Ecdysone 20-monooxygenase: characterization of an insect cytochrome p-450 dependent steroid hydroxylase.
Ecdysone 20-monooxygenase, the enzyme system that hydroxylates ecdysone at C-20 of the side-chain to form ecdysterone, has been characterized in the fat body of early last instar larvae of the tobacco hornworm, Manduca sexta, using a radioenzymological assay. Ecdysterone was demonstrated to be the product of the enzyme system by high-pressure liquid chromatography, gas-liquid chromatography and mass spectrometry. Differential centrifugation, sucrose-gradient centrifugation, electron microscopy and organelle-marker enzyme analysis revealed that ecdysone 20-monooxygenase activity is associated with the mitochondria. The enzymatic properties of ecdysone 20-monooxygenase are that it is most active in a 0.05 M phosphate buffer, is inhibited by Mg2+ and exhibits pH and temperature optima at 7.5 and 30 degrees C, respectively. The enzyme complex has an apparent Km for ecdysone of 1.60 x 10(-7) M and is competitively inhibited by its product, ecdysterone, with an apparent Ki of 2.72 x 10(-5) M. The cytochrome P-450 nature of this insect steroid hydroxylase was initially suggested by its obligate requirement for NADPH and its inhibition by carbon monoxide, p-chloromercuribenzoate, metyrapone and p-aminoglutethimide but not by cyanide. Difference spectroscopy revealed the presence of cytochrome P-450 in the fat-body mitochondrial fraction. A photochemical action spectrum of ecdysone 20-monooxygenase activity confirmed the involvement of cytochrome P-450 in this monooxygenase system.
D000818:Animals; D002248:Carbon Monoxide; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004440:Ecdysone; D005216:Fat Body / Q000201:enzymology; D007700:Kinetics; D007814:Larva / Q000201:enzymology; D007915:Lepidoptera / Q000201:enzymology*; D008928:Mitochondria / Q000201:enzymology; D009036:Moths / Q000201:enzymology*; D013250:Steroid Hydroxylases / Q000302:isolation & purification / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology
D004440:Ecdysone; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases