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Interactions between solubilized cytochrome P-450 and hepatic microsomes
Abstract:
Solubilized cytochromes P-450 and P-448 have been prepared from liver microsomes of phenobarbital- and 3-methylcholanthrene-pretreated rats, respectively. These hemoproteins can bind to microsomes and increase the microsomal monoxygenase activities. The binding of cytochrome P-450 enhances the microsomal benzphetamine demethylase activity, whereas cytochrome P-448 enhances the ethoxycoumarin dealkylase and benzo[a]pyrene hydroxylase activities. The added cytochrome P-450 is believed to be incorporated into the microsomal membrane, and the enriched microsomes can be separated from the free hemoprotein by gel filtration. A correlation between the increased cytochrome P-450 content and the enhanced catalytic activity of the microsomes is shown. Several lines of evidence suggest that the exogenous cytochrome P-450 molecules become catalytically active only when they are incorporated into the membrane. By measuring the enhanced ethoxycoumarin dealkylase activity, the rate of the proposed incorporation of cytochrome P-448 into microsomes can be measured, and the temperature dependence of the rate is reported. The addition of cytochromes P-448 and P-450 causes a great increase in the monoxygenase activities of microsomes which have been treated with linoleic acid hydroperoxide. The hydroperoxide treatment denatures almost all the cytochrome P-450 molecules in the microsomes but retains most of the NADPH-cytochrome P-450 reductase activity. Experiments with such microsomes indicate that the added cytochrome P-450 molecules, after incorporation into the membrane, have a direct access to the reductase molecules and are able to receive electrons directly from the latter. The present results are consistent with a nonrigid model for the organization of cytochrome P-450 and NADPH-cytochrome P-450 reductase in the microsomal membrane.
Insights
Solubilized cytochromes P-450 and P-448 enhance microsomal enzyme activity upon binding to membranes. These hemoproteins become catalytically active after incorporation, interacting directly with NADPH-cytochrome P-450 reductase.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Cytochromes P-450 (CYP) and P-448 are hemoproteins found in liver microsomes.
- These enzymes play crucial roles in xenobiotic metabolism and detoxification.
- Microsomal enzyme activities can be modulated by the presence and organization of these CYPs.
Purpose of the Study:
- To investigate the binding and catalytic activity of solubilized cytochromes P-450 and P-448 when added to rat liver microsomes.
- To determine the conditions and mechanisms by which these exogenous hemoproteins become active.
- To explore the relationship between CYP incorporation into the microsomal membrane and enhanced enzyme function.
Main Methods:
- Preparation of solubilized cytochromes P-450 and P-448 from rat liver microsomes.
- Incubation of solubilized CYPs with native or hydroperoxide-treated microsomes.
- Assay of microsomal enzyme activities such as benzphetamine demethylase, ethoxycoumarin dealkylase, and benzo[a]pyrene hydroxylase.
- Separation of incorporated CYPs from free hemoproteins using gel filtration.
- Measurement of enzyme kinetics and temperature dependence.
Main Results:
- Solubilized cytochromes P-450 and P-448 bind to microsomes, increasing specific monoxygenase activities.
- Cytochrome P-450 enhances benzphetamine demethylase activity; cytochrome P-448 boosts ethoxycoumarin dealkylase and benzo[a]pyrene hydroxylase activities.
- Exogenous CYPs are incorporated into the microsomal membrane to become catalytically active.
- Hydroperoxide-treated microsomes, depleted of native CYPs but retaining reductase activity, show restored enzyme function upon addition of exogenous CYPs.
- Incorporated CYPs directly interact with NADPH-cytochrome P-450 reductase, receiving electrons for catalytic activity.
Conclusions:
- Exogenous cytochromes P-450 and P-448 can be functionally integrated into the microsomal membrane.
- Membrane incorporation is essential for the catalytic activity of added hemoproteins.
- The results support a dynamic, non-rigid model for the organization of CYPs and NADPH-cytochrome P-450 reductase in the endoplasmic reticulum membrane.