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NO-independent regulatory site of direct sGC stimulators like YC-1 and BAY 41-2272.

Becker EM, Alonso-Alija C, Apeler H, Gerzer R, Minuth T, Pleiss U, Schmidt P, Schramm M, Schröder H, Schroeder W, Steinke W, Straub A, Stasch JP - BMC Pharmacol. (2001)

Bottom Line: The synthesized photoaffinitylabel directly stimulates the purified sGC and shows in combination with NO a synergistic effect on sGC activity.Determination of radioactivity of the single PTH-cycles from the sequencing of this CNBr fragment detected the cysteines 238 and 243 as binding residues of the 3H-meta-PAL.Our data demonstrate that the region surrounding the cysteines 238 and 243 in the alpha1-subunit of the sGC could play an important role in regulation of sGC activity and could be the target of this new type of sGC stimulators.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pharma Research Center, Bayer AG, Wuppertal, Germany. eva.becker@gmx.net

ABSTRACT

Background: The most important receptor for nitric oxide is the soluble guanylate cyclase (sGC), a heme containing heterodimer. Recently, a pyrazolopyridine derivative BAY 41-2272, structurally related to YC-1, was identified stimulating soluble guanylate cyclase in an NO-independent manner, which results in vasodilatation and antiplatelet activity. The study described here addresses the identification of the NO-independent site on soluble guanylate cyclase.

Results: We developed a photoaffinity label (3H-meta-PAL) for the direct and NO-independent soluble guanylate cyclase (sGC) stimulator BAY 41-2272 by introducing an azido-group into the tritium labeled compound. The synthesized photoaffinitylabel directly stimulates the purified sGC and shows in combination with NO a synergistic effect on sGC activity. Irradiation with UV light of 3H-meta-PAL together with the highly purified sGC leads to a covalent binding to the alpha1-subunit of the enzyme. This binding is blocked by unlabeled meta-PAL, YC-1 and BAY 41-2272. For further identification of the NO-independent regulatory site the 3H-meta-PAL labeled sGC was fragmented by CNBr digest. The 3H-meta-PAL binds to a CNBr fragment, consisting of the amino acids 236-290 of the alpha1-subunit. Determination of radioactivity of the single PTH-cycles from the sequencing of this CNBr fragment detected the cysteines 238 and 243 as binding residues of the 3H-meta-PAL.

Conclusions: Our data demonstrate that the region surrounding the cysteines 238 and 243 in the alpha1-subunit of the sGC could play an important role in regulation of sGC activity and could be the target of this new type of sGC stimulators.

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Stimulation of the crude sGC preparation from bovine lung by BAY 41-2272 in the absence and presence of DEA/NO (10 μM). The specific activity of sGC preparation is expressed as x-fold stimulation vs. basal activity (basal activity in the presence of Mg2+: 152 nmol/mg/min). The data presented represent means ± SEM, from 4 independent experiments performed in duplicate.
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Figure 10: Stimulation of the crude sGC preparation from bovine lung by BAY 41-2272 in the absence and presence of DEA/NO (10 μM). The specific activity of sGC preparation is expressed as x-fold stimulation vs. basal activity (basal activity in the presence of Mg2+: 152 nmol/mg/min). The data presented represent means ± SEM, from 4 independent experiments performed in duplicate.

Mentions: The putative target sequence of bovine sGC differs from rat and human by the substitution of an arginine at position 238 instead of cysteine. Therefore we investigated if BAY 41-2272 is active against the bovine enzyme or in isolated bovine vascular preparations. We prepared crude sGC containing fraction from bovine lung by performing the homogenization and ion-exchange steps of the method described earlier [11] with a specific basal activity of 0.15 nmol/mg/min. Using this preparation we examined the characteristic of sGC stimulation by BAY 41-2272 alone and in the presence of high concentrations of DEA/NO (Fig. 10). This study shows that BAY 41-2272 stimulates directly the crude sGC-containing fraction from bovine lung in a concentration dependent manner. In combination, BAY 41-2272 and the NO donor DEA/NO potentiates over a wide range of concentrations.


