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Pulmonary microsomal cytochrome P-450 from 3-methylcholanthrene-treated hamsters: purification, characterization, and
Abstract:
A major form of pulmonary cytochrome P-450 (pulmonary P-450MC) was purified approximately 165-fold from lung microsomes of 3-methylcholanthrene (MC)-treated hamsters. The purified preparation contained 14.2 nmol of cytochrome P-450 (P-450) per mg protein and was essentially free from NADPH-cytochrome P-450 (cytochrome c)-reductase (NADPH-reductase) and epoxide hydrolase. Pulmonary P-450MC exhibits an absorption maximum at 446.5 nm in the difference spectrum of reduced hemoprotein-CO complex, and a low-spin state of ferric iron in the heme. By sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, the molecular weight of pulmonary P-450MC was estimated to be 56,000. In a reconstituted system, pulmonary P-450MC efficiently catalyzed benzo(a)pyrene (BP) hydroxylation, but showed low activities for 7-ethoxycoumarin O-deethylation and benzphetamine N-demethylation. In Ouchterlony double diffusion analysis, hamster pulmonary P-450MC reacted to the antibody prepared against rat hepatic P-450MC to form a faint precipitation line with a spur, indicating that the two P-450MCs have a common antigenic site but are not immunologically identical. When incubated with [14C]BP in a reconstituted system containing NADPH-reductase and epoxide hydrolase, hamster pulmonary P-450MC formed much higher amounts of BP diols, especially 7,8-diol, than were formed by rat pulmonary P-450MC.
Insights
Researchers purified pulmonary cytochrome P-450MC from hamsters, finding it efficiently hydroxylates benzo(a)pyrene. This enzyme shows distinct activity and immunological properties compared to rat P-450MC.
Area of Science:
- Biochemistry
- Pharmacology
- Toxicology
Background:
- Pulmonary cytochrome P-450 (P-450) enzymes play a crucial role in metabolizing xenobiotics in the lungs.
- Understanding specific pulmonary P-450 isoforms, like P-450MC, is vital for assessing lung toxicity and drug metabolism.
Purpose of the Study:
- To purify and characterize a major pulmonary cytochrome P-450 form (P-450MC) from 3-methylcholanthrene-treated hamsters.
- To investigate the catalytic activity and immunological properties of the purified hamster pulmonary P-450MC.
Main Methods:
- Purification of pulmonary P-450MC from hamster lung microsomes.
- Spectroscopic analysis (reduced CO-difference spectrum) and SDS-polyacrylamide gel electrophoresis for characterization.
- Enzyme activity assays in reconstituted systems (e.g., benzo(a)pyrene hydroxylation).
- Immunological analysis using Ouchterlony double diffusion with antibodies against rat hepatic P-450MC.
Main Results:
- Pulmonary P-450MC was purified ~165-fold, exhibiting a CO-binding peak at 446.5 nm and a molecular weight of 56,000.
- The purified enzyme efficiently catalyzed benzo(a)pyrene hydroxylation, forming higher amounts of BP diols, particularly 7,8-diol, compared to rat pulmonary P-450MC.
- Hamster pulmonary P-450MC showed immunological cross-reactivity with rat hepatic P-450MC but was not identical.
Conclusions:
- Hamster pulmonary P-450MC is a distinct enzyme form with potent benzo(a)pyrene hydroxylase activity.
- This enzyme contributes significantly to pulmonary metabolism of polycyclic aromatic hydrocarbons.
- Comparative analysis highlights species- and tissue-specific differences in P-450 enzyme systems.