ID OF ENDOGENOUS LIGAND FOR DIOXAN ACTIVATED RECEPTOR (AH RECEPTOR)

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。二恶英是一种主要的环境毒素，它与AH受体结合，激活一些基因，导致鱼类的毒性和生殖系统的一些变化。在正常情况下，ah受体的生物学作用尚不清楚。利用Ah受体应答报告基因系统和选择性提取技术，我们成功地从猪肺中溶解了该受体的内源配体。我们采用间歇硅胶吸附洗脱、大规模硅胶层析和三步高效液相层析的方法成功地纯化了该配体。这产生了一种在报告基因系统中保持转录活性的同质物质。我们在该配体上有低分辨率和高分辨率的质谱图以及紫外吸收光谱。高分辨质谱图给出了一个经验公式：C18H12N2O4。分子的硼氢化物还原破坏了它的生物活性和发色团。到目前为止，我们还没有确定还原产物的相对分子质量，但无论如何，即使有了这些信息，我们也无法确定配体的结构。很明显，核磁共振波谱将为我们提供缺失的信息，使我们能够识别这种备受追捧的物质。我们相信，在500 MHz仪器上对1微克样品进行核磁共振可以为我们提供缺失的信息。如果这失败了，我们相信，在750兆赫的仪器上运行一小段时间，肯定会提供我们所需的分辨率。一旦鉴定完成，我们将立即开始配体的化学合成，并开始确定其生物学作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Dioxin is a major environmental toxin that binds to the Ah receptor activating a number of genes, resulting in toxicity and a number of changes in the reproductive system of fish. The biological role of the Ah receptor under normal conditions remains unknown. Using an Ah receptor-responsive reporter gene system and a selective extraction technique, we have successfully solubilized the endogenous ligand for this receptor from porcine lung. We have successfully purified this ligand using batch silica gel absorption and elution, large scale silica gel chromatography, followed by three high performance liquid chromatographic steps. This produces a homogenous substance that retains transcriptional activity in the reporter gene system. We have both low and high resolution mass spectra on this ligand as well as an ultraviolet absorption spectra. The high resolution mass spectra has given us an empirical formula which is C18H12N2O4. Borohydride reduction of the mole cule destr oys its biological activity and its chromophore. So far we have not determined the molecular weight of the reduction product, but in any case even with this information we are unable to identify positively the structure of the ligand. It is very clear that nuclear magnetic resonance spectroscopy would provide us the missing information, allowing identification of this much sought-after substance. We believe NMR on a 1 microgram sample in the 500 MHz instrument would provide us the missing information. If this fails, we believe that a short run on the 750 MHz instrument would certainly provide the resolution we need. Once identification has been achieved, we will immediately begin chemical synthesis of the ligand and begin to determine its biological role.