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

Abstract

Two neuroactive peptides, A and B, have been isolated from the atrial gland in the reproductive tract of Aplysia. Each of the two peptides is able to induce egg-laying behavior in reci ient animals. In vitro recordings rom the abdominal ganglion show that both peptides also trigger longlasting discharges in the bag cell neurons at concentrations around 0.1 #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 wi carboxypeptidase Y degradation and analysis of tryptic peptides revealed the following sequence for peptide A: H-Ala-Val-Lys-Leu-Ser-SerAsp-Gly-Asn-Tyr-Pro-Phe-Asp-Leu-Ser-Lys-Glu-Asp-Gly-AlaGln-Pro-Tyr-Phe-Met-Thr-Pro-Arg-Leu-Arg-Phe-Tyr-Pro-Ile.Peptide B differs from A in only four positions. The first nine residues of B are: Ala-Val-Lys-Ser-Ser-Ser-Tyr-Glu-Lys-, whereas residues 10-34 of B are identical to those of A. The calculated M, 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. It is estimated that each atrial gland contains at least 150 jug of peptide A and 50 ,ug of B. Neither peptide resembles the egg-laying hormone isolated from bag cell neurons. It is postu ated 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.The neuropeptidergic bag cells in the abdominal ganglion of Aplysia californica are able to initiate egg laying and its associated behavior by secreting egg-laying hormone (ELH) during a long-lasting afterdischarge (1-3). This afterdischarge may be triggered by electric stimulation of a presumed afferent pathway from the head ganglia (4, 5). Neither the location in the head ganglia nor the transmitter of these input neurons to the bag cells are presently known.When a homogenate of bag cells containing ELH is injected into the hemocoel of a sexually mature animal, a pattern of egg-laying behavior is triggered that results in egg laying within an hour after injection (6, 7). ELH has been purified and found to be a polypeptide of 36 amino acids with a Mr of 4385 and a pI of 9.0-9.2 (8). Copulation may be a natural stimulus for egg laying and it has recently been shown that the atrial glabd in the reproductive tract of Aplysia contains a factor that is able to induce egg-laying behavior, in a similar way to ELH, when injected into recipient Aplysia (9, 10). Moreover, it was shown that the atrial gland factor resembles ELH in apparent molecular weight and pI, although in contrast to bag cell homogenates some bioactive material was at pH 7.4 on isoelectric focusing gels in addition to the pI 9.3 material (9, 10). This paper describes the purification and primary structure of two atrial gland peptides, each of which is able to in...

Nitric oxide induces aspects of egg-laying behavior in Aplysia

“…NO is unlikely to be an intermediary for these effects, since cutting the pleuralabdominal connectives by which neurons in the head ganglia communicate with the bag cells did not block the effect of the NO donor. Bag cell and atrial gland peptides also directly excite the bag cells (Heller et al, 1980;Kauer et al, 1987). NO is unlikely to be an intermediary for these effects, since stimulating the bag cells with the NO donor did not cause a bag cell after-discharge (Fig.…”

“…In some Aplysia species, including in A. fasciata that was used in this study, a distinct atrial gland is not present, and tissue homologous to the atrial gland is present in the anterior portion of the large hermaphroditic duct (Painter et al, 1985). Homogenates of the bag cells or of the atrial gland elicit egg-laying when injected into Aplysia Bernheim and Mayeri, 1995;Heller et al, 1980;Kupfermann, 1967), in part because some peptides act on the gonad and cause the release of yolk-containing eggs. Eggs are then transported via the small hermaphroditic duct from the gonad to the to the fertilization chamber, where they are fertilized by previously stored sperm cells.…”

“…The peptides are synthesized on a single precursor protein, which is then cleaved (Nagle et al, 1989;Scheller et al, 1983). Homologues of the bag-cell peptides are also synthesized in and released from the atrial gland of the genital tract Heller et al, 1980;Nagle et al, 1989;Painter et al, 1988;Scheller et al, 1982). In some Aplysia species, including in A. fasciata that was used in this study, a distinct atrial gland is not present, and tissue homologous to the atrial gland is present in the anterior portion of the large hermaphroditic duct (Painter et al, 1985).…”

A Personal Journey of Discovery: Developing Technology and Changing Biology

“…These are but a few examples of the many other fascinating proteins that we microsequenced during the late 1970s and 1980s (15)(16)(17)(18).…”

Assaying for peptides in individual Aplysia neurons with mass spectrometry

“…In the case of Aplysia, the behavior of egg laying (28) is governed by a family of neuropeptides, including the 36-residue ELH released from bag cells (Fig. 1C) and peptides A and B released from the atrial gland (31). Although a battery of peptides are involved in egg laying, they are all derived from and encoded by a small gene family (24).…”