Characterization of a Rat Lung Microsomal Fraction Obtained by Sepharose 2B UItrafiitration

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 • g supernatant of lung homogehates, followed by ultracentrifugation of the material eluted in the void volume. Microsomes isolated in this manner contain specific levels of cytochromes bs 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 bs 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 3methylcholanthrene 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.