ISOLATION OF 5 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 neurope ptide locustatachykinin I (LomTK I) and a cockroach hindgut contractio n 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 chroma tography (rpHPLC) column systems were required to obtain pure peptides . The sequences of the 5 isolated myostimulatory and LomTK immunoreact ive peptides were determined by Edman degradation. Four of these were confirmed by mass spectrometry and chemical synthesis as: APSGFLGVRami de, NGERAPCSKKAPSGFLGTRamide, APAMGFQGVRamide and APSGFMGMRamide. The fifth peptide, APEESPKRAPSGFLGVRamide, was confirmed only by mass spec trometry. These peptides, which were designated Leucophaea tachykinin- related peptides 1-5 (LemTRP 1-5), are structurally related to tachyki nin-related peptides previously isolated from a locust, blowfly and mo squito species, but showed a somewhat larger variability in their amin o-acid sequence (including the carboxy terminus). The two N-terminally extended forms contain putative cleavage sites (KR and KK, respective ly) and such extended tachykinins have not been previously identified in insects. All 5 LemTRPs are myotropic and induce increases in the to nus and frequency of spontaneous contractions of hindgut muscle in L. maderae. The potency of the different synthetic isoforms is very simil ar; they all have a stimulus threshold concentration of 2.5 x 10(-10) M and an ED(50) of about 10(-9) M. The synthetic peptides were tested in RIA and found to cross react to different degrees with the antiseru m to LomTK I, but it is likely that in immunocytochemistry performed e arlier, all 5 forms were detected in the midgut. It is, however, not c lear which isoforms are located in endocrine cells and neural fibers o f the midgut, respectively.