Oxidative modulation of soluble guanylate cyclase by manganese.
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.
D000818:Animals; D006162:Guanylate Cyclase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology; D008345:Manganese / Q000378:metabolism* / Q000494:pharmacology; D010084:Oxidation-Reduction; D051381:Rats
D008345:Manganese; D006162:Guanylate Cyclase