Metabolism of an insect diuretic hormone by Malpighian tubules studied by liquid chromatography coupled with electrospray ionization mass spectrometry.
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.
Li
H
H
Department of Biochemistry, University of Nevada, Reno, NV 89557, USA.
Wang
H
H
Schegg
K M
KM
Schooley
D A
DA
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
D007301:Insect Hormones; D017382:Reactive Oxygen Species; C109616:diuretic hormone, Manduca sexta; D006861:Hydrogen Peroxide; D010450:Endopeptidases