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 Pb(2+)-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 Pb(2+)-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+.
Srivastava
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College of Optometry, University of Houston, Texas 77204-6052, USA.
Hurwitz
R L
RL
Fox
D A
DA
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002417:Cattle; D006152:Cyclic GMP / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D007854:Lead / Q000494:pharmacology*; D008274:Magnesium / Q000494:pharmacology*; D017948:Retinal Rod Photoreceptor Cells / Q000187:drug effects* / Q000201:enzymology
D007854:Lead; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D006152:Cyclic GMP; D008274:Magnesium; D002118:Calcium