A subcellular fractionation scheme for locust muscle has been developed in attempts to isolate neuromuscular nerve terminals. A vesicular subfraction was isolated by differential and density gradient centrifugation procedures after gentle homogenisation of the tissue in locust saline/sucrose. Electron microscopic observations together with measurement of marker enzyme activities revealed this fraction to consist of a mixed population of membrane vesicles, including large membranous profiles containing smaller vesicles (0.05-0.08mum). Enzyme marker assays suggested that the vesicles were probably derived from several sources and there was evidence that these included plasma membrane and sarcoplasmic reticulum. The vesicle fraction contained occluded L-glutamate and showed uptake of L-glutamate, L-aspartate, D-aspartate, L-glutamine and L-alanine. The uptake was partially dependent on the presence of Na+ ions. It has been tentatively suggested, from preliminary data, that the fraction contained both a high and a low affinity uptake system for all the amino acids except L-alanine which showed only a high affinity component. The suggested high affinity uptake component was specific for L-glutamate and L- and D-aspartate and was susceptible to inhibition by glutamate and aspartate analogues which were not potent amino acid receptor agonists or antagonists. The data, by analogy with the amino acid uptake characteristics of mammalian synaptosomes, are in accordance with the suggestion that presynaptic nerve terminals may be present in the vesicle fraction. The results lend biochemical support to the hitherto predominantly electrophysiological evidence that L-glutamate is the excitatory transmitter at the insect neuromuscular junction and that L-aspartate may function as a neuromodulator at these synapses.
|Date of Award||1981|