A pigment-dispersing hormone (PDH) from eyestalks of the fiddler crab Ucapugilator has been purified by gel filtration, ion-exchange chromatography, partition chromatography, and reversed-phase liquid chromatography. Based on automated gas-phase sequencing and subsequent identification ofcarboxyl-terminal amide, we have assigned the primary structure of this peptide as Asn-Ser-Glu-Leu-Ile-AsnSer-Ile-Leu-Gly-Leu-Pro-Lys-Val-Met-Asn-Asp-Ala-NH2. We have confirmed the sequence by synthesizing this peptide and demonstrating that the synthetic PDH and the native PDH display identical chromatographic behavior and biological activity. This hormone is a member of a family of invertebrate neuropeptides that includes a light-adapting/pigment-dispersing octadecapeptide hormone from the prawn Pandalus borealis. In assays for melanophore pigment dispersion in destalked fiddler crabs, Uca PDH was 21-fold more potent than Pandalus PI)H. These two hormones share a hexapeptide core sequence (residues 5-10: -Ile-Asn-Ser-Ile-Leu-Gly-) as well as the amino-and carboxyl-terminal residues but differ at positions 3, 4, 11, 13, 16, and 17. These results point to speciesrelated or group-specific structural differences among crustacean PDHs.
MATERIALS AND METHODSAnimals. Fiddler crabs (U. pugilator), collected from Panacea, Florida, were maintained in the laboratory in a semiterrestrial system, with sand and recirculating seawater, at 220C under a 14-hr light/10-hr dark regime and were fed on dry catfish food. Eyestalks freshly ablated from Uca were lyophilized and stored in a desiccator at -20'C until use.Bioassay. Aliquots of initial extract and of the fractions from purifications, as well as solutions of purified and synthetic peptides, were assayed for melanophore pigment dispersion in destalked fiddler crabs. Samples to be assayed were dissolved in crustacean physiological saline (15) and injected into destalked crabs (50 gl per crab). The chromatophoral stages were scored and the activity values and relative potencies were calculated as described (11). During purification the desired fractions were each tested on 4 or 5 crabs. In tests for relative potencies of native and synthetic peptides, each dose was assayed on 15 crabs (three assays, 5 crabs per assay).Extraction and Purification. In this separation effort, 8500 eyestalks (17 g) were powdered, added to 200 ml of boiling H20, and stirred for 10 min. After the mixture was cooled and the residue had settled, the supernatant was siphoned off for further processing. The residue was reextracted five times, each with 200 ml of H20 at 70-90'C. The cooled combined extract was acidified to 5% HOAc and centrifuged at 10,000 x g for 20 min at 40C. The supernatant was mixed with pretreated Bio-Rex 70 (1 g of resin/15 ml of extract). The resin pretreatment included sequential equilibration with 0.5 M HC1, 50% HOAc, and 5% HOAc. The mixture was stirred for 6 hr at 20'C and poured into a column. After the unadsorbed material passed through the column, the resin was washed with one c...
