allatostatin-1 and amastatin

The degradation of synthetic cydiastatin 4 by enzymes of the foregut and hemolymph, and transport across the foregut of larvae of the tobacco hawkmoth moth, Manduca sexta, were investigated using reversed-phase high performance liquid chromatography (RP-HPLC) together with matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In the hemolymph in vitro, cydiastatin 4 had a half-life of ca. 30 min. Two degradation products were identified; cydiastatin 4(1-6), due to cleavage of the C-terminal di-peptide GL-amide, and cydiastatin 4(2-8), due to cleavage of the N-terminal A residue. This hydrolysis could be inhibited by up to 93% by 1,10-phenanthroline. Other protease inhibitors had lesser effects (<21% inhibition of degradation) including the aminopeptidase inhibitors amastatin and bestatin, and the chelator EDTA. When incubated with foregut extract in vitro, cydiastatin 4 had a half-life of 23 min, and the hydrolysis products detected were also cydiastatin 4(1-6) and cydiastatin 4(2-8). Similarly, 1-10 phenanthroline inhibited foregut enzyme degradation of cydiastatin 4 by ca. 80%, whereas amastatin, bestatin, and EDTA had very little effect (<10% inhibition). Cydiastatin 4 was transported, intact, from the lumen to the hemolymph side of foregut tissues that were mounted as flat sheets in modified Ussing chambers. This trans-epithelial flux of peptide was dose and time-dependent, but was <3% of the amount of cydiastatin 4 present in the lumen bathing saline. In contrast, no trans-epithelial transport of peptide was apparent across everted foregut sac preparations.

Topics: Animals; Biological Transport; Chromatography, High Pressure Liquid; Digestive System; Hemolymph; Hydrolysis; Larva; Leucine; Manduca; Neuropeptides; Peptides; Phenanthrolines; Protease Inhibitors; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Incubation of Dip-AST 5 (Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-NH2) with membrane preparations of midgut, hindgut, brain, or corpora allata (CA) results in its inactivation in terms of the inhibition of juvenile hormone biosynthesis. Dip-AST 5 is initially cleaved at Gly7-Leu8 to yield the N-terminal heptapeptide (Asp-Arg-Leu-Tyr-Ser-Phe-Gly). At supraphysiological concentration, the half-life of Dip-AST 5 varied from 24 min by membrane preparations of brain to approximately 53 min following incubation with midgut membrane preparations. At more physiological concentrations (nanomolar), Dip-AST 5 was still initially cleaved to yield the inactive N-terminal heptapeptide with a half-life ranging from 23 min with brain membrane preparations to 85 min with membrane preparations of midgut. The fact that Dip-AST 5 is rapidly degraded to an inactive product by membrane preparations or whole tissues (CA) indicates that Dip-AST 5 has a different metabolic fate in tissue preparations than in diluted hemolymph (Garside et al., 1997). These findings demonstrate that the degradation of allatostatins by tissue preparations of D. punctata may play an important role in the termination of their ability to inhibit juvenile hormone biosynthesis by the CA and/or to modulate muscle activity in the hindgut.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Brain; Cell Membrane; Chromatography, High Pressure Liquid; Cockroaches; Corpora Allata; Edetic Acid; Hormone Antagonists; Kinetics; Neuropeptides; Peptide Fragments; Peptides; Phenanthrolines; Protease Inhibitors