title: Metabolism of an insect diuretic hormone by Malpighian tubules studied by liquid chromatography coupled with electrospray ionization mass spectrometry.
abstract: The larger of two diuretic hormones of the tobacco hornworm, Manduca sexta, (Mas-DH) is a peptide of 41 residues. It is one of a family of seven currently known insect diuretic hormones that are similar to the corticotropin-releasing factor-urotensin-sauvagine family of peptides. We investigated the possible inactivation of Mas-DH by incubating it in vitro with larval Malpighian tubules (Mt), the target organ of the hormone. The medium was analyzed, and degradation products were identified, using on-line microbore reversed-phase liquid chromatography coupled to electrospray ionization mass spectrometry (RPLC-ESI-MS). This sensitive technique allows identification of metabolites of Mas-DH (present at an initial level of approximately 1 microM). An accurate Mr value for a metabolite is usually sufficient for unambiguous identification. Mas-DH is cleaved by Mt proteases initially at L29-R30 and R30-A31 under our assay conditions; some Mas-DH is also oxidized, apparently at M2 and M11. The proteolysis can be inhibited by 5 mM EDTA, suggesting that divalent metals are needed for peptide cleavage. The oxidation of the hormone can be inhibited by catalase or 1 mM methionine, indicating that H2O2 or related reactive oxygen species are responsible for the oxidative degradation observed. RPLC-ESI-MS is shown here to be an elegant and efficient method for studying peptide hormone metabolism resulting from unknown proteases and pathways.
authors.affiliation: Department of Biochemistry, University of Nevada, Reno, NV 89557, USA.
authors.forename: H
authors.initials: H
authors.lastname: Li
authors.forename: H
authors.initials: H
authors.lastname: Wang
authors.forename: K M
authors.initials: KM
authors.lastname: Schegg
authors.forename: D A
authors.initials: DA
authors.lastname: Schooley
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid; D004231:Diuresis; D010450:Endopeptidases / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D066298:In Vitro Techniques; D007301:Insect Hormones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007814:Larva / Q000378:metabolism; D008317:Malpighian Tubules / Q000378:metabolism*; D018402:Manduca / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D007301:Insect Hormones; D017382:Reactive Oxygen Species; C109616:diuretic hormone, Manduca sexta; D006861:Hydrogen Peroxide; D010450:Endopeptidases