Novel Neonicotinoid-Agarose Affinity Column for Drosophila and Musca Nicotinic Acetylcholine Receptors |
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Authors: | Motohiro Tomizawa Bachir Latli John E Casida |
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Institution: | Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, University of California, Berkeley, California, U.S.A. |
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Abstract: | 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 K D values of 1–2 n M and B max values of 560–850 fmol/mg of protein. Locusta and Periplaneta nAChRs isolated with an α-bungarotoxin (α-BGT)-agarose affinity column are known to be α-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 n M ) 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-polyacrylamide 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 α-BGT-agarose affinity column. Photoaffinity labeling of the Drosophila and Musca putative subunits from the neonicotinoid column with 125I-α-BGT-4-azidosalicylic acid gives a labeled derivative of 66–69 kDa. The yield is 2–5 µg 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. |
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Keywords: | Affinity chromatography Drosophila Musca Nicotinic acetylcholine receptor Novel affinity column |
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