Isolation and Characterization of a Diuretic Peptide, AP-I, from the Corpora Cardiaca of the House Cricket, Acheta Domesticus
Fractionation of extracts of the corpora cardiaca of the house cricket, Acheta domesticus, by reversed-phase high performance liquid chromatography (RP-HPLC) on octylsilica column packings, revealed a number of peptides which had diuretic activity. One of these peptides, AP-I, was associated with a major peak of UV absorbance. AP-I has been isolated using RP-HPLC, and the purity confirmed using a range of chromatographic procedures including: elution in different solvent systems; the use of octadecyl and phenyl columns; and separation on size-exclusion and ion-exchange columns. In all instances diuretic activity was associated with a peak of UV absorbance corresponding to AP-I. After treatment with pyroglutamate aminopeptidase, AP-I was sequenced by gas-phase Edman-degradation, and determined as <QRDIFHAQTDIFQVPK-OH. This sequence has been confirmed by peptide mapping, and the proposed molecular weight of 1924.98 conforms with that obtained by fast atom bombardment mass-spectrometry (FAB-MS). A peptide with this sequence has been synthesized using FMOC chemistry, and its molecular weight confirmed by FAB-MS. The synthetic peptide co-elutes with AP-I on a Lichrosorb RP8 column, but it does not stimulate fluid secretion in Acheta. Possible reasons for this lack of activity are discussed.
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