Seven tachykinin-related peptides isolated from the brain of the Madeira cockroach: evidence for tissue-specific expression of isoforms.

We have isolated seven tachykinin-related peptides (TRPs) from an acidic extract of 1000 brains of the cockroach Leucophaea maderae. Four different reversed-phase high performance liquid chromatography (RP-HPLC) column systems were required to obtain pure peptides. During the purification the fractions were monitored in a radioimmunoassay (RIA) with an antiserum to locust TRP locustatachykinin I (LomTK I) and a cockroach hindgut muscle contraction bioassay. The sequences of the seven isolated LomTK immunoreactive and myostimulatory peptides were determined by Edman degradation. Six of these were confirmed by mass spectrometry and chemical synthesis as: APSGFLGVRamide, APAMGFQGVRamide, APAAGFFGMRamide, VPASGFFGMRamide, GPSMGFHGMRamide, and APSMGFQGMRamide. The seventh peptide, APEESPKRAPSGFLGVRamide, was confirmed only by mass spectrometry. These peptides were designated Leucophaea maderae tachykinin-related peptides 1, 2, 5-9 (LemTRP 1, 2, 5-9). Two more peptides were isolated using the same assays: SGLDSLSGATFGGNR and ALFEESTVSAEPR. The first shares the C-terminus FX1GX2R with the Lem TRPs, whereas the second one is not related to the TRPs (none of these peptides were synthesized and thus putative C-terminal amidation was not confirmed). Three of the brain peptides, LemTRP 1, 2, and 5, have previously been isolated from the midgut of L. maderae, whereas the others appear to be brain specific. Because the LemTRPs 3 and 4 appear to be unique for the midgut, we have indications for tissue-specific expression of TRPs. All seven confirmed LemTRPs of the brain are myotropic and induce increases in the amplitude and frequency of spontaneous contractions and tonus of hindgut muscle in L. maderae.