of a NO species, as could be demonstrated by the stimulation of soluble guanylate cyclase in a cell free system (e.g. 8a, KM = 72 mumol/L). In vivo the nitrosimines show antithrombotic properties. Two h after a single oral dose of 8g (60 mg/kg) a 57% inhibition of the laser induced thrombus formation in the mesenteric arterioles of rats is observed. After 8 h a 43% inhibition still is seen.
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PMID:New NO-donors with antithrombotic and vasodilating activities, VI: thiazole-2-nitrosimines. 797 24

The peptide hormone guanylin constitutes two topological stereoisomers, which are connected through an equilibrium of interconversion. To investigate the importance of amino acid residues in the central region between the inner cysteines and at the carboxy terminus for this isomerism, synthetic derivatives of guanylin were compared by HPLC, 2D1H NMR spectroscopy and by their guanylyl cyclase-C (GC-C)-activating potency. An increase in the central sterical bulk by introduction of diiodo-Tyr9 had virtually no effect on the isomerization kinetics. Compared to guanylin, carboxy-terminal amidation did not affect the equilibrium between the two isoforms either. In contrast, two significantly stabilized isomers were obtained by extending the carboxy terminus of guanylin with one additional leucine resembling the characteristic of human uroguanylin isomers. This effect was intensified by a further Lys-Lys extension, thus revealing that the conformational exchange between the guanylin isomers is dependent on the extent of the sterical hindrance in the carboxy-terminal region of this peptide. Demonstrated by 2D NMR spectroscopy, the separated isomers of the carboxy-terminally extended derivatives of guanylin exhibit unambiguously closely related structures as found originally for guanylin isomers, which are only detectable as a mixture. Because only one of the stabilized guanylin isomers activates guanylyl cyclase-C, the three-dimensional structure of the GC-C-activating guanylin isomer is now defined. The stabilized isoforms of guanylin described in this study represent suitable tools for the separate functional investigation of the GC-C-agonistic isomer of guanylin as well as of its isomeric counterpart.
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PMID:Carboxy-terminal extension stabilizes the topological stereoisomers of guanylin. 992 96

The effect of nitric oxide radicals (NO) on the activity of porcine aortic endothelial Na(+)-K(+)-ATPase is reported. Measurements were made using an in vitro cell system and 133Cs magnetic resonance (NMR). It is shown that NO, through stimulation of guanylate cyclase, results in a reduction of pump activity. Similar observations were made using 8-Br-cGMP. Measurement of the cytosolic volume indicated no changes in volume during incubation with 8-Br-cGMP. Our measurements indicate a continuous regulation of endothelial Na(+)-K(+)-ATPase activity by endogenous NO. This regulation could be removed by L-NAME, resulting in a small increase in pump activity.
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PMID:Short-term regulation of endothelial Na(+)-K(+)-pump activity by cGMP: a 133Cs magnetic resonance study. 1008 5

Guanylin is a guanylyl cyclase (GC)-activating peptide that is mainly secreted as the corresponding prohormone of 94 amino acid residues. In this study, we show that the originally isolated 15-residue guanylin, representing the COOH-terminal part of the prohormone, is released from the prohormone by cleavage of an Asp-Pro amide bond under conditions applied during the isolation procedures. Thus, the 15-residue guanylin is probably a non-native, chemically induced GC-activating peptide. This guanylin molecule contains two disulfide bonds that are absolutely necessary for receptor activation. We demonstrate that the folding of the reduced 15-residue guanylin results almost completely in the formation of the two inactive disulfide isomers. In contrast, the reduced form of proguanylin containing the entire prosequence folds to a product with the native cysteine connectivity. Because proguanylin lacking the 31 NH2-terminal residues of the prosequence folds only to a minor extent to guanylin with the native disulfide bonds, it is evident that this NH2-terminal region contributes significantly to the correct disulfide-coupled folding. Structural studies using CD and NMR spectroscopy show that native proguanylin contains a considerable amount of alpha-helical and, to a lesser extent, beta-sheet structural elements. In addition, a close proximity of the NH2- and the COOH-terminal regions was found by NOESY. It appears that this interaction is important for the constitution of the correct conformation and provides an explanation of the minor guanylyl cyclase activity of proguanylin by shielding the bioactive COOH-terminal domain from the receptor.
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PMID:Role of the prosequence of guanylin. 1049 86

