Isolation of five tachykinin-related peptides from the midgut of the cockroach Leucophaea maderae: existence of N-terminally extended isoforms.

Using a radioimmunoassay (RIA) with an antiserum to the locust neuropeptide locustatachykinin I (LomTK I) and a cockroach hindgut contraction bioassay as monitors, we isolated 5 tachykinin-related peptides from an acidic extract of 600 midguts of the cockroach Leucophaea maderae. A series of 4 different reversed-phase high performance liquid chromatography (rpHPLC) column systems were required to obtain pure peptides. The sequences of the 5 isolated myostimulatory and LomTK immunoreactive peptides were determined by Edman degradation. Four of these were confirmed by mass spectrometry and chemical synthesis as: APSGFLGVRamide, NGERAPGSKKAPSGFLGTRamide, APAMGFQGVRamide and APSGFMGMRamide. The fifth peptide, APEESPKRAPSGFLGVRamide, was confirmed only by mass spectrometry. These peptides, which were designated Leucophaea tachykinin-related peptides 1-5 (LemTRP 1-5), are structurally related to tachykinin-related peptides previously isolated from a locust, blowfly and mosquito species, but showed a somewhat larger variability in their amino-acid sequence (including the carboxy terminus). The two N-terminally extended forms contain putative cleavage sites (KR and KK, respectively) and such extended tachykinins have not been previously identified in insects. All 5 LemTRPs are myotropic and induce increases in the tonus and frequency of spontaneous contractions of hindgut muscle in L. maderae. The potency of the different synthetic isoforms is very similar; they all have a stimulus threshold concentration of 2.5 x 10(-10) M and an ED50 of about 10(-9) M. The synthetic peptides were tested in RIA and found to cross react to different degrees with the antiserum to LomTK I, but it is likely that in immunocytochemistry performed earlier, all 5 forms were detected in the midgut. It is, however, not clear which isoforms are located in endocrine cells and neural fibers of the midgut, respectively.