Title of article :
Characterization of Ecdysteroid 26-Hydroxylase: An Enzyme Involved in Molting Hormone Inactivation
Author/Authors :
Williams، نويسنده , , Daryl R. and Fisher، نويسنده , , Michael J. and Rees، نويسنده , , Huw H. Rees، نويسنده ,
Issue Information :
روزنامه با شماره پیاپی سال 2000
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°C. The apparent Km 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 Vmax 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 N2) 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.
Keywords :
Ecdysteroid , 26-hydroxylase , cytochrome P450 , Manduca sexta.
Journal title :
Archives of Biochemistry and Biophysics
Journal title :
Archives of Biochemistry and Biophysics