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

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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.