Characterization of a rat lung microsomal fraction obtained by sepharose 2B ultrafiltration.
A new procedure for obtaining rat lung microsomes essentially free of interfering hemoproteins has been developed. The method includes Sepharose 2B column chromatography of the 12,000 X g supernatant of lung homogenates, followed by ultracentrifugation of the material eluted in the void volume. Microsomes isolated in this manner contain specific levels of cytochromes b5 and P-450 and of NADPH-cytochrome c reductase that are among the highest ever reported for a rat lung microsomal fraction. After treatment of rats with 3-methylcholanthrene, the specific content of cytochrome P-450 in lung microsomes is doubled and that of cytochrome b5 increases 1.5 times. Several spectral differences between hepatic and lung microsomal cytochrome P-450 are apparent. In lung microsomes, the maximum of the reduced CO-bound cytochrome complex in a difference spectrum is at 453 nm for the noninduced hemoprotein and shifts to 451 nm after 3-methylcholanthrene induction. In contrast, no significant change in the ethylisocyanide difference spectra of reduced microsomes is obtained after induction; moreover, the spectra obtained with induced and noninduced cytochrome P-450 are similar to the one shown by hepatic microsomes from polycyclic hydrocarbon-treated rats. Furthermore, spectrophotometric studies on n-octylamine binding to control and induced lung cytochrome P-450 yielded results different from those previously obtained with rabbit liver microsomes. It is concluded that the cytochrome P-450 present in rat lung microsomes before and after 3-methylcholanthrene treatment of the animals is distinctly different from the liver hemoprotein.
D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002847:Chromatography, Agarose; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification; D003580:Cytochromes / Q000302:isolation & purification; D008168:Lung / Q000648:ultrastructure*; D008297:Male; D008748:Methylcholanthrene / Q000494:pharmacology; D008861:Microsomes / Q000378:metabolism* / Q000648:ultrastructure; D008862:Microsomes, Liver / Q000032:analysis; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification; D010634:Phenobarbital / Q000494:pharmacology; D011335:Proadifen / Q000494:pharmacology; D051381:Rats; D013057:Spectrum Analysis; D013268:Stimulation, Chemical; D014461:Ultracentrifugation
D003580:Cytochromes; D008748:Methylcholanthrene; D003577:Cytochrome P-450 Enzyme System; D011335:Proadifen; D009251:NADPH-Ferrihemoprotein Reductase; D010634:Phenobarbital