Characterization of hepatic and pulmonary cytochromes P-450 in 3-methylcholanthrene-treated hamsters

Abstract

Two major forms of hepatic cytochrome P-450 (hepatic P-450_MCI and P-450_MCII) were purified approximately 5-fold from liver microsomes in Syrian golden hamsters treated with 3-methylcholanthrene (MC). The purified preparations of hepatic P-450_MCI and P-450_MCII contained 9.6 and 8.3 nmol cytochrome P-450 (P-450) per mg protein, respectively, and were essentially free from NADPH-cytochrome c (P-450) reductase (fpT), NADH-cytochrome b₅ reductase and cytochrome b₅. By sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weights of hepatic P-450_MCI and P-450 _mMCII were estimated to be 56 000 and 53 500. Further, a major form of pulmonary P-450 (P-450_MC) were purified from lung microsomes of MC-treated hamster, and contained 14.2 nmol P-450 per mg protein, and estimated to be 56 000 in monomeric molecular weight, indicating the similar molecular weight to hepatic P-450_MCI in the hamster. From the absorption spectra the oxidized forms of hepatic P-450_MCI and P-450_MCII were high- and low-spin ferric hemoproteins, respectively, and pulmonary P-450_MC was similar to hepatic P-450_MCII in their hemoprotein spin state. No difference, however, was observed in the CO-reduced forms among hepatic P-450_MCI, P-450_MCII and pulmonary P-450_MC, all exhibiting 446.5 nm Soret bands. In a reconstituted system containing fpT and dilauroylphos-phatidylcholine (DLPC), pulmonary P-450_MC efficiently catalyzed benzo[a]pyrene (BP) hydroxylation at a rate of 11.4 mol formed per min per mol P-450, but hepatic P-450_MCI and P-450_MCII both exhibited lower levels, e. g., 0.49 and 0.54, respectively. These findings indicated a clear tissue difference in the activity of BP hydroxylation between lung and liver in MC-treated hamsters.