Purification and characterization of Aplysia atrial gland secretory granules containing egg-laying prohormone-related peptides

A purification scheme is described for the isolation of secretory granules containing egg-laying prohormone-related peptides from the atrial gland of Aplysia californica, an exocrine organ in the reproductive tract. Granules were purified by differential centrifugation of atrial gland homogenates followed by centrifugation on continuous Percoll-sucrose gradients. Quantitative enzyme assays in conjunction with electron microscopic analyses demonstrated that secretory granules thus isolated were significantly purified with respect to other subcellular organelles such as mitochondria and lysosomes. Immunoelectron microscopy demonstrated that the majority (∼85%) of the purified secretory granules were immunoreactive for A-NTP (N-terminal peptide), a cleavage product of the egg-laying prohormone-related A and A′ precursors (residues 22-34). The purified granules represented an enriched source of peptides that were readily resolved by reversed-phase high performance liquid chromatography.