Properties and affinity purification of the mixed-type putative acetylcholine receptor from wild and a mutant strain of house flies

Abstract

Binding of decamethonium to a soluble preparation from house fly head (either wild or a mutant strain) showed a single kind of binding with values for wild strain of K d = 0.095 μM and B max = 0.22 nmol/mg protein. The mutant had a four-fold greater affinity and a seven-fold lesser amount. The binding was blocked by both nicotinic and muscarinic drugs. The decamethonium binding migrated in sucrose gradients as a single peak, with sedimentation coefficient s 20,w = 12.5 S and therefore a molecular weight of 342 000. Purification by affinity chromatography was achieved with only partial loss of activity, and the purified material demonstrated a single band on analytical disc gel electrophoresis. Electrophoresis in sodium dodecyl sulphate gels showed two subunits of molecular weights 94 000 and 64 000. Both subunits had an isoelectric point of 4.8.