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)

An extremely rapid and sensitive assay for guanylate cyclase utilizing [alpha-32P]-GTP has been developed. It involves incubation of 5-100 mug of enzyme protein with 1 mM [alpha-32P]-GTP in 40 mM Tris HC1 buffer (pH 7.4) containing 3-3 mM MnSO2, 10 mM theophylline and 1 mM cyclic GMP. The reaction is terminated by addition of EDTA, and [32P]-cyclic GMP formed is isolated by sequential chromatography on Dowex-50-H+ and alumina. Recovery of 75-85% of [3H]-cyclic GMP and a blank of 0.001-0.003% of added [32P]-GTP was routinely obtained. The [32P] radioactivity isolated was shown to be cyclic GMP by a variety of techniques. The assay has also been shown to be applicable for a variety of tissues.
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PMID:A rapid method for the assay of guanylate cyclase. 0 69

A method for the assay of guanylate cyclase is described utilizing alpha-[32P]-GTP as substrate for the enzyme reaction. 100-150 microgram of enzyme protein is incubated in a 15.6 mM Tris-HCl buffer incubation mixture, pH 7.6. The reaction is stopped by the addition of EDTA. The [32P]-cyclic GMP formed is separated by a two-step column chromatography on Dowex 50W-X4 ion-exchange resin and neutral alumina. The recovery for cyclic GMP was about 70%. The blank values ranged from 0.001-0.003% of the added alpha-[32P]-GTP which had been purified by Dowex 50W-X4 column chromatography. This method was employed for the assay of guanylate cyclase activities in different tissues.
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PMID:A sensitive method for the assay of guanylate cyclase activity. 3 7

Atrial natriuretic factor (ANF) is a peptide hormone from the heart atrium with potent natriuretic and vasorelaxant activities. The natriuretic activity of ANF is, in part, mediated through the adrenal gland, where binding of ANF to the 130-kDa ANF receptor causes suppression of aldosterone secretion. Incubation of bovine adrenal membranes at pH < 5.6 caused a rapid and spontaneous cleavage of the 130-kDa ANF receptor, yielding a 65-kDa polypeptide that could be detected by photoaffinity labeling by 125I-labeled N alpha 4-azidobenzoyl-ANF(4-28) followed by SDS/PAGE under reducing conditions. Within 20 min of incubation at pH 4.0, essentially all the 130-kDa receptor was converted to a 65-kDa ANF binding protein. This cleavage reaction was completely inhibited by inclusion of 5 mM EDTA. When SDS/PAGE was carried out under non-reducing conditions, the apparent size of the ANF receptor remained unchanged at 130 kDa, indicating that the 65-kDa ANF-binding fragment was still linked to the remaining part(s) of the receptor polypeptide through a disulfide bond(s). The disappearance of the 130-kDa receptor was accompanied by a parallel decrease in guanylate cyclase activity in the membranes. Inclusion of EDTA in the incubation not only prevented cleavage of the 130-kDa receptor, but also protected guanylate cyclase activity, indicating that proteolysis, but not the physical effects of the acidic pH, causes inactivation of guanylate cyclase. The 130-kDa ANF receptor in adrenal membranes was competitively protected from photoaffinity labeling by ANF(1-28) or ANF(4-28), but not by atriopeptin I [ANF(5-25)] or C-ANF [des-(18-22)-ANF(4-23)-NH2]. On the contrary, the 65-kDa ANF-binding fragment generated after incubation at pH 4.0 was protected from labeling by any of the above peptides, indicating broader binding specificity. After incubation in the presence of EDTA, the 130-kDa ANF receptor, which was protected from proteolysis, retained binding specificity identical to that of the 130-kDa receptor in untreated membranes. The results indicate that the broadening of selectivity is caused by cleavage, but not by the physical effect of acidic pH. Spontaneous proteolysis of ANF receptor by an endogenous metalloendopeptidase, occurring with concomitant inactivation of guanylate cyclase activity and broadening of ligand-binding selectivity, may be responsible for the generation of low-molecular-mass receptors found in the adrenal gland and other target organs of ANF. The proteolytic process may play a role in desensitization or down-regulation of the ANF receptor.
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PMID:Proteolytic cleavage of atrial natriuretic factor receptor in bovine adrenal membranes by endogenous metalloendopeptidase. Effects on guanylate cyclase activity and ligand-binding specificity. 135 9

