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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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Coomassie-blue stained Western Blot and autoradiogram of photoaffinity labeled sGC fragments after CNBr digest, SDS-PAGE on a TRIS-Tricin-gel (16.5%) (48 h exposition on Imaging-Plate screened by a folio of contamination monitors). Shown are a 14C-labeled rainbow molecular weightmarker (14C-M), further molecular weightmarkers, and photoaffinity labeled sGC (200 μg) after CNBr digest (sGC CNBr). The arrows show the molecular weightmarkers with their molecular weights (kDa) or the 3H-meta-PAL labeled sGC fragments (CNBr VI-VIII) with their molecular weights, as calculated after sequencing.
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Figure 7: Coomassie-blue stained Western Blot and autoradiogram of photoaffinity labeled sGC fragments after CNBr digest, SDS-PAGE on a TRIS-Tricin-gel (16.5%) (48 h exposition on Imaging-Plate screened by a folio of contamination monitors). Shown are a 14C-labeled rainbow molecular weightmarker (14C-M), further molecular weightmarkers, and photoaffinity labeled sGC (200 μg) after CNBr digest (sGC CNBr). The arrows show the molecular weightmarkers with their molecular weights (kDa) or the 3H-meta-PAL labeled sGC fragments (CNBr VI-VIII) with their molecular weights, as calculated after sequencing.

Mentions: To identify the binding residues of the 3H-meta-PAL at the α1-subunit, sGC was labeled with 3H-meta-PAL and the labeled protein was fragmented by CNBr cleavage. The resulting protein fragments were separated by electrophoresis (10–20% SDS-PAGE), transferred to a PVDF membrane, stained with Coomassie-blue and exposed to Imaging-Plates (Fig. 6). The resulted autoradiogramm shows five highly labeled protein fragments (CNBr I-V) with molecular weights of 51.8 / 35.9 / 30.2 / 11.7 and 6.2 kDa (Fig. 6) which were identified and excised from the Coomassie-blue stained Western blots. Sequencing of this bands showed, that they contain different fragments of both the α1- as the β1-subunit (Tab. 4). For better separation of peptides in the lower molecular weight range, the same fragments were separated on a TRIS-Tricine-gel. In this autoradiogramm we detected three highly labeled protein fragments (CNBr VI-VIII) with molecular weights of 14.7 / 11.7 and 6.2 kDa (Fig. 7). Before sequencing of the different labeled sGC fragments, both intact subunits of sGC were sequenced. We observed, that the first 20 coded amino acids of the α1-subunit probably belong to the presequence. The β1-subunit corresponded to the published sequence. Because of the selective labeling of the α1-subunit by the 3H-meta-PAL (Fig. 4 and 5), in Tab. 3 only the theoretically CNBr fragments of the α1-subunit (AS 20-690) are aligned and signed after their position. It is known that CNBr fragments show different migration properties in SDS-PAGE related to the used molecular weightmarkers. Therefore in the figures we used the calculated molecular weights from sequencing to mark the labeled CNBr fragments. The sequenced bands are marked in Fig. 6 and 7. The results of the sequencing of the different fragments are shown in Tab. 4. The overview of the determined α1-sequences shows, that the 3H-meta-PAL specifically binds to the CNBr VIII = CNBr-2 fragment of the α1-subunit (Tab. 3 and 4) with the amino acids 236–290.


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)

Coomassie-blue stained Western Blot and autoradiogram of photoaffinity labeled sGC fragments after CNBr digest, SDS-PAGE on a TRIS-Tricin-gel (16.5%) (48 h exposition on Imaging-Plate screened by a folio of contamination monitors). Shown are a 14C-labeled rainbow molecular weightmarker (14C-M), further molecular weightmarkers, and photoaffinity labeled sGC (200 μg) after CNBr digest (sGC CNBr). The arrows show the molecular weightmarkers with their molecular weights (kDa) or the 3H-meta-PAL labeled sGC fragments (CNBr VI-VIII) with their molecular weights, as calculated after sequencing.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC64637&req=5

Figure 7: Coomassie-blue stained Western Blot and autoradiogram of photoaffinity labeled sGC fragments after CNBr digest, SDS-PAGE on a TRIS-Tricin-gel (16.5%) (48 h exposition on Imaging-Plate screened by a folio of contamination monitors). Shown are a 14C-labeled rainbow molecular weightmarker (14C-M), further molecular weightmarkers, and photoaffinity labeled sGC (200 μg) after CNBr digest (sGC CNBr). The arrows show the molecular weightmarkers with their molecular weights (kDa) or the 3H-meta-PAL labeled sGC fragments (CNBr VI-VIII) with their molecular weights, as calculated after sequencing.
Mentions: To identify the binding residues of the 3H-meta-PAL at the α1-subunit, sGC was labeled with 3H-meta-PAL and the labeled protein was fragmented by CNBr cleavage. The resulting protein fragments were separated by electrophoresis (10–20% SDS-PAGE), transferred to a PVDF membrane, stained with Coomassie-blue and exposed to Imaging-Plates (Fig. 6). The resulted autoradiogramm shows five highly labeled protein fragments (CNBr I-V) with molecular weights of 51.8 / 35.9 / 30.2 / 11.7 and 6.2 kDa (Fig. 6) which were identified and excised from the Coomassie-blue stained Western blots. Sequencing of this bands showed, that they contain different fragments of both the α1- as the β1-subunit (Tab. 4). For better separation of peptides in the lower molecular weight range, the same fragments were separated on a TRIS-Tricine-gel. In this autoradiogramm we detected three highly labeled protein fragments (CNBr VI-VIII) with molecular weights of 14.7 / 11.7 and 6.2 kDa (Fig. 7). Before sequencing of the different labeled sGC fragments, both intact subunits of sGC were sequenced. We observed, that the first 20 coded amino acids of the α1-subunit probably belong to the presequence. The β1-subunit corresponded to the published sequence. Because of the selective labeling of the α1-subunit by the 3H-meta-PAL (Fig. 4 and 5), in Tab. 3 only the theoretically CNBr fragments of the α1-subunit (AS 20-690) are aligned and signed after their position. It is known that CNBr fragments show different migration properties in SDS-PAGE related to the used molecular weightmarkers. Therefore in the figures we used the calculated molecular weights from sequencing to mark the labeled CNBr fragments. The sequenced bands are marked in Fig. 6 and 7. The results of the sequencing of the different fragments are shown in Tab. 4. The overview of the determined α1-sequences shows, that the 3H-meta-PAL specifically binds to the CNBr VIII = CNBr-2 fragment of the α1-subunit (Tab. 3 and 4) with the amino acids 236–290.

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