Preparation and properties of partially purified pulmonary cytochrome P-450 from rabbits.
Cytochrome P-450 from rabbit pulmonary microsomes was purified approximately 32-fold. The purification method involved solubilization of microsomes using sodium cholate, and recovery of cytochrome P-450 in the precipitate formed between 25 to 42% saturation of the digested microsomes with ammonium sulfate in the absence of glycerol. Further purification was achieved by chromatography on DEAE-cellulose and hydroxylapatite using Emulgen 913 as an eluent. Partially purified preparations containing up to 7.4 nmol of cytochrome P-450 per mg of protein were essentially free of NADPH-cytochrome c reductase activity and cytochromes b5 and P-420. However, epoxide hydrase was found to co-purify with cytochrome P-450. The CO-difference spectrum of dithionite-reduced purified cytochrome showed the expected peak at 450 nm. However, the magnitude of the peak was dependent on added microsomal lipid fraction in the assay medium. Purified pulmonary cytochrome P-450 formed typical types I and II substrate difference spectra with benzphetamine and pyridine, respectively. Sodium dodecyl sulfate-gel electrophoresis of partially purified cytochrome P-450 gave two major bands when stained with Coomassie blue. The faster moving band which contained peroxidase activity had an estimated molecular weight of 49,000 +/- 1,200. The cytochrome P-450 fraction, when combined with solubilized pulmonary microsomal NADPH-cytochrome c reductase and lipid fractions, was active in the O-deethylation of 7-ethoxycoumarin and the N-demethylation of benzphetamine.
D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism; D008168:Lung / Q000201:enzymology*; D008297:Male; D008861:Microsomes / Q000201:enzymology*; D008970:Molecular Weight; D010088:Oxidoreductases / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism; D011817:Rabbits; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet
D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D010089:Oxidoreductases, N-Demethylating