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

The chromatography of soluble human and rat platelet guanylate cyclases (105000 g supernatants) on DEAE-cellulose in 50 mM Tris HCl buffer, containing 0.22 M NaCl, has yielded virtually identical elution profiles, each with two protein peaks (I and II). Only peak II was found to have guanylate cyclase activity. Experiments with human platelets showed that inactive protein peak I inhibited the activity of guanylate cyclase preparation (peak II) and restored the already lost ability of the enzyme to be activated by sodium nitroprusside. In experiments with rat platelets, inactive fraction I had no effect on guanylate cyclase activity (peak II), and the enzyme was not activated by sodium nitroprusside either before or after DEAE-cellulose. 105000g supernatant of human platelets had an absorbance maximum at 415 nm (Soret band), which disappeared from the spectrum of the active fraction (II) but was found in the spectrum of the inactive (inhibitory) fraction I. Experiments with rat platelets demonstrated the absence of Soret band in the corresponding spectra. It was concluded that, contrary to the generally accepted notion, heme is not a prosthetic group of the soluble rat platelet guanylate cyclase.
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PMID:Role of heme in the regulation of human and rat platelet soluble guanylate cyclases. 168 34

Chromatography of soluble human platelet guanylate cyclase (105,000 g supernatant) on DEAE-cellulose in a linear gradient of NaCl (0-0.5 M) in 50 mM Tris-HCl buffer pH 7.6 gave two protein peaks, I and II, of which only peak II possessed the guanylate cyclase activity (0.18-0.22 M NaCl). The protein fraction I was found to possess an inhibiting activity; its addition to the partially purified enzyme decreased the guanylate cyclase activity by 60-70% in the presence of Mg2+ with no effect on the enzyme activity in the presence of Mn2+. The isolated enzyme lost (by approximately 80%) its ability to be activated by sodium nitroprusside; the latter was reconstituted after addition of the inhibiting fraction. The data obtained testify to the heme origin of the endogenous inhibitor of human platelet guanylate cyclase.
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PMID:[An endogenous regulator of the guanylate cyclase activity of human platelets]. 198 50

Homogeneous or partially purified soluble guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) from rat liver exhibited variable sensitivity to assay pH that was dependent upon buffer composition and the cation cofactor. Enzyme activity with 3 mM Mn2+ in excess of Mn2+-GTP was considerably less in Tris buffers above pH 8.0 than in glycine buffer. In the pH range of 6.0-7.6, however, manganese-supported activity was greater in Tris buffers than in imidazole or cacodylate buffers of corresponding pH. The differences in activity seen with various buffers were not apparent when Mg2+ was the sole cation cofactor but were dependent upon Mn2+ concentrations in excess of Mn2+-GTP. The effects of excess Mn2+ on guanylate cyclase varied with assay pH and buffer composition. At pH 7.6 in Tris-HCl buffer, excess Mn2+ increased guanylate cyclase activity with an apparent Ka of 0.25 mM and concentrations above 3 mM were slightly inhibitory. At pH 9.0 in Tris-HCl buffer, however, concentrations of excess Mn2+ above 0.1 mM were strongly inhibitory. By comparison, in cacodylate (pH 7.6) or glycine (pH 9.0) buffers, high concentrations of excess Mn2+ were considerably less inhibitory and the apparent Ka values for excess Mn2+ were greater than in Tris-HCl buffer at equivalent pH. The variable effects of Mn2+ on enzyme activity as a function of buffer pH and composition were qualitatively similar to its effects on catecholamine oxidation. Furthermore, the inhibition of guanylate cyclase by excess Mn2+ was partially prevented by dithiothreitol and the stimulation of enzyme activity by excess cation was completely blocked by the antioxidant hydroquinone. The studies suggest that the apparent requirement and preference of soluble guanylate cyclase for excess Mn2+ as cation cofactor, as well as the inhibition of enzyme activity by excess Mn2+ may be mediated by oxidative events associated with changes in the oxidation state of the free cation.
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PMID:Oxidative modulation of soluble guanylate cyclase by manganese. 610 32

