Ecdysone 20-mono-oxygenase in the desert locust, Schistocerca gregaria.
The enzyme catalysing the hydroxylation of ecdysone to 20-hydroxyecdysone, ecdysone 20-mono-oxygenase (EC 1.14.99.22), was investigated in the Malpighian tubules of fifth-instar locusts, Schistocerca gregaria. Enzyme activity was optimal at 35 degrees C and pH 6.8-8.0. Under these conditions the mono-oxygenase exhibited an apparent Km for ecdysone of 7.1 X 10(-7) M, a maximal specific activity of 1.1 nmol/h per mg of protein and was competitively inhibited by 20-hydroxyecdysone with an apparent Ki of 6.3 X 10(-7) M. Enzyme activity was decreased in the presence of Ca2+, Mg2+, EDTA and non-ionic detergents. The Malpighian tubule ecdysone 20-mono-oxygenase was localized primarily in the subcellular fraction sedimenting at 7500 g and, on the basis of marker enzyme profiles, was assigned mainly to the mitochondria. NADPH was required for activity, although addition of NADH together with NADPH had a synergistic effect. NADP+-dependent isocitrate dehydrogenase (EC 1.1.1.42) and an energy-dependent NAD(P) transhydrogenase (EC 1.6.1.1.) appeared to be the major sources of reducing equivalents, with the contribution from the 'malic enzyme' (EC 1.1.1.40) being less important. The monooxygenase was characterized as a cytochrome P-450-containing mixed-function oxidase from the inhibition patterns with metyrapone, CO and cyanide; CO inhibition was reversible with monochromatic light at 450 nm. However, the ecdysone 20-mono-oxygenase shows much lower sensitivity to CO inhibition and to photodissociation of the CO-inhibited complex than do vertebrate cytochrome P-450-dependent hydroxylation systems. The concentration of cytochrome P-450 in the Malpighian tubule mitochondria was 30 pmol/mg of protein. The properties of the mono-oxygenase are discussed in relation to hydroxylation enzymes from other sources.
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002413:Cations, Divalent / Q000494:pharmacology; D002498:Centrifugation; D002988:Cloaca / Q000201:enzymology*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D006110:Grasshoppers / Q000201:enzymology*; D008317:Malpighian Tubules / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology
D002413:Cations, Divalent; D009243:NAD; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D004492:Edetic Acid; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C020095:ecdysone 20-hydroxylase