A key challenge in studying protein/protein interactions is to accurately identify contact surfaces, i.e. regions of two proteins that are in direct physical contact. Aside from x-ray crystallography and NMR spectroscopy few methods are available that address this problem. Although x-ray crystallography often provides detailed information about contact surfaces, it is limited to situations when a co-crystal of proteins is available. NMR circumvents this requirement but is limited to small protein complexes. Other methods, for instance protection from proteolysis, are less direct and therefore less informative. Here we describe a new method that identifies candidate contact surfaces in protein complexes. The complexes are first stabilized by cross-linking. They are then digested with a protease, and the cross-linked fragments are analyzed by mass spectrometry. We applied this method, referred to as COSUMAS (contact surfaces by mass spectrometry), to two proteins, retinal guanylyl cyclase 1 (RetGC1) and guanylyl cyclase-activating protein-1 (GCAP-1), that regulate cGMP synthesis in photoreceptors. Two regions in GCAP-1 and three in RetGC1 were identified as possible contact sites. The two regions of RetGC1 that are in the vicinities of Cys(741) and Cys(780) map to a kinase homology domain in RetGC1. Their identities as contact sites were independently evaluated by peptide inhibition analysis. Peptides with sequences from these regions block GCAP-1-mediated regulation of guanylyl cyclase at both high and low Ca2+ concentrations. The two regions of GCAP-1 cross-linked to these peptides were in the vicinities of Cys(17) and Cys(105) of GCAP-1. Peptides with sequences derived from these regions inhibit guanylyl cyclase activity directly. These results support a model in which GCAP-1 binds constitutively to RetGC1 and regulates cyclase activity by structural changes caused by the binding or dissociation of Ca2+.
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PMID:Identification of proximate regions in a complex of retinal guanylyl cyclase 1 and guanylyl cyclase-activating protein-1 by a novel mass spectrometry-based method. 1138 42

Nitric oxide (*NO) and *NO-derived reactive species rapidly react with lipids during both autocatalytic and enzymatic oxidation reactions to yield nitrated derivatives that serve as cell signaling molecules. Herein we report the synthesis, purification, characterization, and bioactivity of nitrolinoleate (LNO2). Nitroselenylation of linoleic acid yielded LNO2 that was purified by solvent extraction, silicic acid chromatography, and reverse-phase HPLC. Structural characterization was performed by IR spectroscopy, 15N-NMR, LC-negative ion electrospray mass spectroscopy (MS), and chemiluminescent nitrogen analysis. Quantitative MS analysis of cell and vessel LNO2 metabolism, using L[15N]O2 as an internal standard, revealed that LNO2 is rapidly metabolized by rat aortic smooth muscle (RASM) monolayers and rat thoracic aorta, resulting in nitrite production and up to 3-fold increases in cGMP (ED50 = 30 microM for RASM, 50 microM for aorta). LNO2 induced endothelium-independent relaxation of preconstricted rat aortic rings, which was unaffected by L(G)-nitro-l-arginine methyl ester addition and inhibited by the guanylate cyclase inhibitor 1H-[1,2,4] oxadiazole[4,3-a]quinoxalin-1-one and the *NO scavenger HbO2. These results reveal that synthetic LNO2, identical to lipid derivatives produced biologically by the reaction of *NO and *NO-derived species with oxidizing unsaturated fatty acids (e.g., linoleate), can transduce vascular signaling actions of *NO.
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PMID:Nitrolinoleate, a nitric oxide-derived mediator of cell function: synthesis, characterization, and vasomotor activity. 1244 58


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