Receptor site, purification

As pointed out above, the bioassay design depends on the objective(s) of the study. A bioassay to determine allelopathic interactions in the field or in an ecological setting may have a quite different design than one used to determine PGR activity of a compound or to determine its molecular mode of action. Specific bioassays can be used to follow the isolation/purification of allelochemicals, evaluate their phytotoxic (or growth simulation) effects (i.e., visual effects), determine their host range/selectivity, evaluate allelopathic action of volatile compounds, or examine physiological/biochemical effects, such as photodynamic and membrane effects, effects on photosynthesis, specific enzyme sites, and effects at the ultrastructural level to locate receptor sites or sites of injury. Several examples of useful bioassays will be presented later. 

Enzyme purification Binding proteins and receptor sites Insolubilized nucleotides and nucleic acids Insolubilized lectins Immunochemical applications... 

Simon EJ, Gioannini TL. Opioid receptor multiplicity isolation, purification and chemical characterization of binding sites. In Opioids I. Handbook of Experimental Pharmacology. Vol. 104 (Herz A, ed). Springer, Heidelberg, 1993 3-26. 

In another example, ligands can be biotinylated with a cleavable biotinylation reagent and then incubated with receptor molecules. The resulting complex can be isolated by affinity chromatography on immobilized (strept)avidin. Final purification of the ligand-receptor can be accomplished by cleaving the biotin modification sites while the complex is still bound to the support. The receptor complex thus can be eluted from the column without the usual harsh conditions required to break the avidin-biotin interaction. 

In this way n.c.a. [18F]FUB 272 was obtained (no specific activity mentioned) after 146 minutes in a 65% yield (corrected for decay) after a triple HPLC purification, due to impurities with a retention time very close to the desired product. Biodistribution studies showed that brain uptake was again too low for use in PET. The study also showed an uptake into the cerebellum, where no H3 receptors are present, so [18F]FUB 272 also binds to a non H3 receptor binding site in the brain. 

Simon EJ, Hiller JM. Solubilization and Purification of opioid binding sites. In Pasternak GW, ed. The Opiate Receptors. Clifton, NJ Humana Press, 1988 165-194. 

---