TY - JOUR
T1 - Inhibition of purified soluble guanylyl cyclase by copper ions
AU - Schrammel, Astrid
AU - Koesling, Doris
AU - Gorren, Antonius C.F.
AU - Chevion, Mordechai
AU - Schmidt, Kurt
AU - Mayer, Bernd
PY - 1996/10/11
Y1 - 1996/10/11
N2 - The aim of the present study was to investigate the effect of Cu(II) ions on soluble guanylyl cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2; sGC] and to test for a possible physiological role of this putative cofactor of the enzyme [Gerzer et al., FEBS Lett. 132: 71-74, 1981]. CuSO4 was found to inhibit NO-stimulated sGC with an IC50 of 2.2 ± 0.3 μM. Virtually complete inhibition of guanosine-3',5'-cyclic monophosphate (cGMP) formation was observed at 10 μM of the copper salt. Presence of CuSO4 (2 μM) did not significantly affect the potency of 2,2-diethyl-1-nitroso-oxyhydrazine (DEA/NO) but did markedly decrease maximal cyclase activity from 3.71 ± 0.2 μmol cGMP x mg-1 x min-1 to 1.75 ± 0.2 μmol cGMP x mg-1 x min-1. The nonstimulated enzyme was also sensitive to CuSO4 (IC50 of 6.2 ± 1.2 μM). Addition of glutathione, which potently complexes Cu(I) ions, induced a pronounced rightward shift of the concentration-response curves for inhibition by CuSO4 of both DEA/NO-stimulated and nonstimulated guanylyl cyclase. The inhibitory effect of CuSO4 was completely antagonized by the specific Cu(I) chelator neocuproine, with a half-maximal effect at 5.9 ± 0.2 μM. In contrast, the Cu(II) chelator cuprizone and several thiols, which do not form stable Cu(I) complexes, were far less protective. Our results suggest that inhibition of soluble guanylyl cyclase by CuSO4 is unrelated to heme-mediated enzyme stimulation and may arise from the reversible high affinity binding of Cu(I) ions to a site of the protein that is critically involved in enzyme catalysis.
AB - The aim of the present study was to investigate the effect of Cu(II) ions on soluble guanylyl cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2; sGC] and to test for a possible physiological role of this putative cofactor of the enzyme [Gerzer et al., FEBS Lett. 132: 71-74, 1981]. CuSO4 was found to inhibit NO-stimulated sGC with an IC50 of 2.2 ± 0.3 μM. Virtually complete inhibition of guanosine-3',5'-cyclic monophosphate (cGMP) formation was observed at 10 μM of the copper salt. Presence of CuSO4 (2 μM) did not significantly affect the potency of 2,2-diethyl-1-nitroso-oxyhydrazine (DEA/NO) but did markedly decrease maximal cyclase activity from 3.71 ± 0.2 μmol cGMP x mg-1 x min-1 to 1.75 ± 0.2 μmol cGMP x mg-1 x min-1. The nonstimulated enzyme was also sensitive to CuSO4 (IC50 of 6.2 ± 1.2 μM). Addition of glutathione, which potently complexes Cu(I) ions, induced a pronounced rightward shift of the concentration-response curves for inhibition by CuSO4 of both DEA/NO-stimulated and nonstimulated guanylyl cyclase. The inhibitory effect of CuSO4 was completely antagonized by the specific Cu(I) chelator neocuproine, with a half-maximal effect at 5.9 ± 0.2 μM. In contrast, the Cu(II) chelator cuprizone and several thiols, which do not form stable Cu(I) complexes, were far less protective. Our results suggest that inhibition of soluble guanylyl cyclase by CuSO4 is unrelated to heme-mediated enzyme stimulation and may arise from the reversible high affinity binding of Cu(I) ions to a site of the protein that is critically involved in enzyme catalysis.
KW - Cu(I) ions
KW - CuSO-induced enzyme inhibition
KW - cuprizone
KW - glutathione
KW - neocuproine
KW - soluble guanylyl cyclase
UR - http://www.scopus.com/inward/record.url?scp=0030580091&partnerID=8YFLogxK
U2 - 10.1016/0006-2952(96)00425-X
DO - 10.1016/0006-2952(96)00425-X
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C2 - 8831723
AN - SCOPUS:0030580091
SN - 0006-2952
VL - 52
SP - 1041
EP - 1045
JO - Biochemical Pharmacology
JF - Biochemical Pharmacology
IS - 7
ER -