The effect of substrate on the expression of activity catalyzed by cytochrome P-450: metabolism mediated by rabbit isozyme 6 in pulmonary microsomal and reconstituted monooxygenase systems.
The content of cytochrome P-450, isozyme 6, in the rabbit pulmonary microsomal fraction was estimated by immunochemical methods to be 1 to 3% of the total cytochrome P-450. Following treatment of rabbits with 2,3,7,8-tetrachlorodibenzo-p-dioxin, the pulmonary microsomal concentration of isozyme 6 increased 16-fold. Isozyme 6 was also detected by immunochemical methods, but not by electrophoresis and staining for protein, in preparations of isozyme 5 isolated from the pulmonary microsomal fraction of untreated rabbits. The metabolism of benzo[a]pyrene in these preparations was found to be catalyzed by isozyme 6, not by isozyme 5 as previously concluded. Cytochrome P-450, isozyme 4, was not detected in the pulmonary microsomal fraction from untreated or 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rabbits. Although benzo[a]pyrene and 7-ethoxyresorufin are both substrates for isozyme 6, the pulmonary microsomal metabolism of these compounds was not increased to the same extent by treatment of rabbits with 2,3,7,8-tetrachlorodibenzo-p-dioxin (about 13-fold for 7-ethoxyresorufin and less than 2-fold for BP). However, lack of agreement between increases in isozyme 6 content and activity, and between the relative increases of the activities with the two substrates, was overcome by the addition of purified NADPH-cytochrome P-450 reductase to the microsomal incubations. When alpha-naphthoflavone, at the minimum concentration required for greater than 90% inhibition of isozyme 6 catalysis, was present in the incubations, no increases in activity were obtained by the addition of purified reductase. The turnover numbers of isozyme 6 in microsomal preparations incubated with purified reductase were similar to those of the purified isozyme in a reconstituted monooxygenase system. The relevance of our results to determinations of the substrate specificities and the microsomal concentrations and activities of isozymes of cytochrome P-450 is discussed. In addition, these parameters are used to assess the extent to which the catalytic potential of isozyme 6 is expressed in the rabbit pulmonary microsomal fraction.
D000818:Animals; D001564:Benzo(a)pyrene / Q000378:metabolism; D001571:Benzoflavones / Q000494:pharmacology; D002384:Catalysis; D002848:Chromatography, DEAE-Cellulose; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008168:Lung / Q000201:enzymology*; D008297:Male; D008861:Microsomes / Q000201:enzymology; D008862:Microsomes, Liver / Q000201:enzymology; D010078:Oxazines / Q000378:metabolism; D010105:Oxygenases / Q000378:metabolism; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D011817:Rabbits
D001571:Benzoflavones; D065607:Cytochrome P-450 Enzyme Inhibitors; D007527:Isoenzymes; D010078:Oxazines; D000072317:Polychlorinated Dibenzodioxins; D001564:Benzo(a)pyrene; C007649:ethoxyresorufin; C011512:alpha-naphthoflavone; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases