7 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 acid ic extract of 1000 brains of the cockroach Leucophaea maderae. Four di fferent reversed-phase high performance liquid chromatography (RP-HPLC ) column systems were required to obtain pure peptides. During the pur ification the fractions were monitored in a radioimmunoassay (RIA) wit h an antiserum to the locust TRP locustatachykinin I (LomTK I) and a c ockroach hindgut muscle contraction bioassay. The sequences of the sev en isolated LomTK immunoreactive and myostimulatory peptides were dete rmined by Edman degradation. Six of these were confirmed by mass spect rometry and chemical synthesis as: APSGFLGVRamide, APAMGFQGVRamide, AP AAGFFGMRamide, VPASGFFGMRamide, GPSMGFHGMRamide, and APSMGFQGMRamide. The seventh peptide, APEESPKRAPSGFLGVRamide, was confirmed only by mas s spectrometry. These peptides were designated Leucophaea maderae tach ykinin-related peptides 1, 2, 5-9 (LemTRP 1, 2, 5-9). Two more peptide s were isolated using the same assays: SGLDSLSGATFGGNR and ALFEESTVSAE PR. The first shares the C-terminus FX(1)GX(2)R with the LemTRPs, wher eas the second one is not related to the TRPs (none of these peptides were synthesized and thus putative C-terminal amidation was not confir med). Three of the brain peptides, LemTRP 1, 2, and 5, have previously been isolated from the midgut of L. maderae, whereas the others appea r to be brain specific. Because the LemTRPs 3 and 4 appear to be uniqu e for the midgut, we have indications for tissue-specific expression o f TRPs. All seven confirmed LemTRPs of the brain are myotropic and ind uce increases in the amplitude and frequency of spontaneous contractio ns and tonus of hindgut muscle in L. maderae. (C) 1997 Elsevier Scienc e Inc.