Lead- and calcium-mediated inhibition of bovine rod cGMP phosphodiesterase: interactions with magnesium.
Lead- and calcium-mediated inhibition of bovine rod cGMP phosphodiesterase: interactions with magnesium.
复制标题
铅和钙介导的牛杆 cGMP 磷酸二酯酶抑制：与镁的相互作用。
DOI：
10.1006/taap.1995.1167
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Fox,DA
中科院分区：
文献类型：
--
作者：
Srivastava,D;Hurwitz,RL;Fox,DA
以前，我们表明，cGMP水解大鼠全视网膜匀浆表现出剂量依赖性抑制发展铅暴露和浓度依赖性抑制与直接铅暴露。此外，发育铅暴露导致视网膜cGMP和视杆细胞Ca 2+水平呈剂量依赖性增加。为了确定Pb 2+或Ca 2+是否直接抑制视杆特异性cGMP磷酸二酯酶（PDE）并检查这种抑制的动力学机制，在不同浓度的cGMP和Mg 2+、Pb 2+和/或Ca 2+存在下测定纯化的牛视杆cGMP PDE。增加底物cGMP的浓度导致Pb 2+和Ca 2+浓度-响应曲线向左移动，表明Pb 2+的半最大抑制浓度从纳摩尔降低到皮摩尔水平。增加浓度的辅因子，Mg 2+，导致移动的Pb 2+和Ca 2+的浓度-响应曲线的权利，表明减少的抑制PDE活性的Pb 2+或Ca 2+。1/速度与1/Mg 2+作为Pb 2+的函数的图显示，相对于毫摩尔浓度的Mg 2+，皮摩尔浓度的Pb 2+竞争性抑制PDE。与这一发现一致，Mg 2+逆转了Pb 2+诱导的PDE抑制。我们最近的动力学分析表明，Mg 2+和cGMP结合在PDE上的相互作用位点以随机顺序。结果表明，Pb ~（2+）与cGMP的结合位点相同，但亲和力比Mg ~（2+）高4-6个对数单位，从而阻止cGMP的水解。这些发现为理解Pb 2+诱导的cGMP PDE抑制提供了一种新的机制。这些结果可能对使用Mg 2+作为辅因子的其他酶具有影响，并且表明Mg 2+可能在这些情况下用于逆转Pb 2+的抑制。
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+.