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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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Autoradiogram of 3H-meta-PAL labeled sGC after separation by SDS-PAGE. Lane1: 12.5 μg sGC with 3H-meta-PAL without irradiation; Lane 2: 12.5 μg sGC with 3H-meta-PAL after irradiation; Lane 3: as 2 but in the presence of 100 μM unlabeled meta-PAL; Lane 4: as 2 but in the presence of 10 μM unlabeled meta-PAL; Lane 5: as 2 but in the presence of 100 μM ODQ; Lane 6: as 2 but in the presence of 100 μM YC-1.
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Figure 5: Autoradiogram of 3H-meta-PAL labeled sGC after separation by SDS-PAGE. Lane1: 12.5 μg sGC with 3H-meta-PAL without irradiation; Lane 2: 12.5 μg sGC with 3H-meta-PAL after irradiation; Lane 3: as 2 but in the presence of 100 μM unlabeled meta-PAL; Lane 4: as 2 but in the presence of 10 μM unlabeled meta-PAL; Lane 5: as 2 but in the presence of 100 μM ODQ; Lane 6: as 2 but in the presence of 100 μM YC-1.

Mentions: In addition, we examined the effects of different sGC modulators on binding of 3H-meta-PAL on purified sGC (Fig. 5). Both unlabeled meta-PAL (100 μM and 10 μM), YC-1 (100 μM), BAY 41-2272 [22] but also ODQ (100 μM) diminished concentration-dependently the binding of 3H-meta-PAL to the α1-subunit. ODQ inhibit the binding of the 3H-meta-PAL to the α1-subunit, but in this case weak unspecific labeling of the β1-subunit and the α1-subunit was detected. 3H-meta-PAL processed with sGC without irradiation showed no insertion of radioactivity (Fig. 5).


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)

Autoradiogram of 3H-meta-PAL labeled sGC after separation by SDS-PAGE. Lane1: 12.5 μg sGC with 3H-meta-PAL without irradiation; Lane 2: 12.5 μg sGC with 3H-meta-PAL after irradiation; Lane 3: as 2 but in the presence of 100 μM unlabeled meta-PAL; Lane 4: as 2 but in the presence of 10 μM unlabeled meta-PAL; Lane 5: as 2 but in the presence of 100 μM ODQ; Lane 6: as 2 but in the presence of 100 μM YC-1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC64637&req=5

Figure 5: Autoradiogram of 3H-meta-PAL labeled sGC after separation by SDS-PAGE. Lane1: 12.5 μg sGC with 3H-meta-PAL without irradiation; Lane 2: 12.5 μg sGC with 3H-meta-PAL after irradiation; Lane 3: as 2 but in the presence of 100 μM unlabeled meta-PAL; Lane 4: as 2 but in the presence of 10 μM unlabeled meta-PAL; Lane 5: as 2 but in the presence of 100 μM ODQ; Lane 6: as 2 but in the presence of 100 μM YC-1.
Mentions: In addition, we examined the effects of different sGC modulators on binding of 3H-meta-PAL on purified sGC (Fig. 5). Both unlabeled meta-PAL (100 μM and 10 μM), YC-1 (100 μM), BAY 41-2272 [22] but also ODQ (100 μM) diminished concentration-dependently the binding of 3H-meta-PAL to the α1-subunit. ODQ inhibit the binding of the 3H-meta-PAL to the α1-subunit, but in this case weak unspecific labeling of the β1-subunit and the α1-subunit was detected. 3H-meta-PAL processed with sGC without irradiation showed no insertion of radioactivity (Fig. 5).

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