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Purification and characterization of a form of P450 from horse liver microsomes
Abstract:
A form of P450 [termed P450(h-1)] was purified from the liver microsomes of a male horse to electrophoretic homogeneity. The specific content of the final P450(h-1) preparation was 14.8 nmol/mg of protein and the recovery was 0.38% of the microsomal P450. The apparent molecular weight of P450(h-1) was 52,000 Da. The absorption spectra of P450(h-1) indicated that P450(h-1) was a low- and high-spin mixed type P450 in the oxidized form. The reconstituted system containing P450(h-1) could catalyze benzphetamine N-demethylation, 7-ethoxycoumarin O-deethylation, and testosterone 16 alpha-hydroxylation. In the horse hepatic microsomes, aniline p-hydroxylation and testosterone 6 beta-hydroxylation, in addition to the above reactions, were detected. The N-terminal amino acid sequence of P450(h-1) was highly homologous to that of rat P450 2C11. Western blot analysis using anti-P450(h-1) antibody revealed that this antibody most strongly recognized P450 2C13 among ten rat P450s belonging to eight different subfamilies involved in hepatic drug metabolism. This anti-P450(h-1) antibody inhibited the testosterone 16 alpha-hydroxylase activity in horse liver microsomes. These results suggest that P450(h-1) belongs to the P450 2C subfamily and contributes to the testosterone 16 alpha-hydroxylation in horse liver microsomes.
Insights
Researchers purified a horse liver enzyme, P450(h-1), identifying its role in testosterone metabolism. This cytochrome P450 enzyme is crucial for specific hydroxylation reactions in horses.
Area of Science:
- Biochemistry
- Enzymology
- Pharmacology
Background:
- Cytochrome P450 enzymes are vital for drug and xenobiotic metabolism.
- Understanding species-specific P450s is crucial for comparative pharmacology and toxicology.
- Horse liver P450s involved in drug metabolism remain incompletely characterized.
Purpose of the Study:
- To purify and characterize a specific cytochrome P450 enzyme from horse liver microsomes.
- To determine the catalytic activities and substrate specificities of the purified enzyme.
- To elucidate the P450 subfamily and its role in equine drug metabolism.
Main Methods:
- Purification of P450(h-1) from male horse liver microsomes to electrophoretic homogeneity.
- Spectroscopic analysis to determine the spin state of the oxidized P450(h-1).
- Reconstitution assays to assess catalytic activities (N-demethylation, O-deethylation, hydroxylation).
- N-terminal amino acid sequencing and Western blot analysis using specific antibodies.
- Comparison with known rat P450 enzymes.
Main Results:
- P450(h-1) was purified with a specific content of 14.8 nmol/mg protein and an apparent molecular weight of 52,000 Da.
- The enzyme exhibited catalytic activity in benzphetamine N-demethylation, 7-ethoxycoumarin O-deethylation, and testosterone 16 alpha-hydroxylation.
- N-terminal sequencing showed high homology to rat P450 2C11.
- Antibody against P450(h-1) cross-reacted strongly with rat P450 2C13 and inhibited testosterone 16 alpha-hydroxylase activity in horse microsomes.
Conclusions:
- P450(h-1) is a low- and high-spin mixed type P450 enzyme from horse liver.
- The enzyme belongs to the P450 2C subfamily, based on sequence homology and antibody cross-reactivity.
- P450(h-1) significantly contributes to testosterone 16 alpha-hydroxylation in horses.