Cytochrome P-450-catalyzed formation of 20-hydroxy-ecdysone in larval housefly mitochondria.
Six forms of cytochrome P-450 in the mitochondria of larvae from Musca domestica were isolated by solubilization with CHAPS followed by ammonium sulfate fractionation and HPLC on an anion-exchange column. Forms 1, 2, 3, 5, and 6 catalyzed the formation of 20-hydroxy-ecdysone from ecdysone in the presence of NADPH and pig adrenal adrenodoxin and adrenodoxin reductase at rates not much different that observed in mitochondria; whereas, fraction 4 showed an activity which was about 10-fold higher than mitochondria. Forms 4 and 5 were further purified by HPLC on a cation-exchange column followed by removal of excess detergent by hydroxyl apatite column chromatography. In vitro reconstitution of the monooxygenase activity confirmed that form 4 is primarily involved in the formation of 20-hydroxy-ecdysone from ecdysone. SDS-polyacrylamide gel electrophoresis indicated a high degree of purity of both forms 4 and 5, with molecular weights of 56 and 58 KDa, respectively.
Srivatsan
J
J
Department of Zoology, University of Georgia, Athens 30602.
Weirich
M
M
Agosin
M
M
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D004441:Ecdysterone / Q000096:biosynthesis*; D006793:Houseflies / Q000378:metabolism*; D007814:Larva; D008928:Mitochondria / Q000378:metabolism*; D008970:Molecular Weight; D013250:Steroid Hydroxylases / Q000302:isolation & purification / Q000378:metabolism*
D004441:Ecdysterone; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C020095:ecdysone 20-hydroxylase