Identification of an Acidic Dipeptide, β‐Aspartylglycine, in the CNS of Aplysia

Abstract

A novel dipeptide, beta-aspartylglycine (beta-DG), has been isolated from tissues of the marine gastropod mollusc Aplysia californica. This compound was detected only in Aplysia and not in other molluscs, such as Helix or Mercenaria, or in lobster or frog. Among the Aplysia tissues, the highest levels of beta-DG were in nervous tissue and in the reproductive tract. beta-DG was assayed by HPLC as the o-phthaldialdehyde derivative and found to be present in all individual, identified neurons at a concentration of approximately 40 pmol/microgram protein. The peptide was identified as beta-DG by gas chromatography-mass spectrometry (GCMS) using trimethylsilyl derivatives prepared before and after acid hydrolysis. It was further characterized as the beta-isomer by TLC, including Rf, atypical blue-gray color with ninhydrin, and a violet color with Cu2+-ninhydrin. A fractionation scheme is described whereby acid-soluble tissue constituents can be divided into acidic, neutral, and basic components using mini ion-exchange columns. This partial purification prior to TLC analysis was necessary to remove compounds that interfered with the isolation of beta-DG.

Confirmation of d-aspartic acid in the novel dipeptide β-aspartylglycine isolated from tissue extract of Aplysia kurodai

“…analysis can be attributed to incomplete separation from tissue extract and/or to seasonal variation. /J-Asp-Gly has been identified previously in human urine (Buchanan et al, 1962;Gejyo et al, 1978), in collagen (Pisano et al, 1966), in caecal contents of mammals (Welling & Groen, 1978;Welling, 1982) and also in tissues of Aplysia californica (McCaman & Stetzler, 1984). However, there is no information in these reports on the configuration of aspartic acid of /,-Asp-Gly.…”

Synthesis of γ‐Glutamyldopamine and Other Peptidoamines in the Nervous System of Aplysia californica

“…From each sample, basic, neutral, and acidic amino acid (AA) fractions were prepared using a series of miniion-exchange resins (Bio Rex 70, Na'; SP-Sephadex, C25-Hf; and AGI-formate) and each fraction was analyzed by TLC using cellulose sheets and n-butanol-acetic acid-water (BAW), 4: 1: 1, as described earlier (McCaman and Stetzler, 1984). Appropriate AAs and y-Glu-amines were co-chromatographed as standards along with tissue samples.…”

“…Considerable amounts of aspartate and glycine were also taken up and recovered unchanged in ganglion extracts. In both cases, radioactivity was also recovered from an area co-chromatographic with paspartylglycine (P-DG), R, = 0.12, and, in the case of glycine, co-chromatographic with glutathione (y-ECG), R, = 0.05 (McCaman and Stetzler, 1984). However, neither AA appears to form an amine conjugate, i.e., no radioactivity was found in areas with an R, higher than the parent compound and no amines were produced after hydrolysis of the acidic AA fraction.…”