LEAD-MEDIATED AND CALCIUM-MEDIATED INHIBITION OF BOVINE ROD CGMP PHOSPHODIESTERASE - INTERACTIONS WITH MAGNESIUM

Previously we showed that cGMP hydrolysis in rat whole retinal homogen ates exhibited a dose-dependent inhibition following developmental lea d exposure and a concentration-dependent inhibition with direct Pb2+ e xposure. Additionally, developmental lead exposure resulted in a dose- dependent increase in retinal cGMP and rod Ca2+ levels. To determine w hether Pb2+ or Ca2+ directly inhibited the rod-specific cGMP phosphodi esterase (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 concen trations of the substrate, cGMP, resulted in a shift of the Pb2+ and C a2+ concentration-response curves to the left, indicating a decrease i n the half-maximal inhibitory concentrations of Pb2+ from nanomolar to picomolar levels. Increasing concentrations of the cofactor, Mg2+, re sulted in a shift of the Pb2+ and Ca2+ concentration-response curves t o the right, indicating a decrease in the inhibition of PDE activity b y Pb2+ or Ca2+. A plot of 1/velocity vs 1/Mg2+ as a function of Pb2+ r evealed that picomolar concentrations of Pb2+ competitively inhibited PDE relative to millimolar concentrations of Mg2+. Consistent with thi s finding, Mg2+ reversed the Pb2+-induced inhibition of PDE. Our recen t kinetic analysis showed that Mg2+ and cGMP bind at interacting sites on the PDE in a random order. The present results reveal that Pb2+ ma y bind at the same site but with 4-6 log units higher affinity than Mg 2+, thus preventing the hydrolysis of cGMP. These findings provide a n ovel mechanism for understanding the Pb2+-induced inhibition of cGMP P DE. These results may have implications for other enzymes using Mg2+ a s a cofactor and suggest that Mg2+ may be useful in these situations f or reversing the inhibition by Pb2+. (C) 1995 Academic Press, Inc.