Tomizawa M, Latli B, Casida J E
Department of Environmental Science, Policy and Management, University of California, Berkeley 94720-3112, USA.
J Neurochem. 1996 Oct;67(4):1669-76. doi: 10.1046/j.1471-4159.1996.67041669.x.
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.
新烟碱类杀虫剂如吡虫啉（IMI）可作为昆虫烟碱型乙酰胆碱受体（nAChR）的激动剂。黑腹果蝇和家蝇的头部膜对[3H]IMI有一个单一的高亲和力结合位点，解离常数（KD）值为1 - 2 nM，最大结合量（Bmax）值为560 - 850 fmol/mg蛋白质。已知用α-银环蛇毒素（α-BGT）-琼脂糖亲和柱分离的飞蝗和蜚蠊nAChR是α亚基同聚体。本研究使用1-[N-（6-氯-3-吡啶甲基）-N-乙基]氨基-1-氨基-2-硝基乙烯++ +（其在2 - 3 nM时抑制[3H]IMI与果蝇和家蝇头部膜的结合）来制备新烟碱类-琼脂糖亲和柱。该方法是将用曲拉通溶解的果蝇或家蝇头部膜引入基于新烟碱类的柱中，用IMI洗脱，然后通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳进行分析，结果仅得到三种蛋白质（69、,66和61 kDa），暂定为nAChR的假定亚基；使用α-BGT-琼脂糖亲和柱从家蝇中也得到了相同的三种蛋白质。用125I-α-BGT-4-叠氮基水杨酸对来自新烟碱类柱的果蝇和家蝇假定亚基进行光亲和标记，得到一种66 - 69 kDa的标记衍生物。从1 g果蝇或家蝇头部可获得2 - 5微克的受体蛋白。用新烟碱类亲和色谱法分离天然果蝇和家蝇受体将有助于对昆虫nAChR的结构和功能进行研究。
