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.
KeywordsAmino Acid Analysis Capillary Zonal Electrophoresis Fluid Secretion Malpighian Tubule Diuretic Activity
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- 1.W. Mordue and P. J. Morgan, Chemistry of peptide hormones, in:“Comprehensive Insect Physiology, Biochemistry and Pharmacology”, G. A. Kerkut and L. I. Gilbert, eds., Pergamon Press, Oxford, Vol. 7, pp 153–183 (1985).Google Scholar
- 3.J. E. Phillips, Endocrine control of salt and water balance: excretion, in:“Endocrinology of Insects”, R. G. H. Downer and H. Laufer, Alan Liss, New York, pp 411–425(1983).Google Scholar
- 6.J. A. Ramsay, Active transport of water by the Malpighian tubules of the stick insect, Dixippus morosus (Orthoptera; Phasmidae), J. Exp. Biol, 31:104–113 (1954).Google Scholar
- 7.C. H. Wheeler and G. M. Coast, Assay and characterization of diuretic factors in insects, J. Insect Physiol, in press (1989).Google Scholar
- 12.G. M. Coast and C. H. Wheeler, The distribution and relative potency of diuretic peptides in the house cricket, Acheta domesticus, Physiol Entomol., in press (1989).Google Scholar
- 13.C. W. Clifford, R. M. Roe and J. P. Woodring, Rearing methods for obtaining house crickets, Acheta domesticus, of known age, sex and instar, Ann. Entomol Soc. America, 70:69–74 (1977).Google Scholar
- 15.D. Dupont, P. Keim, A. Chui, I. Harding and K. J. Wilson, Gas-phase hydrolysis for PTC-amino acid analysis, Derivatizer-Analyzer Bulletin No. 2, Applied Biosystems Inc. (1987).Google Scholar
- 16.P. J. Morgan and W. Mordue, Separation and characteristics of diuretic hormone from the corpus cardiacum of Locusta, Comp. Biochem. Physiol, 75B:75–80 (1983).Google Scholar