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NADH-dependent aryl hydrocarbon hydroxylase in rat liver mitochondrial outer membrane
Abstract:
NADH-dependent 3,4-benzpyrene hydroxylase activity was detected in the purified mitochondrial outer membrane fraction from the livers of rats treated with 3-methylcholanthrene. The specific activity in the outer membrane fraction is nearly equal to that of microsomes, a level too high to be accounted for only by the microsomal contamination. On the other hand, the NADPH-dependent 3,4-benzpyrene hydroxylase activity in the outer membrane fraction is about 50% of that of microsomes. The ratio of the specific activity of NADPH- to NADH-dependent 3,4-benzpyrene hydroxylase in microsomal fraction was about 3.5, while that of the outer membrane fraction was about 1.5. Moreover, it was found that NADH-dependent 3,4-benzpyrene hydroxylase activity in mitochondrial outer membrane from control rat liver was cyanide-insensitive, while that in microsomes was cyanide-sensitive. These results suggest the presence in the mitochondrial outer membrane fraction of aryl hydrocarbon hydroxylase activity which uses as electron donor NADH nearly to the same extent as NADPH. The hydroxylase system is composed of cyanide-insensitive cytochrome P-450 and is inducible markedly by 3-methylcholanthrene treatment. The probable electron transfer pathways in the mitochondrial outer membrane cytochrome P-450 oxidase system are discussed.
Insights
Mitochondrial outer membranes contain NADH-dependent 3,4-benzpyrene hydroxylase activity, similar to microsomes. This enzyme system, inducible by 3-methylcholanthrene, uses both NADH and NADPH as electron donors.
Area of Science:
- Biochemistry
- Cell Biology
- Toxicology
Background:
- Microsomal fractions are known to possess aryl hydrocarbon hydroxylase activity.
- The role of mitochondrial outer membranes in xenobiotic metabolism is less understood.
- 3-methylcholanthrene is a known inducer of cytochrome P-450 enzymes.
Purpose of the Study:
- To investigate the presence and characteristics of 3,4-benzpyrene hydroxylase activity in purified rat liver mitochondrial outer membrane fractions.
- To compare the kinetic properties and cofactor dependencies of this activity with those found in the microsomal fraction.
- To determine the inducibility and cyanide sensitivity of the mitochondrial outer membrane hydroxylase system.
Main Methods:
- Purification of mitochondrial outer membrane fractions from rat liver.
- Assay of NADH- and NADPH-dependent 3,4-benzpyrene hydroxylase activity.
- Determination of enzyme kinetics and cofactor preferences.
- Assessment of cyanide sensitivity and inducibility by 3-methylcholanthrene.
Main Results:
- NADH-dependent 3,4-benzpyrene hydroxylase activity was detected in mitochondrial outer membrane fractions at levels comparable to microsomes.
- The ratio of NADPH- to NADH-dependent activity was significantly lower in the outer membrane (1.5) compared to microsomes (3.5).
- Mitochondrial outer membrane NADH-dependent activity was cyanide-insensitive, unlike the cyanide-sensitive microsomal activity, suggesting a distinct cytochrome P-450 system.
Conclusions:
- The mitochondrial outer membrane possesses a functional aryl hydrocarbon hydroxylase system inducible by 3-methylcholanthrene.
- This system utilizes NADH as an electron donor to a significant extent, alongside NADPH.
- The cyanide-insensitive nature points to a unique cytochrome P-450 component within the mitochondrial outer membrane involved in xenobiotic metabolism.