The basal activity of soluble rat myocardial guanylate cyclase (105000 x g supernatant) is stimulated by 2 mM dithiothreitol (2-fold). In the presence of 2 mM dithiothreitol, sodium nitroprusside enhances the enzyme activation up to 26.5-fold. Addition of heme-containing proteins (hemoglobin or myoglobin) produces further stimulation of the enzyme--by 44% and 69%, respectively. Ion-exchange chromatography of rat myocardial 105000 x g supernatant by stepwise elution with 50 mM Tris-HCl buffer pH 7.6 containing 0.22 M NaCl revealed two protein peaks (I and II), of which only peak II possessed the guanylate cyclase activity. The spectrum of the 105000 x g supernatant had an absorption maximum at 415 nm (Soret band) which disappeared from the spectrum of the protein peak II but was detected in the inactive protein peak I. The guanylate cyclase preparation (peak II) lost its ability to be activated by sodium nitroprusside. All the attempts to reconstitute the nitroprusside-induced activation of the enzyme by adding the inactive protein peak I or the heme-containing proteins (hemoglobin or myoglobin) to peak II were unsuccessful. The possible mechanism of rat myocardial guanylate cyclase activation by sodium nitroprusside is discussed.
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PMID:[The role of heme in activating rat myocardial guanylate cyclase by sodium nitroprusside]. 777 74

Effects of aminoethylisothiuronium bromide (AET), known as radioprotector, on human platelet soluble guanylate cyclase and on ADP-induced human platelets aggregation were studied. It was shown that AET - in Tris buffer and at certain pH values - is converted, via transguanidine rearrangement, to mercaptoethylguanidine. The latter contains in its molecule both the guanidine and SH groups which act as donor and acceptor of nitric oxide (NO), respectively. It was demonstrated that AET, after its rearrangement to mercaptoethylguanidine, is able to activate human platelet soluble guanylate cyclase, as well as to inhibit ADP-induced human stimulatory effect of AET is dependent on the effectiveness of its transguanidine rearrangement to mercaptoethylguanidine. The molecular mechanism of the hypotensive by - effect of AET is proposed.
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PMID:Guanidine thiol--a new activator of soluble guanylate cyclase with antihypertensive and antiaggregatory properties. 852 55

The effects of aminoethylisothiuronium bromide known as a radioprotector on the activity of human platelet soluble guanylate cyclase and on ADP-induced aggregation of human platelets have been studied. It has been shown that in Tris-buffer and at definite pH values aminoethylisothiuronium bromide is converted into mercaptoethylguanidine as a result of a transguanidine rearrangement. The latter contains in its molecule both guanidine and SH-groups which appear to be the donor and acceptor of nitric oxide, respectively. It was found that after its rearrangement into mercaptoethylguanidine, aminoethylisothiuronium bromide activates human platelet soluble guanylate cyclase, inhibits ADP-induced aggregation of human platelets and accelerates their spontaneous disaggregation. The stimulating effect of aminoethylisothiuronium bromide depends on the effectiveness of its transguanidine rearrangement into mercaptoethylguanidine. A molecular mechanism of the hypotensive side effect of aminoethylisothiuronium bromide is proposed.
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PMID:[A new activator of soluble guanylate cyclase generated by nitric oxide and having antihypertensive and anti-aggregation properties]. 856 54

Previous studies have showed that SIN-1, a nitric oxide (NO) donor, injected into the dorsolateral column of the periaqueductal gray (dlPAG) induces flight reactions. This drug, however, can also produce peroxynitrite, which may interfere in this effect. In addition, it is also unknown if this effect is mediated by local activation of soluble guanylate cyclase (sGC). The aims of this study, therefore, were (1) to investigate if NOC-9 (6-(2-Hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hexanamine), a NO donor that does not produce peroxynitrite, would produce flight reactions after intra-dlPAG administration similar to those induced by SIN-1; (2) to verify if these responses could be prevented by local injection of a selective guanylate cyclase inhibitor (ODQ). Male Wistar rats (n=5-12) with cannulae aimed at the dlPAG received injections of TRIS (pH 10.0, 0.5 microl), NOC-9 (75 and 150 nmol), saline or SIN-1 (200 nmol) and were placed in an open arena for 10 min. In a subsequent experiment animals (n=7-8) were pretreated with ODQ (1 nmol/0.5 microl) before receiving NOC-9 150 nmol. NOC-9 induced a significant dose-dependent increase in flight reactions in the first minute after injection (% of animals displaying flight: vehicle=0%, NOC 75=67%, NOC 150=75%). SIN-1 had a similar effect (100% of animals showing flight) but the effects lasted longer (10 min) than those of NOC-9. The effect of NOC-9 (150 nmol) was prevented by pretreatment with ODQ (% of animals displaying flight: vehicle+NOC 150=71%, ODQ+NOC 150=37%). The results suggest that NO donors injected into the dlPAG induce defensive responses that are not mediated by secondary peroxynitrite production. Moreover, they also indicate that these defensive responses depend on activation of local sGC. The data strengthen the proposal that NO can modulate defensive reactions in the dlPAG.
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PMID:NOC-9, a selective nitric oxide donor, induces flight reactions in the dorsolateral periaqueductal gray of rats by activating soluble guanylate cyclase. 1942 60