The bag cell neurons of Aplysia california synthesize and secrete a peptide hormone, egg-laying hormone (ELH), that acts peripherally to induce the assembly and transport of an egg cordon, and centrally to induce behaviors that serve the goal of egg deposition. The hormone is synthesized as part of a larger precursor molecule that is known to contain sequences that could be expressed as individual peptides. One of these peptides (AP) has been shown to be produced coordinately with ELH during precursor processing by the cells. Both eLH and AP are transported in vesicles to the secretory terminals of the bag cells and released upon depolarizing stimulation. Studies in progress have demonstrated that ELH and AP synthesized at the same time do not enter transport coordinately, AP appears before ELH of the same age. This differential is retained and expressed at the time of secretion as well. The array of possible hypotheses to account for this phenomenon can be simplified by posing the first-order question: """"""""Where does precursor cleavage occur?"""""""" The experiments proposed in this application will approach this question by addressing the specific issue of whether precursor processing occurs before vesicle enclosure of AP and ELH, or takes place within the vesicle. To examine this problem, the ELH-AP precursor will be synthesized in vitro from bag cell RNA. It will be exposed to subcellular fractions from the bag cells either cotranslationally or after isolation from the translation mixture. Gel electrophoretic and peptide-mapping procedures will be used to assay the products derived from precursor exposure to subcellular fractions. Attempts will then be made to characterize the nature of the processing steps further through modification of the precursor, or its components, and blocking of the putative enzymatic activities of the subcellular fractions. Since these studies will concentrate on the secretory vesicle, additional studies will be undertaken to begin characterization of the vesicle and its contents. These include continuation of the use of bifunctional cross-linkers to determine if ELH and AP are enclosed in single vesicle type. Monoclonal antibody production will be begun as well. Text exceeds capacity.