The action of ANF is, at least in part, mediated by the activation of particulate guanylate cyclase. Increases in plasma ANF levels induce a marked increase in the plasma levels and urinary excretion of cyclic GMP. In contrast to agents that stimulate particulate guanylate cyclase, activators of soluble guanylate cyclase, such as the bioactive molsidomine metabolite, SIN 1, induce only a modest, not significant, increase in plasma cyclic GMP levels. Thus, increases in plasma cyclic GMP levels appear to be specific for the activation of particulate guanylate cyclase. Cyclic GMP is stable in whole blood in the presence of EDTA and can easily be measured in plasma and urine. It may therefore be a valuable alternative for ANF measurement in the clinical routine. In contrast to urinary ANF excretion, the urinary excretion of cyclic GMP sensitively reflects increases in plasma ANF levels. Measurement of cyclic GMP excretion may therefore be an alternative for plasma ANF and plasma cyclic GMP measurement especially in situations where blood drawing is difficult, e.g. in newborns. Measurement of basal cyclic GMP followed by determination of increases in cyclic GMP levels after injection of a small ANF bolus dose tests the cellular sensitivity to ANF. This may give further insight in the mechanism of the regulation of ANF effects. Therefore, cyclic GMP in many cases appears to be a sensitive marker for the action of ANF in man.
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PMID:Is cyclic GMP a clinically useful marker for ANF action? 284 15

Incubation of the adrenal membranes at pH 3.5-5.6 resulted in apparent proteolysis of 140 kDa protein to yield a 70 kDa polypeptide containing an ANF-binding site, which could be photoaffinity labeled by [125I]4-azidobenzoyl monoiodo ANF-(4-28). This 70 kDa fragment was found to be disulfide-linked to the remaining segment(s) of the molecule, giving a total apparent Mr of 140,000 when not reduced. The acidic pH-dependent proteolysis was rapid even at 0 degree C, suggesting close association of an endopeptidase with ANF receptor. The proteolysis was inhibited by EDTA, but not by phenylmethanesulfonyl fluoride, N-ethylmaleimide or pepstatin, indicating that the enzyme is a metalloendopeptidase. The inhibition was reversed by ZnCl2 or MnCl2, but not CaCl2 or MgCl2. The adrenal membranes contained guanylate cyclase activity of 1.1 nmol/min/mg protein using Mn-GTP as a substrate, which could be stimulated by 0.1 microM ANF to 2.7 nmol/min/mg. The membranes showed high affinity to ANF-(1-28) and ANF-(4-28), but little affinity to the truncated peptides ANF-(5-25) and ANF-(7-23). After treatment at pH 3.5 and 0 degrees C for 15 min, the membranes retained ANF-binding activity but with broader specificity, exhibiting high affinity to all four peptides above. It was suggested that an acidic metalloendopeptidase in the adrenal membranes may be involved in ANF receptor cleavage.
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PMID:Acidic pH- and metal ion (Zn++ or Mn++)-dependent proteolysis of 140 kDa atrial natriuretic factor receptor in bovine adrenal cortex plasma membranes: evidence for membrane-bound acidic metalloendopeptidase. 289 2

A guanylate cyclase preparation partially purified from supernatant of a pig lung extract was subjected to affinity chromatography on an Agarose-GTP column. The major portion of the cyclase activity was adsorbed on the column and then eluted with 50 mM EDTA and 0.5 M KCl, whereas the fractions non-adsorbed on the column contained a factor which enhanced the cyclase activity. Addition of the activating factor to a cyclase reaction mixture increase the enzyme activity without a time lag, and this enhancement by the factor was dose-dependent. With concomitant presence of cyclase and the factor in the reaction mixture the apparent Km value for GTP-Mn2+ of the enzyme was 56 microM, this value being the same as in absence of the factor, however, here the maximum velocity increased 4-fold. The factor was nondiffusable, heat-labile, partially sensitive to trypsin, and resistant to acid or alkali. As estimated by gel filtration, this factor had an apparent molecular weight of 85 000.
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PMID:Involvement of a macromolecular activating factor in activity of guanylate cyclase partially purified from supernatant of a pig lung extract. 610 82

