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Characterization of a Rat Lung Microsomal Fraction Obtained by Sepharose 2B Ultrafiltration¹

Department of Forensic Medicine, Karolinska Institutet, S-104 01 Stockholm 60, Sweden

² To whom reprint requests should be addressed.

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.

¹ This work was supported by NIH Contract = CP 33363, "Studies on Polycyclic Hydrocarbon Metabolism in the Respiratory Tract."

³ Present address: Farmaceuticka fakulta UK, Bratislava, CSSR.

Received 2/27/75. Accepted 6/ 3/75.