Purification and primary structure of two neuroactive peptides that cause bag cell afterdischarge and egg-laying in Aplysia

Abstract

Neuroactive peptides, A and B, were isolated from the atrial gland in the reproductive tract of A. californica. Each of the peptides induces egg-laying behavior in recipient animals. In vitro recordings from the abdominal ganglion show that both peptides also trigger long-lasting discharges in the bag cell neurons at concentrations around 0.1 .mu.M. The peptides were purified by a combination of ammonium sulfate precipitation, agarose gel filtration and cation exchange chromatography. Each peptide has 34 amino acid residues. Microsequencing together with carboxypeptidase Y degradation and analysis of tryptic peptides revealed the following sequence for peptide A: H-Ala-Val-Lys-Leu-Ser-Ser-Asp-Gly-Asn-Tyr-Pro-Phe-Asp-Leu-Ser-Lys-Glu-Asp-Gly-Ala-Gln-Pro-Tyr-Phe-Met-Thr-Pro-Arg-Leu-Arg-Phe-Tyr-Pro-Ile. Peptide B differs from A in 4 positions. The 1st 9 residues of B are: Ala-Val-Lys-Ser-Ser-Ser-Tyr-Glu-Lys-; residues 10-34 of B are identical to those of A. The calculated MW of A is 3924 and that of B is 4032. The pI of peptide A as determined by isoelectric focusing in polyacrylamide gels is 7.9-8.1 and that of peptide B is 9.0-9.2. Each atrial gland contains at least 150 .mu.g of peptide A and 50 .mu.g of B. Neither peptide resembles the egg-laying hormone isolated from bag cell neurons. It is postulated that the atrial gland peptides are released during copulation, and then by interacting with neuronal receptors in the head ganglia and pleuroabdominal connectives they cause the bag cells to afterdischarge, thereby releasing egg-laying hormone.