title: Characterization of ecdysteroid 26-hydroxylase: an enzyme involved in molting hormone inactivation.
abstract: Insect molting hormone (ecdysteroid) inactivation occurs by several routes, including 26-hydroxylation and further oxidation to the 26-oic acids. Thus, the ecdysteroid 26-hydroxylase is a critical enzyme involved in precise regulation of ecdysteroid titers during insect development. Administration of the ecdysteroid agonist, RH-5849 (1,2-dibenzoyl, 1-tert-butyl hydrazone), or 20-hydroxyecdysone to the tobacco hornworm, Manduca sexta, results in induction of ecdysteroid 26-hydroxylase activity in midgut mitochondria and microsomes. The biochemical and kinetic properties of the ecdysteroid 26-hydroxylase were investigated. The mitochondrial enzyme was found to have optimal activity at a pH of 7. 5 in a Hepes or sodium phosphate buffer at 30-37 degrees C. The apparent K(m) of the microsomal 26-hydroxylase for 20-hydroxyecdysone substrate was lower than that of the mitochondrial enzyme for either 20-hydroxyecdysone or ecdysone substrate. The V(max) of the 26-hydroxylase in both subcellular fractions was slightly higher using 20-hydroxyecdysone as substrate compared to ecdysone. Demonstration that activity of the mitochondrial 26-hydroxylase was inhibited by incubation in a CO (or N(2)) atmosphere, taken together with the requirement for reducing cofactor and the efficacy of the P450 inhibitors, ketoconazole and fenarimol, provided strong evidence that the hydroxylase is cytochrome P450-dependent. Indirect evidence suggested that the mitochondrial and microsomal ecdysteroid 26-hydroxylase(s) could exist in a less active dephosphorylated state or more active phosphorylated state. Using Escherichia coli alkaline phosphatase to remove covalently bound phosphate groups, the activity of the 26-hydroxylase was decreased and, conversely, activity was enhanced using a cAMP-dependent protein kinase with appropriate cofactors. In addition, the protein kinase was shown to reactivate the 26-hydroxylase activity in alkaline phosphatase-treated fractions.
authors.affiliation: Cellular Regulation and Signaling Group, School of Biological Sciences, University of Liverpool, Life Sciences Building, Crown Street, Liverpool, L69 7ZB, United Kingdom.
authors.forename: D R
authors.initials: DR
authors.lastname: Williams
authors.forename: M J
authors.initials: MJ
authors.lastname: Fisher
authors.forename: H H
authors.initials: HH
authors.lastname: Rees
mesh_terms: D000227:Adenine Nucleotides / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002248:Carbon Monoxide / Q000378:metabolism / Q000494:pharmacology; D053493:Cholestanetriol 26-Monooxygenase; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004440:Ecdysone / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D004441:Ecdysterone / Q000378:metabolism; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D018402:Manduca / Q000166:cytology / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology; D008928:Mitochondria / Q000201:enzymology; D009584:Nitrogen / Q000378:metabolism / Q000494:pharmacology; D010100:Oxygen / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation; D011190:Potassium Cyanide / Q000494:pharmacology; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013696:Temperature
chemical_list: D000227:Adenine Nucleotides; D065607:Cytochrome P-450 Enzyme Inhibitors; D004440:Ecdysone; D004441:Ecdysterone; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D053493:Cholestanetriol 26-Monooxygenase; D017868:Cyclic AMP-Dependent Protein Kinases; D000469:Alkaline Phosphatase; D011190:Potassium Cyanide; D009584:Nitrogen; D010100:Oxygen