Axonemes were isolated from purified bovine retinal rod outer segments by dissolving the outer segment membranes in detergent and separating the axonemes by centrifugation on a linear detergent-containing sucrose density gradient. Guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.61.2) activity was concentrated in the axoneme fraction. Guanylate cyclase eluted in the void volume when detergent-solubilized rod outer segments were subjected to exclusion chromatography on Sepharose 4B. Attempts to extract guanylate cyclase from isolated axonemes with salt, EDTA, base and other reagents were successful.
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PMID:Association of guanylate cyclase with the axoneme of retinal rods. 610 15

The partially purified soluble guanylate cyclase (GTP pyrophosphatelyase(cyclizing), EC 4.6.1.2) from human caudate nucleus is stimulated from 2 to 4-fold by metal chelating agents. EDTA (K 1/2 - 4.8 microM) is more potent than CDTA (K 1/2 = 13.2 microM) or EGTA (K 1/2 = 21.8 microM) at stimulating activity. Stimulation by chelating agents is apparently not due to removal of inhibitory divalent cations which contaminate the enzyme or reaction mixture. EDTA increases guanylate cyclase activity in part by increasing the affinity of the enzyme for the substrate (MgGTP) 10-fold. Dopamine inhibits partially purified guanylate cyclase in the presence or absence of EDTA. Dopamine increases the Ka of guanylate cyclase for the activator, free Mn2+, more than 50-fold, from 3 to 150 microM.
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PMID:Stimulation of guanylate cyclase by EDTA and other chelating agents. 611 47

The membraneous guanylate cyclase of cilia from Paramecium tetraurelia used MgGTP and MnGTP as substrate with Michaelis constants for GTP of 71.5 microM and 36 microM, respectively. A linear Arrhenius plot indicated that a single enzyme entity exists not sensitive to possible phase transitions of membrane lipids. Guanylate cyclase is activated by low concentrations (less than 100 microM) and inhibited by high concentrations (greater than 100 microM) of calcium, half-maximal effects were obtained with 8 microM and 500 microM Ca2+, respectively. Only strontium ions displayed partial activating and inhibiting potency, all other divalent cations tested, Ba2+, Fe2+, Co2+, Mn2+, Sn2+ and Ni2+ had no effect on guanylate cyclase activity. Ca2+ activation increased V; Km remained identical. The Ca2+ stimulated activity was not inhibited by trifluoperazine, tentatively suggesting that the stimulation may not be mediated by calmodulin. Ca2 inhibition was due to a single binding site of Ca2+ at the guanylate cyclase as evidence by a Hill coefficient h = -1 and was noncompetitive. The lanthanides La3+, Ce3+ and Tb3+ were powerful inhibitors of guanylate cyclase, with La3+ the half-maximal effect was obtained with 0.6 microM, it was kinetically a mixed-type inhibition. La3+ and CA2+ competed for the same binding site on the guanylate cyclase as determined by detailed kinetic analysis. Addition of EDTA reversed the activation and inhibition by Ca2+ and the inhibition by La3+. It is discussed that guanylate cyclase may be the initial target enzyme in the cilia for the calcium transient of the calcium-potassium action potential of Paramecium.
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PMID:Characterization of a Ca2+-dependent guanylate cyclase in the excitable ciliary membrane from Paramecium. 612 19

A procedure was developed for the large scale preparation of membranes from pig atria which are enriched 10-13 fold in the muscarinic acetylcholine receptor. The procedure involved differential centrifugation and sucrose-gradient centrifugation in solutions containing 150 mM-NaClO4 and 5 mM-EDTA to minimize membrane aggregation. The final membrane preparation bound about 1.1 pmol of L-quinuclidinyl benzilate/mg of protein. Comparable results were obtained with either fresh or frozen tissue. About the same yield (120 pmol of L-quinuclidinyl benzilate sites/100 g of tissue) and specific activity of membranes were obtained from different regions of the atria. The final preparation was stable at -80 degrees C in buffered sucrose solutions. The membranes appeared mostly as sheets or fragments and partly as closed vesicles in the electron microscope and were heterogeneous in isopycnic Percoll gradients. Marker enzyme studies showed that the receptor was enriched in parallel with the plasma membrane markers guanylate cyclase (particulate form) and (Na+ + K+)-activated ATPase. Some contamination by mitochondrial outer and endoplasmic reticulum membranes was evident from the distribution of monoamine oxidase and glucose-6-phosphatase activity, but the preparation was largely free of sarcoplasmic reticulum, mitochondrial inner, and lysosomal membranes.
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PMID:Preparation and characterization of muscarinic-acetylcholine-receptor-enriched membranes from pig atria. 709 26


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