Reconstitution of the rabbit pulmonary microsomal mixed-function oxidase system from solubilized components

Area of Science:

• Biochemistry
• Enzymology
• Pharmacology

Background:

• Microsomal enzymes, including cytochrome P-450 and NADPH-cytochrome c reductase, are crucial for drug metabolism.
• Understanding the reconstitution of these enzyme systems is vital for studying drug interactions and toxicity.

Purpose of the Study:

• To solubilize and purify lung cytochrome P-450 and NADPH-cytochrome c reductase.
• To reconstitute enzyme activities and investigate factors influencing drug hydroxylation.
• To compare the effectiveness of different reductase preparations and identify essential cofactors.

Main Methods:

• Solubilization and purification of lung microsomal cytochrome P-450 and NADPH-cytochrome c reductase using sonication, detergent digestion, and chromatography.
• Reconstitution assays combining purified enzyme fractions to measure benzphetamine N-demethylase and 7-ethoxycoumarin deethylase activities.
• Investigation of the role of different reductase preparations, lipid fractions, and NADH in enzyme activity.

Main Results:

• Purified NADPH-cytochrome c reductase fractions (4-6 fold) and cytochrome P-450 fractions (2 fold) were obtained.
• Reconstitution of benzphetamine N-demethylase and 7-ethoxycoumarin deethylase activities was achieved by combining enzyme fractions.
• Reductase fractions from sodium cholate digestion were more effective than those from sodium deoxycholate.
• Higher concentrations of reductase were needed for 7-ethoxycoumarin deethylase compared to benzphetamine N-demethylase.
• A heat-stable lipid fraction was essential for maximal hydroxylation activity.

Conclusions:

• Successful reconstitution of lung drug-metabolizing enzymes was demonstrated.
• The choice of detergent and the presence of a lipid cofactor significantly impact enzyme activity.
• Differential cofactor requirements suggest distinct properties of the reconstituted enzyme systems for different substrates.