The objective of this research is to understand the mechanism by which glutamate is released from nerve endings in a calcium-dependent manner, in particular to investigate a proteinaceous cytosolic factor required for optimal release. Glutamate is widely recognized as the strongest candidate for the major excitatory neurotransmitter in the central nervous system. There is now good evidence that glutamate is accumulated into synaptic vesicles in an energy-dependent manner, supporting the neurotransmitter role. This uptake system is highly specific for glutamate and is considered to play an important role in glutamate synaptic transmission. However, the mechanism underlying glutamate release is not well understood. Recently, we have devised an approach to studying glutamate release in a cell-free system and obtained evidence that vesicular glutamate is released in a calcium-dependent manner from permeabilized synaptosomes. Our evidence indicates that a cytosolic-proteinaceous substance is required for optimal release. In this application, it is proposed to (a) purify the stimulatory (or obligatory) proteinaceous factor by classical chromatography and high pressure liquid chromatography; (b) characterize the glutamate release from permeabilized synaptosomes with respect to (i) sensitivity to the factor, (ii) sensitivity to calcium, (iii) requirement for the plasma membrane, and (iv) metal ion specificity; (c) determine physicochemical properties of the purified factor by SDS-polyacrylamide gel electrophoresis, gel filtration, sucrose determine the site of action of the factor by examining the interaction of the factor with calcium and specific proteins in the permeabilized synaptosomes; and (e) determine whether the factor is required for release of other amino acid neurotransmitters by examining the effect of the factor on the release of GABA and glycine from permeabilized synaptosomes. This research will not only advance our understanding of the molecular mechanism of glutamate release but also may ultimately provide new insights into the investigation of those types of neuropathophysiology where abnormal glutamate neurotransmission is implicated.