Structural characterization of a diuretic peptide from the central nervous system of the leech Erpobdella octoculata: Angiotensin II amide

Abstract

Purification of a material immunoreactive to an anti-serum against angiotensin II and present in the central nervous system of the pharyngobdellid leech Erpobdella octoculata was performed by reversed-phase high pressure liquid chromatography combined with both enzyme-linked immunosorbent assay and dot immuno-binding assays for angiotensin II. Establishment of the amino acid sequence by Edman degradation, electro- spray, and fast atom bombardement mass spectrometry measurements and enzymatic treatment by carboxypeptidase A indicated that this 'central' angiotensin H-like material, the first one fully characterized in the animal kingdom, is an angiotensin II amide. This finding constitutes also the first biochemical characterization of a peptide of the angiotensin family in an invertebrate. Synthetic angiotensin II amide exerts, when injected in leeches, a diuretic effect and is, 1 and 2 h postinjection, 100-fold more potent than vertebrate angiotensin II. An identification of the proteins immunoreactive to an antiserum against angiotensin II performed at the level of both central nervous system extracts and in vitro central nervous system- translated RNA products indicated that in the two cases, two proteins were detected. Their molecular masses, which were, respectively, ~14 and ~18 kDa for the central nervous system extracts and ~15 and ~19 kDa for in vitro central nervous system-translated RNA products, differ from that of angiotensinogen (~60 kDa), the precursor of vertebrate angiotensin II.