NO-independent regulatory site of direct sGC stimulators like YC-1 and BAY 41-2272.

Becker EM, Alonso-Alija C, Apeler H, Gerzer R, Minuth T, Pleiss U, Schmidt P, Schramm M, Schröder H, Schroeder W, Steinke W, Straub A, Stasch JP - BMC Pharmacol. (2001)

Stimulation of the crude sGC preparation from bovine lung by BAY 41-2272 in the absence and presence of DEA/NO (10 μM). The specific activity of sGC preparation is expressed as x-fold stimulation vs. basal activity (basal activity in the presence of Mg2+: 152 nmol/mg/min). The data presented represent means ± SEM, from 4 independent experiments performed in duplicate.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC64637&req=5

Figure 10: Stimulation of the crude sGC preparation from bovine lung by BAY 41-2272 in the absence and presence of DEA/NO (10 μM). The specific activity of sGC preparation is expressed as x-fold stimulation vs. basal activity (basal activity in the presence of Mg2+: 152 nmol/mg/min). The data presented represent means ± SEM, from 4 independent experiments performed in duplicate.
Mentions: The putative target sequence of bovine sGC differs from rat and human by the substitution of an arginine at position 238 instead of cysteine. Therefore we investigated if BAY 41-2272 is active against the bovine enzyme or in isolated bovine vascular preparations. We prepared crude sGC containing fraction from bovine lung by performing the homogenization and ion-exchange steps of the method described earlier [11] with a specific basal activity of 0.15 nmol/mg/min. Using this preparation we examined the characteristic of sGC stimulation by BAY 41-2272 alone and in the presence of high concentrations of DEA/NO (Fig. 10). This study shows that BAY 41-2272 stimulates directly the crude sGC-containing fraction from bovine lung in a concentration dependent manner. In combination, BAY 41-2272 and the NO donor DEA/NO potentiates over a wide range of concentrations.

Bottom Line: The synthesized photoaffinitylabel directly stimulates the purified sGC and shows in combination with NO a synergistic effect on sGC activity.Determination of radioactivity of the single PTH-cycles from the sequencing of this CNBr fragment detected the cysteines 238 and 243 as binding residues of the 3H-meta-PAL.Our data demonstrate that the region surrounding the cysteines 238 and 243 in the alpha1-subunit of the sGC could play an important role in regulation of sGC activity and could be the target of this new type of sGC stimulators.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pharma Research Center, Bayer AG, Wuppertal, Germany. eva.becker@gmx.net

ABSTRACT

Background: The most important receptor for nitric oxide is the soluble guanylate cyclase (sGC), a heme containing heterodimer. Recently, a pyrazolopyridine derivative BAY 41-2272, structurally related to YC-1, was identified stimulating soluble guanylate cyclase in an NO-independent manner, which results in vasodilatation and antiplatelet activity. The study described here addresses the identification of the NO-independent site on soluble guanylate cyclase.

Results: We developed a photoaffinity label (3H-meta-PAL) for the direct and NO-independent soluble guanylate cyclase (sGC) stimulator BAY 41-2272 by introducing an azido-group into the tritium labeled compound. The synthesized photoaffinitylabel directly stimulates the purified sGC and shows in combination with NO a synergistic effect on sGC activity. Irradiation with UV light of 3H-meta-PAL together with the highly purified sGC leads to a covalent binding to the alpha1-subunit of the enzyme. This binding is blocked by unlabeled meta-PAL, YC-1 and BAY 41-2272. For further identification of the NO-independent regulatory site the 3H-meta-PAL labeled sGC was fragmented by CNBr digest. The 3H-meta-PAL binds to a CNBr fragment, consisting of the amino acids 236-290 of the alpha1-subunit. Determination of radioactivity of the single PTH-cycles from the sequencing of this CNBr fragment detected the cysteines 238 and 243 as binding residues of the 3H-meta-PAL.

Conclusions: Our data demonstrate that the region surrounding the cysteines 238 and 243 in the alpha1-subunit of the sGC could play an important role in regulation of sGC activity and could be the target of this new type of sGC stimulators.

Show MeSH
Related in: MedlinePlus