Inactivation of neuropeptide hormones (AKH I and AKH II) studied in vivo and in vitro

Rayne, Richard C.; O’Shea, Michael

doi:10.1016/0965-1748(92)90096-w

Cited by 51 publications

(28 citation statements)

References 22 publications

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“…However, such information in insect systems is quite limited. Adipokinetic hormones are degraded by metallopeptidases from tissue and membrane preparations of various species (10,11,(35)(36)(37). Proctolin (14,25) and pheromone biosynthesis-activating neuropeptide (38) are degraded by metalloproteases in different insect tissues.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, Mt have been shown to degrade some other insect neuropeptides (10)(11)(12)(13). Peptide hormone degradation products have been routinely analyzed by using separation by reversed-phase liquid chromatography (RPLC) and identified by comparing retention properties of the products to those of a number of synthetic peptide standards representing possible metabolites.…”

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confidence: 99%

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Metabolism of an insect diuretic hormone by Malpighian tubules studied by liquid chromatography coupled with electrospray ionization mass spectrometry

Li¹,

Wang²,

Schegg³

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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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 corticotropinreleasing 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 . 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 H 2 O 2 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.Water balance and ion balance in insects are regulated by the actions of diuretic hormones and antidiuretic hormones. The larger of two diuretic hormones (DH) of the tobacco hornworm Manduca sexta (Mas-DH), RMPSLSIDLPMSVL-RQKLSLEKERKVHALRAAANRNFLNDI-NH 2 , is an amidated peptide with 41 residues (1). It was the first peptide identified of a family of insect diuretic peptides related to corticotropin-releasing factor (CRF), sauvagine, and urotensin I (2, 3). Subsequently, a 30-amino acid DH homologous to Mas-DH was identified from the same species (4). Mas-DH activates urine production in M. sexta Malpighian tubules (Mt) by stimulating cAMP production and, eventually, KCl and NaCl cotransport, causing osmotically driven fluid excretion from hemolymph into the lumen of Mt (5, 6). A G-proteincoupled Mas-DH receptor with seven putative transmembrane domains has been cloned from M. sexta Mt and functionally expressed (7,8). Termination of Mas-DH-activated diuresis is believed to be accomplished by the cessation of Mas-DH production coupled with its rapid removal from hemolymph by unknown mechanisms.Here we report on the metabolism of Mas-DH by M. sexta Mt after incubation in vitro. Mt were chosen because they are the putative target organ of Mas-DH, and they have been found to decrease diuretic activities of crude tissue extracts in four insect species (9). In addition, Mt have been shown to degrade some other insect neuropeptides (10-13). Peptide hormone degradation products have been routinely analyzed by using separation by reversed-phase liquid chromatography (RPLC) and identified by comparing retention properties of the products to those of a number of synthetic peptide standards representing possible metabolites. The products have been detected by UV absorbance (14), by ELISA or RIA (15), by radioactivity (12, 16), from fluorescently labeled or chromogen-modified peptides (15), by amino acid analysis (AAA) of fractions (17), or by Edman degradat...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Metabolism of an insect diuretic hormone by Malpighian tubules studied by liquid chromatography coupled with electrospray ionization mass spectrometry

Li¹,

Wang²,

Schegg³

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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“…In fact, the sum total areas of substrate, product(s), and endogenous AKH I declines approximately 9-12%/hour of incubation. This decline is most likely attributable to an endogenous AKH-degrading endopeptidase present in the corpora cardiaca extract (Isaac, 1987(Isaac, , 1988Rayne and O'Shea, 1992) which cleaves the Asn-Phe bond.…”

Section: Characterization Of Carboxypeptidase Activitymentioning

confidence: 99%

Reconstitution of adipokinetic hormone biosynthesis in vitro indicates steps in prohormone processing

