Insects have low pressure circulatory systems and require diuretic hormones to control production by Malpighian tubules of primary urine. The first insect diuretic hormone (DH) identified (1987) is homologous to arginine vasopressin. The second identified, Manduca sexta DH (1989), is structurally homologous to the corticotropin releasing factor/sauvagine/urotensin I family of neuropeptides. In 1991 several additional insect DHs have been identified, which are homologous to M. sexta DH. An extremely novel aspect of the sequences of the M. sexta DH family of peptides is that they have homologous regions for the amino terminal half of the 41-46 residue sequence, and homology at the carboxyl terminus of the molecule, but gaps are required in the less homologous part of the molecules for good alignment of both termini. These gaps in the sequence should cause profound differences in the three dimensional conformation of the related insect DHs; curiously, the form with the biggest gap in its sequence still has biological activity in Malpighian tubules of the species with the smallest gap in its sequence. We propose studies to isolate and identify additional members of this family of neuropeptides, using an existing enzyme-linked immunosorbent assay to monitor peptide isolations. We also plan to study the relative three dimensional conformation of the members of this family by both computer modeling and by physico-chemical methods (600 MHz NMR and circular dichroism spectrometry on synthetic samples). We will also try to correlate structure with function by studying the relative affinity of the peptides for receptor preparations from two different species of insects, and also study their activity in bioassays. Such studies on this unique family of neuropeptides will provide conformational information which may be of general utility in planning synthesis of neuropeptide analogues. Usually chemical analogue synthesis of neuropeptides is geared to truncations and amino acid substitutions. In addition, it is possible that diuretic peptides may be used in biotechnological approaches for control of mosquitoes and other blood-feeding disease vectors, as very rapid diuresis is essential for survival of blood-feeders. Analogues of insect juvenile hormones (sesquiterpenoids) are currently in commercial use as extremely safe, specific insect control agents.
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