Myotropic, isolation, structural

Schoofs L., Holman G. M., Hayes T. K., Nachman R. J. and DeLoof A. (1991) Isolation, primary structure and synthesis of locustapyrokinin a myotropic peptide of Locusta migratoria. Gen. Comp. Endocrinol. 81, 97-104. 

The 1-125 protein column (operated in a normal-phase mode) was chosen as the final purification step for two reasons 1) the small (sometimes invisible) amount of residue remaining after the previous purification steps was easily dissolved in 95% acetonitrile 5% water (the initial-conditions solvent for this system) and 2) the peaks collected from this column were in a solution (75%-85% acetonitrile) suitable for peptide stabilization and required no further manipulation (other than capping) prior to storage. The structures of the 11 myotropic cockroach peptides isolated with this method are listed, along with appropriate references, in Table I (leucokinins... 

In addition to the L. maderae myotropic peptides, a series of five myotropic peptides were isolated and structurally characterized from head extracts of the cricket, Acheta domesticus. with the same purification system and bioassay (Holman, G. M., et al. Chromatography and Isolation of Insect Hormones and Pheromones, in press.). The structures of these five myotropic peptides, the achetakinins, are shown in Table 1. Like the leucokinins, the achetakinins (Ak s) contain a C-terminal pentapeptide core which is responsible for the myotropic activity (12). In Ak s III and V, the C-terminal pentamer is the same as the leucokinin pentamer (Phe-X-Ser-Trp-Gly-NH2, where X = Asn, His, Ser, or Tyr) but in Ak s I, II, and IV a slightly different pentamer (Phe-X-Pro-Trp-Glv-NH is present. 

Since the publication in 1986 (13,16) of the gastrin/cholecystokinin-like leucosulfakinins (LSK s), several additional insect sulfakinins have been structurally characterized (Table II). The structures of the Drosophila sulfakinins (DSK s) were deduced from a gene sequence which was isolated from Drosophila genomic DNA and head cDNA libraries (17). Although expression of the DSK s remains to be demonstrated, sulfated synthetic replicas are biologically active on the isolated cockroach hindgut (Holman, unpublished observation) as predicted by a study (18) which demonstrated that the hexamer, Tyr(S03)-Gly-His-Met-Arg-Phe-NH2, was the "core structure required for myotropic activity. 

Barium hydroxide hydrolysis followed by amino acid analysis revealed a sulfated Tyr residue. Subsequently, the myotropic nature of PSK was demonstrated on the isolated hindgut preparation of P. americana where the threshold of activity concentration was determined to be 0.25 nM, virtually the same as the threshold concentration (0.22 nM) of LSK on the isolated L. maderae hindgut (13). In addition to PSK, the non-sulfated form of LSK-II was isolated and structurally characterized from the P. americana cc extracts, but the biologically active sulfated form was not found (19). 

The octapeptide leucopyrokinin (LPK) was the eleventh and final myotropic peptide isolated from L. maderae (11). Unlike the leucokinins and LSK s, LPK exhibits a myotropic activity on the muscles of the cockroach foregut and oviduct (Holman, G. M. and Nachman, R. J., unpublished observation.). LPK was the highest titered of the Leucophaea myotropins (1.36 pmol/head) but was the least potent on the isolated hindgut preparation (threshold concentration -0.6 nM). LPK shares a 50% sequence identity with Pea-HTH-II at the 1, 4, 5, and 6 positions and is amidated at the C-terminus. Yet LPK does not contain Trp, which is present at position 8 in every AKH/RPCH peptide. Furthermore, LPK is positively charged with a residue of Arg at position 8. These structural observations show that LPK is not a member of the AKH/RPCH family. A study of LPK analogs supports this view (20). 

Beginning with the initial success of Brown and Starratt (7,8) and continuing to the present, myotropic bioassays have played a major role in the isolation and structural characterization of insect neuropeptides. The success will likely continue since myotropic bioassays are sensitive, rapid, reliable, and reproducible characteristics desirable to the isolation specialist. Insect neuropeptide isolation projects utilizing a myotropic bioassay have been responsible for the discovery of four structurally unique peptide families and the initial demonstration of the existence of natural-analog series which may be quite common in insects. 

Additional myotropic/inhibitory neuropeptide structures will be characterized on the basis of immunological similarity to vertebrate and invertebrate peptide structures, and also with the techniques of molecular biology. Two initial successes with those methods are discussed in this report (17,25). In addition, antibodies raised against FMRFamide were used to isolate and structurally characterize a nonapeptide containing C-terminal FMRFamide from head extracts of Drosophila (26). Subsequently, the gene that codes for this nonapeptide and eight other FMRFamide-related structures was isolated and sequenced (27,28). 

Structure-activity relationship studies for three myotropic families of insect neuropeptides first isolated from Leucophaea maderae are discussed. 

Holman, G M., Nachman, R J, and Wright, M S (1990) A strategy for the isolation and structural characterization of certain insect myotropic peptides that modify the spontaneous contractions of the isolated cockroach hindgut, in Chromatography and Isolation of Insect Hormones and Pheromones (MacCaffery, A R and Wilson, I. D., eds), Plenum, New York, pp 195-204. 

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