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

Arch Biochem Biophys. 2000 Apr 15;376(2):389-98. doi: 10.1006/abbi.2000.1731.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenine Nucleotides / metabolism
  • Alkaline Phosphatase / metabolism
  • Animals
  • Carbon Monoxide / metabolism
  • Carbon Monoxide / pharmacology
  • Cholestanetriol 26-Monooxygenase
  • Cyclic AMP-Dependent Protein Kinases / antagonists & inhibitors
  • Cyclic AMP-Dependent Protein Kinases / metabolism
  • Cytochrome P-450 Enzyme Inhibitors
  • Cytochrome P-450 Enzyme System / metabolism
  • Ecdysone / antagonists & inhibitors*
  • Ecdysone / metabolism*
  • Ecdysterone / metabolism
  • Enzyme Activation
  • Hydrogen-Ion Concentration
  • Kinetics
  • Manduca / cytology
  • Manduca / enzymology*
  • Microsomes / enzymology
  • Mitochondria / enzymology
  • Nitrogen / metabolism
  • Nitrogen / pharmacology
  • Oxygen / metabolism
  • Oxygen / pharmacology
  • Phosphorylation
  • Potassium Cyanide / pharmacology
  • Steroid Hydroxylases / antagonists & inhibitors
  • Steroid Hydroxylases / metabolism*
  • Temperature

Substances

  • Adenine Nucleotides
  • Cytochrome P-450 Enzyme Inhibitors
  • Ecdysone
  • Ecdysterone
  • Carbon Monoxide
  • Cytochrome P-450 Enzyme System
  • Steroid Hydroxylases
  • Cholestanetriol 26-Monooxygenase
  • Cyclic AMP-Dependent Protein Kinases
  • Alkaline Phosphatase
  • Potassium Cyanide
  • Nitrogen
  • Oxygen