Probing the presence of the ligand-binding haem in cellular nitric oxide receptors
Probing the presence of the ligand-binding haem in cellular nitric oxide receptors
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DOI：
10.1038/sj.bjp.0707687
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发表时间：
2008-04-01
影响因子：
7.3
通讯作者：
Garthwaite, J.
中科院分区：
文献类型：
--
作者：
Roy, B.;Mo, E.;Garthwaite, J.
背景与目的：一氧化氮(NO)作用于与鸟苷酸环化酶(GC)偶联的受体，导致cGMP积聚。NO结合部位是一个血红素基团，氧化或失去它会减弱NO刺激的活性。据报道，使用这两种非不敏感形式的激动剂已经出现。在这里，我们表征了一个原型化合物(BAY 58-2667)的作用，并用它来评估细胞GC的血红素状态。实验方法：对纯化的蛋白和大鼠血小板进行GC活性测量。关键结果：对纯化的GC进行的实验表明，BAY 58-2667的靶标是无血型的GC，而不是血氧化形式的GC。BAY 58-2667的药效约为NO的一半。在血小板方面，BAY 58-2667是一种有效的GC激活剂(EC(50)类似于15 nM)，但最大作用仅为NO的1%左右。然而，它足以引起cGMP依赖的蛋白质磷酸化。对非诱发GC活性的深刻(85%)脱敏并不改变Bay 58-2667的有效性。然而，血红素氧化使Bay 58-2667的效果提高了22倍，这意味着大约一半的细胞GC是不含血红素的。氧化似乎提高了血红素从纯化的ITC中解离的速度。结论和启示：像Bay 58-2667这样的化合物有助于探测细胞中NO受体在血红素口袋中的占据情况，但不能区分氧化和还原的血红蛋白。在体内，这种化合物可能在血红素结合不足或增加血红素丢失的条件下特别有效。
Background and purpose: Nitric oxide ( NO) acts on receptors coupled to guanylyl cyclase ( GC), leading to cGMP accumulation. The NO binding site is a haem group, oxidation or loss of which diminishes NO-stimulated activity. Agonists reportedly engaging both these NO-insensitive forms have emerged. Here we characterize the effect of a prototype compound ( BAY 58-2667) and use it to assess the haem status of cellular GC.Experimental approach: GC activity measurements were made on the purified protein and on rat platelets.Key results: Experiments on purified GC showed that the target for BAY 58-2667 is the haem-free GC, not the haem-oxidized form. The efficacy of BAY 58-2667 was about half that shown normally by NO. In platelets, BAY 58-2667 was a potent GC activator ( EC(50)similar to 15 nM) but the maximum effect was only about 1% of that achievable with NO. Nevertheless, it was enough to evoke cGMP-dependent protein phosphorylation. Profound ( 85 %) desensitization of NO-evoked GC activity did not alter the effectiveness of BAY 58-2667. Haem oxidation, however, increased the efficacy of BAY 58-2667 by 22-fold, implying that about half the cellular GC was then haem-free. Oxidation appeared to enhance the rate of haem dissociation from purified GC.Conclusions and implications: Compounds such as BAY 58-2667 are useful for probing the occupancy of the haem pocket of NO receptors in cells but not for distinguishing oxidized from reduced haem. In vivo, such compounds are likely to be particularly effective in conditions where there is deficient haem incorporation or enhanced haem loss.