title: Novel neonicotinoid-agarose affinity column for Drosophila and Musca nicotinic acetylcholine receptors.
abstract: Neonicotinoids such as the insecticide imidacloprid (IMI) act as agonists at the insect nicotinic acetylcholine receptor (nAChR). Head membranes of Drosophila melanogaster and Musca domestica have a single high-affinity binding site for [3H]IMI with KD values of 1-2 nM and Bmax values of 560-850 fmol/mg of protein. Locusta and Periplaneta nAChRs isolated with an alpha-bungarotoxin (alpha-BGT)-agarose affinity column are known to be alpha-subunit homooligomers. This study uses 1-[N-(6-chloro-3-pyridylmethyl)-N-ethyl]amino-1-amino-2-nitroethene++ + (which inhibits [3H]IMI binding to Drosophila and Musca head membranes at 2-3 nM) to develop a neonicotinoid-agarose affinity column. The procedure-introduction of Triton-solubilized Drosophila or Musca head membranes into this neonicotinoid-based column, elution with IMI, and analysis by lithium dodecyl sulfate-polyacrylamicle gel electrophoresis-gives only three proteins (69, 66, and 61 kDa) tentatively assigned as putative subunits of the nAChR; the same three proteins are obtained with Musca using the alpha-BGT-agarose affinity column. Photoaffinity labeling of the Drosophila and Musca putative subunits from the neonicotinoid column with 125I-alpha-BGT-4-azidosalicylic acid gives a labeled derivative of 66-69 kDa. The yield is 2-5 micrograms of receptor protein from 1 g of Drosophila or Musca heads. Neonicotinoid affinity chromatography to isolate native Drosophila and Musca receptors will facilitate studies on the structure and function of insect nAChRs.
authors.affiliation: Department of Environmental Science, Policy and Management, University of California, Berkeley 94720-3112, USA.
authors.forename: M
authors.initials: M
authors.lastname: Tomizawa
authors.forename: B
authors.initials: B
authors.lastname: Latli
authors.forename: J E
authors.initials: JE
authors.lastname: Casida
mesh_terms: D000818:Animals; D002038:Bungarotoxins; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D004331:Drosophila melanogaster; D006257:Head; D006793:Houseflies; D007093:Imidazoles / Q000378:metabolism*; D007306:Insecticides / Q000378:metabolism*; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D000073943:Neonicotinoids; D009574:Nitro Compounds; D011869:Radioligand Assay; D011978:Receptors, Nicotinic / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D002038:Bungarotoxins; D007093:Imidazoles; D007306:Insecticides; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D000073943:Neonicotinoids; D009574:Nitro Compounds; D011978:Receptors, Nicotinic; C082359:imidacloprid