A peptide that strongly stimulates the secretion of juvenile hormone from corpora allata in vitro (allatotropin) has been purified from extracts of heads of pharate adult Manduca sexta. The primary structure of this 13-residue peptide has been determined: H-Gly-Phe-Lys-Asn-Val-Glu-Met-Met-Thr-Ala-Arg-Gly-Phe-NH(2). This neurohormone has no sequence similarity with any known neuropeptide from other organisms. Synthetic allatotropin, as well as truncation fragments, including one with the five amino terminal residues deleted, showed in vitro activity indistinguishable from that of native allatotropin.

A peptide (Manduca sexta ailatostatin) that strongly inhibits juvenile hormone biosynthesis in vitro by the corpora allata from fifth-stadium larvae and adult females has been purified from extracts of heads of pharate adult M. sexta by a nine-step purification procedure. The primary structure of this 15-residue peptide has been determined: pGlu-Val-ArgPhe-Arg-Gln-Cys-Tyr-Phe-Asn-Pro-Ile-Ser-Cys-Phe-OH, where pGlu is pyroglutamate). To our knowledge, this neurohormone has no sequence similarity with any known neuropeptide from other organisms. The synthetic free acid and amide forms showed in vitro activity indistihable from that of native M. sexta aliatostatn. The EDsO of synthetic M. sexta allatostatin on early fifth stadium larval corpora allata in vitro was =2 nM. This inhibition was reversible. In a cross-species study, M. sexta allatostatin also inhibited the corpora allata of adult female Heliothis virescens but had no effect on the activity of corpora allata ofadult females of the beetle Tenebrio molitor, the grasshopper Melanoplus sanguinipes, or the cockroach Periplaneta americana.Juvenile hormone (JH), which is synthesized and released by the retrocerebral corpora allata (CA), plays a vital role in insect development, primarily in the control of metamorphosis, adult sexual maturation, and reproduction (1, 2). Environmental and physiological factors influence neurosecretory centers in the brain that affect the activity of CA through peptidergic materials either transported directly to the CA by axons or released into the blood (3). These neuropeptides may be stimulatory (allatotropins, e.g., refs. 4-8) (27). However, instead of assaying for JH production by chloroform extraction followed by TLC analysis, we used a rapid isooctane partitioning assay for JH (28). In the cross-species studies, we used the same in vitro technique (one or two pair of CA per incubation).Extraction and Preliminary Purification (Steps 14). Thirty thousand trimmed heads of M. sexta (fresh weight, "'.1460 g) were processed in three batches of 10,000. Each batch was defatted by homogenizing in 2 liters of ice-cold acetone and filtered. Residues were extracted with 1.4 liters of 1 M HOAc/20 mM HCO (containing 0.1 mM phenylmethylsulfonyl fluoride and 0.01 mM pepstatin A, prepared immediately before use) and centrifuged at 10,000 x g for 30 min at 40C. The pellet was extracted and centrifuged twice using a total volume of 2.8 liters of the same solution. The combined supernatants were stirred with swollen SP-Sephadex C-25 resin (300 ml) overnight. The resin was allowed to settle for 2 hr, subsequently poured into a Bio-Rad column (50 x 300 mm), and equilibrated with 1 M HOAc. Material was eluted from the column sequentially with 1-liter volumes of 0.05 M NH4OAc (pH 4.0) and 0.05 M, 0.1 M, 0.2 M, 0.4 M, and 0.8 M NH4OAc (pH 7.0). The 0.1 M and 0.2 M fractions, which had M. sexta AS (Mas-AS) activity, were applied directly to 10 g of reversed-phase Vydac C4 packing material (20-30 pAm, contained in a 75-ml polypropylene syr...

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 ...

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