Preliminary characterisation of locust diuretic peptide (DP-1) and another corpus cardiacum peptide (LCCP)

A simple two step HPLC purification protocol is described for one of the locust diuretic peptides (DP-1) and a second corpus cardiacum peptide (LCCP) of unknown function. DP-1 and LCCP are extracted from corpora cardiaca in 20% aqueous methanol (v/v), and applied to a high performance size-exclusion chromatography (HPSEC) column, from which DP-1 and LCCP co-elute in a fraction designated F7/8; this corresponds to a relative molecular mass of ca 6000-7000. Without sample concentration, F7/8 (2 ml) is applied directly onto a wide-pore reversed-phase HPLC column; LCCP and DP-1 are eluted in ca 12 and 20 min respectively on an increasing linear gradient of 0.5% min, starting at 30% acetonitrile. The diuretic activity of the DP-1 material was confirmed using the cAMP assay (Morgan and Mordue, 1985a, Insect Biochem. 15, 247-257). DP-1 and LCCP were isolated from ca 650 locusts using the above procedures for sequence analysis by gas-phase sequencing. The low amount of DP-1 purified allowed only a partial and fragmentary sequence to be determined; insufficient material was available for amino acid analysis. The higher amount of LCCP allowed a partial sequence determination of residues 1-40. The titre of DP-1 in locust CC is calculated to be 1.7-2.1 pmol/CC.