Oxidative modulation of soluble guanylate cyclase by manganese

Abstract

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 Mn 2+ in excess of Mn 2+-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 Mg 2+ was the sole cation cofactor but were dependent upon Mn 2+ concentrations in excess of Mn 2+-GTP. The effects of excess Mn 2+ on guanylate cyclase varied with assay pH and buffer composition. At pH 7.6 in Tris-HCl buffer, excess Mn 2+ increased guanylate cyclase activity with an apparent K a of 0.25 mM and concentrations above 3 mM were slightly inhibitory. At pH 9.0 in Tris-HCl buffer, however, concentrations of excess Mn 2+ above 0.1 mM were strongly inhibitory. By comparison, in cacodylate (pH 7.6) or glycine (pH 9.0) buffers, high concentrations of excess Mn 2+ were considerably less inhibitory and the apparent K a values for excess Mn 2+ were greater than in Tris-HCl buffer at equivalent pH. The variable effects of Mn 2+ 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 Mn 2+ 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 Mn 2+ as cation cofactor, as well as the inhibition of enzyme activity by excess Mn 2+ may be mediated by oxidative events associated with changes in the oxidation state of the free cation.