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In urodeles, chemical stimuli contribute to sex recognition and courtship behavior (1). It has been suggested that males emit olfactory attractants or pheromones to lure females (2). The abdominal gland of the cloaca has been thought to be the site where these substances are produced (3). We have found that the water in which sexually active male newts (Cynops pyrrhogaster) were kept attracted conspecific females (4). The attractant pheromone was secreted by or through the abdominal gland of the cloaca because the water in which abdominal gland-ablated males had been kept did not attract females (4).
We report here the isolation and characterization of the female-attracting pheromone from the abdominal glands of male newts. Female-attracting pheromone activity was monitored by a preference test (5). An aqueous extract of the abdominal glands had a female-attracting pheromone activity. The minimum effective amount of extract in a sponge block that attracted a female placed in a container filled with 3000 ml of water was the equivalent of 0.1% of the abdominal gland content (Fig. 1). (Fig. 1 omitted) The active substance in the abdominal gland was soluble in water but not in organic solvent. When the water-soluble fraction was subjected to gel-filtration column chromatography, the female-attracting pheromone activity emerged in a fraction with a relative molecular mass below 5000. Pronase digestion eliminated the activity, indicating that the active substance is a peptide.
To isolate the active peptide from an aqueous extract of the abdominal glands, we used two purification cycles of reversed-phase high-performance liquid chromatography (HPLC) (Fig. 2). (Fig. 2 omitted) Direct sequencing of the final product with a pulsed gas-liquid phase protein sequencer revealed that it is a decapeptide with amino acid sequence Ser-Ile-Pro-Ser-Lys-Asp-Ala-Leu-Leu-Lys. Its amino acid composition, determined by acid hydrolysis, was Asp, 1.10; Ser, 2.01; Pro, 1.03; Ala, 1.10; Ile, 1.09; Leu, 2.21; and Lys, 2.13. Thus, the amino acid composition data are in agreement with the amino acid residues deduced by peptide sequencing. COOH-terminal analysis by carboxypeptidase-P digestion revealed that the terminus consisted of a free Lys residue. The relative molecular mass of 1071.2, estimated from fast atom bombardment mass spectrometry (6), corresponds with that calculated from the amino acid sequence.
The peptide skowed no sequence homology with any known peptide (7). Thus, the...