Reconstitution of the rabbit pulmonary microsomal mixed-function oxidase system from solubilized components.
Lung microsomal cytochrome P-450 was solubilized and purified 2-fold. NADPH-cytochrome c reductase (EC 1.6.2.3) was solubilized and purified 4-6 fold by three methods with use of sonication and detergent digestion followed by either DEAE-cellulose chromatography or ammonium sulfate fractionation. Benzphetamine N-demethylase and 7-ethoxycoumarin deethylase activities were reconstituted when NADPH-cytochrome c reductase and cytochrome P-450 fractions were combined. Reductase fractions prepared by sodium cholate digestion of microsomes were highly active in supporting the hydroxylation activity in the reconstituted systems, whereas those prepared with sodium deoxycholate were not. About twice as much NADPH-cytochrome c reductase was required for saturation of the 7-ethoxycoumarin deethylase activity as for saturation of the benzphetamine N-demethylase activity. A heat-stable lipid fraction was necessary for maximum hydroxylation activity. NADH did not support benzphetamine N-demethylation in the reconstituted system or increase the rate of reaction when added with NADPH.
D000818:Animals; D002848:Chromatography, DEAE-Cellulose; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism; D003579:Cytochrome Reductases / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D050356:Lipid Metabolism; D008168:Lung / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000302:isolation & purification / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*; D011817:Rabbits; D012995:Solubility
D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D003579:Cytochrome Reductases