Electrophoretic, spectral, catalytic and immunochemical properties of highly purified cytochrome P-450 from sheep lung.
1. Cytochrome P-450LgM2 was purified from sheep lung microsomes in the presence of detergents, Emulgen 913 and cholate. 2. The purification procedure involved the chromatography of the detergent solubilized microsomes on DEAE-cellulose and hydroxylapatite. 3. Cytochrome P-450LgM2 was further purified on second DEAE-cellulose and hydroxylapatite columns. 4. The specific content of the highly purified P-450LgM2 was 16-18 nmol P-450/mg protein and purified 164-fold. 5. The yield was 16% of the initial content in microsomes. 6. The SDS-polyacrylamide slab gel electrophoresis (PAGE) of the purified lung cytochrome P-450LgM2 showed one protein band having the monomer molecular weight of 49,500. 7. The absolute CO-difference spectrum of dithionate-reduced P-450LgM2 gave a peak at 451 nm. 8. When sheep lung cytochrome P-450LgM2 and P-450LM2 purified from liver of phenobarbital (PB)-induced rabbit were subjected to Western Blotting and visualized immunochemically with anti-P-450LM2, they showed identical mobilities. 9. P-450LgM2 was found to be very active in N-demethylation of benzphetamine in a reconstituted system containing purified sheep lung reductase and synthetic lipid. 10. Turnover numbers (min-1) for benzphetamine, aniline, ethylmorphine and p-nitrophenol were determined to be 273, 1.2, 15.5 and 1.05, respectively, in a reconstituted microsomal lung monooxygenase system. 11. Spectral, electrophoretic, biocatalytic and immunochemical properties of sheep lung P-450LgM2 were found to be similar to those of P-450 isozyme 2, purified from PB-treated rabbit liver and of rabbit lung microsomes.
Adali
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O
Department of Biology, Middle East Technical University, Ankara, Turkey.
Arinç
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E
D000818:Animals; D015153:Blotting, Western; D002848:Chromatography, DEAE-Cellulose; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*; D003580:Cytochromes / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005037:Ethylmorphine-N-Demethylase / Q000378:metabolism; D007150:Immunohistochemistry; D008168:Lung / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology*; D008862:Microsomes, Liver / Q000201:enzymology; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification; D011817:Rabbits; D012756:Sheep; D013056:Spectrophotometry, Ultraviolet
D003580:Cytochromes; D003577:Cytochrome P-450 Enzyme System; D005037:Ethylmorphine-N-Demethylase; D009251:NADPH-Ferrihemoprotein Reductase