Atrial gland cells synthesize a family of peptides that can induce egg laying in Aplysia

1. Endogenous peptides induced egg laying in the marine mollusc Aplysia in two ways: egg-laying hormone (ELH) from the neuroendocrine bag cells acts directly, causing the release of eggs from the ovotestis; peptides A and B from the atrial gland act indirectly, activating the bag cells to release ELH. Another atrial gland peptide (egg-releasing hormone; ERH) is a structural and functional hybrid of ELH and peptides A and B; it can act both directly and indirectly to induce egg laying. 2. Atrial glands were incubated in a mixture of³H-amino acids for 18 h, and the biosynthetically labelled peptides isolated using sequential Sephadex G-50 column chromatography and isoelectric focusing. Radiolabelled peaks were localized and bioassayed in intact animals. Bioactive peaks were then characterized functionally using two additional assays: egg laying in bag cell-less animals (ELH-like peptides) and in vitro induction of bag cell discharge (A- and B-like peptides). ERH-like molecules are active in both assays. Homogeneity of bioactive IEF peaks was assessed by SDS-PAGE. 3. Sephadex G-50 gel filtration of biosynthetically labelled atrial gland extracts reveals two major peptide peaks. Peak D (apparent M _r 6,000) is strongly radiolabelled and contains most of the egg-laying activity, but has a low absorbance at 274 nm. Peak E (apparent M _r 3,500) is weakly labelled and contains a small proportion of the total egg-laying activity, but has a large absorbance at 274 nm. 4. Isoelectric focusing of radiolabelled peptides in peak D reveals seven distinct ELH-like species (pI 5.5, 7.5, 8.5, 8.7, 8.9, 9.1, 9.4), and two peaks (pI 5.9, 8.1) that have both ELH-like and A-/B-like activity. The pI 8.1 peak may result from the comigration of peptide A with ERH or with an unidentified ELH-like peptide. It is not yet clear whether the pI 5.9 activity results from comigration of distinct peptides or from the presence of a previously uncharacterized ERH-like molecule. 5. Isoelectric focusing of radiolabelled peptides in peak E reveals five distinct ELH-like species (pI 7.3, 8.5, 8.7, 9.1, 9.4), and one peak (pI 8.9) with both ELH-like and A-/B-like activity. The pI 8.9 peak may result from the comigration of an ELH-like peptide with peptide B. Three of the ELH-like peptides (pI 8.5, 8.9, 9.1) found in peak E are probably identical to the ELH-like peptides found at the same pI's in peak D. 6. The ELH-like pI 7.5 species is a peptide complex held together by a disulfide linkage and hydrophobic interactions. The complex dissociates when treated with 2-mercaptoethanol and pyridine, resulting in a reduction in apparent molecular weight, and the appearance of one basic (pI 9.4) and two acidic (pI 3.3, 3.8) peptides. The pI 9.4 peptide presumably accounts for the ELH-like activity of the parent pI 7.5 complex but differs, based on its mobility on SDS-PAGE, from the pI 9.4 ELH-like peptide(s) described above. 7. These studies, utilizing a two-step purification procedure, functional bioassays, and SDS-PAGE to assess peptide homogeneity, indicate that atrial gland cells synthesize a surprisingly large number of peptides that can induce egg laying in Aplysia.