Lead- and calcium-mediated inhibition of bovine rod cGMP phosphodiesterase: Interactions with magnesium

Previously we showed that cGMP hydrolysis in rat whole retinal homogenates exhibited a dose-dependent inhibition following developmental lead exposure and a concentration-dependent inhibition with direct Pb²⁺ exposure. Additionally, developmental lead exposure resulted in a dose-dependent increase in retinal cGMP and rod Ca²⁺ levels. To determine whether Pb²⁺ or Ca²⁺ directly inhibited the rod-specific cGMP phosphodiesterase (PDE) and to examine the kinetic mechanism of this inhibition, purified bovine rod cGMP PDE was assayed in the presence of varying concentrations of cGMP, and Mg²⁺, Pb²⁺, and/or Ca²⁺. Increasing concentrations of the substrate, cGMP, resulted in a shift of the Pb²⁺ and Ca²⁺ concentration-response curves to the left, indicating a decrease in the half-maximal inhibitory concentrations of Pb²⁺ from nanomolar to picomolar levels. Increasing concentrations of the cofactor, Mg²⁺, resulted in a shift of the Pb²⁺ and Ca²⁺ concentration-response curves to the right, indicating a decrease in the inhibition of PDE activity by Pb²⁺ or Ca²⁺. A plot of 1/velocity vs 1/Mg²⁺ as a function of Pb²⁺ revealed that picomolar concentrations of Pb²⁺ competitively inhibited PDE relative to millimolar concentrations of Mg²⁺. Consistent with this finding, Mg²⁺ reversed the Pb²⁺-induced inhibition of PDE. Our recent kinetic analysis showed that Mg²⁺ and cGMP bind at interacting sites on the PDE in a random order. The present results reveal that Pb²⁺ may bind at the same site but with 4-6 log units higher affinity than Mg²⁺, thus preventing the hydrolysis of cGMP. These findings provide a novel mechanism for understanding the Pb²⁺-induced inhibition of cGMP PDE. These results may have implications for other enzymes using Mg²⁺ as a cofactor and suggest that Mg²⁺ may be useful in these situations for reversing the inhibition by Pb²⁺.