Isolation, characterization and biological activity of a diuretic myokininneuropeptide from the housefly, Musca domestica

A competitive ELISA employing a polyclonal antiserum raised against leucoki nin-I was used to isolate and purify a myokinin (muscakinin) from 1.05 kg o f adult houseflies (Musca domestica). Following solid-phase purification, s even HPLC column steps were used to purify 4.8 nmol of leucokinin-immunorea ctive material. Sequence analysis and mass spectrometry were consistent wit h the structure Asn-Thr-Val-Val-Leu-Gly Lys-Lys Gln-Arg-Phe-His-Ser-Trp-Gly NH2. This peptide was synthesized and co-eluted with the natural peptide o n three different HPLC columns. The activities of natural and synthetic mus cakinin were identical, with both producing a 4-5 fold increase in fluid se cretion by housefly Malpighian tubules at nanomolar concentrations. The pre sence of a pair of basic residues (Lys-Lys) suggested muscakinin might be p rocessed further, with the peptide pGlu-Arg-Phe-His-Ser-Trp-Gly NH2 being p roduced by conversion of an N-terminal glutamine to pyroglutamic acid. Howe ver, this analog was 1000-fold less active than the intact peptide, compara ble to the activity of AK-V which shares the same C-terminal pentapeptide s equence. The diuretic activity of muscakinin is more than double that of a previously identified CRF-related diuretic peptide (Musca-DP) from the hous efly, and the two peptides act synergistically in stimulating fluid secreti on. Muscakinin also increased the frequency and amplitude of contractions b y housefly hindgut which might further contribute to the excretory process. (C) 1999 by Elsevier Science Inc.