Rayne

O’Shea

1994

European Journal of Biochemistry

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We have used a complete, synthetic precursor to adipokinetic hormone I (AKH I) and oligopeptides derived from this precursor as substrates for prohormone-processing enzymes extracted from AKH-synthesizing neurosecretory cells to reconstitute the post-translational steps in AKH biosynthesis in vitro. The results demonstrate the existence of endoproteolytic activity which cleaves the precursor only at the appropriate processing site (at the C-terminal side of Argl3). Further proteolytic processing of C-terminally extended AKH I (AKH-Gly-Lys-Arg) by a carboxypeptidase H-like activity removes the basic residues producing AKH-Gly-Lys, followed by AKH-Gly. Finally, a peptidylglycine-a-amidating-monooxygenase activity produces the amidated bioactive product from the glycine-extended peptide in a two-step process, the first of which requires ascorbate and Cu". Our results show that all steps in AKH precursor processing can be reconstituted and studied in vitro, providing a system to characterize the processing enzymes and to investigate the development of enzyme inhibitors for use as potential insecticides.Neuropeptides are produced by specific, limited proteolysis of larger precursor polypeptides, or prohormones (Docherty and Steiner, 1982;Sossin et al., 1989;Darby and Smyth, 1990). Prohormones are cleaved by endoproteases, often at sites adjacent to pairs of basic amino acid residues (e.g. lysine, arginine). This initial step has been attributed in some cases to members of a recently identified superfamily of subtilisin-like endopeptidases referred to as prohormone convertases (Steiner et al., 1992). Endoproteolytic cleavage is followed by removal of basic residues from C-terminally extended processing intermediates by carboxypeptidase H (also referred to as carboxypeptidase E; Fricker, 1988;Skidgel, 1988). For neuropeptides which require C-terminal amidation, glycine is normally the residue which, via the action of the peptidylglycine-a-amidating-monooxygenase (PAM) enzymes, contributes its amino group to form an amidated C-terminus (Eipper et al., 1992).

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“…Since these primary degradation products are devoid of any adipokinetic activity (22)(23)(24) (25)(26)(27)(28).…”

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confidence: 99%

“…Studies on the degradation of AKH-I and -II in the desert locust, Schistocerca gregaria, performed to get insight into effective inactivation and to obtain quantitative data for their half-lives, have resulted in the elucidation of their complete degradation pathways (22); The first step is the cleavage of the N-terminal tripeptide pGlu-Leu-Asn, which they have in common, resulting in a corresponding hepta-or pentapeptide. Since these primary degradation products are devoid of any adipokinetic activity (22)(23)(24) (25)(26)(27)(28).…”

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confidence: 99%

Locust adipokinetic hormones: carrier-independent transport and differential inactivation at physiological concentrations during rest and flight.

Oudejans

Vroemen²,

Jansen³

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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Since concomitant release of structurally related peptide hormones with apparently similar functions seems to be a general concept in endocrinology, we have studied the dynamics of the lifetime of the three known adipokinetic hormones (AKHs) of the migratory locust, which control flight-directed mobilization of carbohydrate and lipid from fat body stores. Although the structure of the first member of the AKHs has been known for 20 years, until now, reliable data on their inactivation and removal from the hemolymph are lacking, because measurement requires AKHs with high specific radioactivity. Employing tritiated AKHs with high specific radioactivity, obtained by catalytic reduction with tritium gas of the dehydroLeu2 analogues of the AKHs synthesized by the solid-phase procedure, studies with physiological doses of as low as 1.0 pmol per locust could be conducted. The AKHs appear to be transported in the hemolymph in their free forms and not associated with a carrier protein, despite their strong hydrophobicity. Application of AKHs in their free form in in vivo and in vitro studies therefore now has been justified. We have studied the degradation of the three AKHs during rest and flight. The first cleavage step by an endopeptidase is crucial, since the resulting degradation -products lack any adipokinetic activity. Half-lives for AKH-I, -II and -m were 51, 40, and 5 min, respectively, for rest conditions and 35, 37, and 3 min, respectively, during flight.

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Inactivation of neuropeptide hormones (AKH I and AKH II) studied in vivo and in vitro

Cited by 51 publications

References 22 publications

Metabolism of an insect diuretic hormone by Malpighian tubules studied by liquid chromatography coupled with electrospray ionization mass spectrometry

Metabolism of an insect diuretic hormone by Malpighian tubules studied by liquid chromatography coupled with electrospray ionization mass spectrometry

Reconstitution of adipokinetic hormone biosynthesis in vitro indicates steps in prohormone processing

Locust adipokinetic hormones: carrier-independent transport and differential inactivation at physiological concentrations during rest and flight.

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