Characterization of ecdysteroid 26-hydroxylase: An enzyme involved in molting hormone inactivation

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 r egulation of ecdysteroid titers during insect development. Administration o f the ecdysteroid agonist, RH-5849 (1,2-dibenzoyl, 1-tert-butyl hydrazone), or 20-hydroxyecdysone to the tobacco hornworm, Manduca sexta, results in i nduction of ecdysteroid 26-hydroxylase activity in midgut mitochondria and microsomes. The biochemical and kinetic properties of the ecdysteroid 26-hy droxylase were investigated. The mitochondrial enzyme was found to have opt imal 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-hydrox yecdysone substrate was lower than that of the mitochondrial enzyme for eit her 20-hydroxyecdysone or ecdysone substrate. The V-max of the 26-hydroxyla se in both subcellular fractions was slightly higher using 20-hydroxyecdyso ne as substrate compared to ecdysone, Demonstration that activity of the mi tochondrial 26-hydroxylase was inhibited by incubation in a CO (or N-2) atm osphere, taken together with the requirement for reducing cofactor and the efficacy of the P450 inhibitors, ketoconazole and fenarimol, provided stron g evidence that the hydroxylase is cytochrome P450-dependent. Indirect evid ence suggested that the mitochondrial and microsomal ecdysteroid 26-hydroxy lase(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 pro tein kinase with appropriate cofactors, In addition, the protein kinase was shown to reactivate the 26-hydroxylase activity in alkaline phosphatase-tr eated fractions. (C) 2000 academic Press.