NeurobiologyPurification and primary structure of the neuropeptide egg-laying hormone of Aplysia californica ( Communicated by Theodore H. Bullock, August 15,1979 ABSTRACT Egg-laying hormone (ELH), a neuropeptide synthesized by the bag cell neurons, induces egg laying and its correlated behavior in Aplysia californica. In present study, ELH has been purified to homogeneity and its primary structure has been determined. We find this molecule to have 36 amino acid residues with a M, of 4385 and a calculated isoelectric point of 9.7. Direct microsequence analysis revealed a single amino acid sequence that is in agreement with the amino acid composition determined after acid hydrolysis of ELH: HRIleSer-Ile-Asn-Gln-Asp-Leu-Lys-Ala-Ile-Thr-Asp-Met-Leu-LeuThr-Glu-Gln-Ile-Arg-Glu-Arg-Gln-Arg-Tyr-Leu-Ala-Asp-Leu-

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...

The egg vitelline envelope of the marine invertebrate Megathura crenulata is a glycoprotein composed of 37.3 mol % protein and 62.7 mol % carbohydrate. Of the total amino acid content, 61 mol % consists of a single amino acid, threonine. The carbohydrate content includes galactosamine, galactose, and fucose. The molar ratio of threonine to galactosamine is about 1:1. Most of the threonine residues are linked to galactosamine residues via O-glycosidic bonds. A single peptide that was purified following alkaline borohydride treatment of the vitelline envelope had the structure: Abu-Pro-Abu-(Abu6, Pro1, Thr1), where Abu is 2-aminobutyric acid. Several sugar residues have been isolated following the alkaline hydrolysis of the vitelline envelope that include an octasaccharide Gal4Fu4, an hexasaccharide Gal3Fu3, a trisaccharide Gal3, fucose, and galactose. It is proposed that the vitelline envelope of Megathura crenulata eggs is composed of polypeptide chains built to a large extent of closely spaced threonine residues. Almost every threonine residue is linked to a saccharide moiety.

Three enzymes which lyse the egg-membrane of the marine invertebrate Megathura crenulata were obtained from the sperm of a single individual as well as from pooled samples of sperm from several males of the same species. The three enzymes ("lysins") were purified to homogeneity by a combination of gel filtration and ion-exchange chromatography. The three egg-membrane lysins were different as .judged by chromatographic behavior, gel electrophoresis

The egg vitelline envelope of the marine invertebrate, Megathura crenulata, was lyzed either by sperm lysins A, B, C or by dithiothreitol. In each case the lysis mixture consisted of two major fractions, I and II, that could be separated by hydroxylapatite chromatography and had different electrophoretic mobilities on cellulose acetate strips. The amino acid, amino sugar, and neutral sugar compositions of fractions I and II were similar and resembled that of the intact vitelline envelope. Fractions I and II of each lysis mixture emerged in the exclusion volume of a Sepharose 6B column. A vitelline envelope fragment enzymatically formed by lysin was further degraded by dithiothreitol to form smaller fragments. A model of the vitelline envelope of the Megathura crenulata egg is suggested whereby the envelope is composed of polypeptide chains cross-linked by disulfide bonds and built to a large extent of closely spaced threonine residues. Most of the threonine residues are linked to carbohydrate units. Dithiothreitol dissolves the envelope by reducing disulfide bonds, whereas lysins most likely dissolve the envelope by degrading polypeptide chains.