A diuretic hormone (DH) has been isolated from pharate adult heads of Manduca sexta by a nine-step purification procedure. The primary structure of the aminoterminal 40 residues was determined by sequence analysis of intact DH. The structure of an amidated carboxyl-terminal tryptic hexapeptide was characterized by sequence analysis of the peptide, and this hexapeptide was later compared by reversed-phase liquid chromatography with two synthetic hexapeptides with the free acid or amide at the carboxyl terminus. The complete structure of M. sexta DH was established as a 41-residue peptide without disulfide bonds: H-ArgMet-Pro-Ser-Leu-Ser-Ile-Asp-Leu-Pro-Met-Ser-Val-Leu-ArgGln-Lys-Leu-Ser-Leu-Glu-Lys-Glu-Arg-Lys-Val-His-AlaLeu-Arg-Ala-Ala-Ala-Asn-Arg-Asn-Phe-Leu-Asn-Asp-Ile-NH2. M. sexta DH was synthesized and shown to have chromatographic and biological properties identical with those of the native material. Synthetic DH stimulated fluid excretion in vivo upon injection into larval M. sexta and newly emerged adult Pieris rapae. M. sexta DH has considerable sequence homology with corticotropin-releasing factor, urotensin I, and sauvagine.Insects regulate the osmotic composition of their blood within relatively narrow limits in spite of an unfavorable surface-to-volume ratio (1). The major organs responsible for fluid and ion secretion are the Malpighian tubules (Mt). Primary urine from the Mt moves into the gut and eventually to the rectum, where selective resorption of essential metabolites and water typically occurs (for reviews see refs. 1-3).It is generally believed that regulation of fluid secretion in insects is controlled by one or more peptidic diuretic hormones (DHs), while resorption may be regulated by antidiuretic hormones (1, 2, 4, 5). By using in vitro Mt assays, substances with diuretic activity have been found in ganglia from the head, thorax, and abdomen, and in a glandular tissue, the corpora cardiaca (CC) (4-8). Partial purifications of DH from several species indicate that there may be several DHs which differ in size and possibly in modes of action (3,5,(8)(9)(10)(11)(12).To date, isolation of two insect DHs, both from Locusta migratoria, has been reported (13,14 Several species of butterflies exhibit significant diuresis soon after adult eclosion. This phenomenon allowed development of a facile in vivo DH assay (16). Though the well-studied tobacco hornworm moth, Manduca sexta, does not show diuresis at this stage, we were able to utilize the butterfly Pieris rapae in a similar bioassay and to purify a DH from trimmed head tissue of pharate adult M. sexta. We here report the isolation and structure identification of a M. sexta DH, which has homology with sauvagine, corticotropinreleasing factor (CRF), and urotensin I.
MATERIALS AND METHODSInsects. M. sexta were reared on an artificial diet (17). Pharate adult M. sexta were beheaded 24-48 hr before adult eclosion, and the heads were frozen. A posterior section of these frozen heads containing the brain and the CC/corpora allata complex ...
