1. A membranous sub-fraction has been prepared from locust (Schistocerca gregaria) muscle. The fraction shows some enrichment in the specific binding of L-glutamate compared with the total homogenate of the muscle. Electron microscopy and enzyme distribution studies suggest that the fraction is composed mainly of membrane and disrupted mitochondria. Two methods have been used to measure the specific binding of L-glutamate based on rapid filtration through Whatman GF/C filters and a rapid centrifugation procedure using a Beckman Airfuge. The latter method gave better reproducibility within any one study but both assay methods showed great variation between preparations. There appears to be no metabolism of L-glutamate by the membrane fraction during the time over which the binding studies were made. Preliminary experiments with three marine toxins - suberitine and alpha and beta cephalotoxin suggest that these agents may prove to be useful in elucidating the nature of the glutamate binding sites on the muscle membranes. The variability of the binding activities of the muscle membranes has so far precluded any unequivocal designation of the L-glutamate binding sites as synaptic receptors. 2. The specific binding of [125I]-alpha-bungarotoxin to a membrane fraction of locust (Schistocerca gregaria) supra-oesophageal ganglia showed the following characteristics:- a) binding increased linearly with protein b) binding saturated at concentrations of [125I]-alpha-bungarotoxin greater than 2 nM c) bound [125I]-alpha-bungarotoxin dissociated in a biphasic manner d) binding was preferentially inhibited by nicotinic ligands. When the membrane fraction was treated with the co-valent affinity label [3H]-MBTA and subjected to SDS-PAGE most of the radioactivity was recovered as a single peak, corresponding to an Mr of 58,000. The binding characteristics (KD and Bmax) of the membrane fraction did not change significantly on solubilisation in the non-ionic detergent Triton X-100. Up to a 1000 fold purification of the alpha-bungarotoxin binding component was achieved after affinity chromatography on immobilised alpha-bungarotoxin. The of the partially purified binding component was unchanged relative to the membrane fraction, when measured from equilibrium binding studies but determination of the k-1 showed dissociation of [125I]-alpha-bungarotoxin to be monophasic. The partially purified alpha-bungarotoxin binding component sedimented as a single component after centrifugation in a sucrose density gradient (14.2s; Mr 400,000 - 450,000) and SDS PAGE showed two major protein bands of Mr 's 60,000 and 41,000. No acetylcholine esterase activity was detected in this partially purified binding component fraction.
|Date of Award||1981|