Purification and characterization of a form of P450 from horse liver microsomes.

The research describes the purification and characterization of a form of P450 enzyme (named P450(h-1)) from the liver of a male horse. The study also explores the reaction capabilities of P450(h-1) and suggests it belongs to the P450 2C subfamily and plays a role in testosterone 16 alpha-hydroxylation in horse liver microsomes.

Purification of P450(h-1)

• The researchers purified a version of the P450 enzyme, specifically P450(h-1), from the liver microsomes (tiny enzyme-rich structures in cells) of a male horse to the point of ‘electrophoretic homogeneity’, meaning all the proteins present had a uniform distribution of electric charge.
• The final preparation of P450(h-1) had a specific content of 14.8 nmol/mg protein and represented a 0.38% recovery of the P450 present in the horse’s microsomes.

Characterization of P450(h-1)

• The apparent molecular weight of P450(h-1) was found to be 52,000 Daltons (Da), a unit used to measure molecular weight of atomic level particles.
• The study observed that P450(h-1) exhibited behaviors of both low- and high-spin P450 enzymes in its oxidized form.

Functional Abilities of P450(h-1)

• Researchers tested if the reconstituted P450(h-1) enzyme could catalyze certain reactions. It was found able to catalyze benzphetamine N-demethylation, 7-ethoxycoumarin O-deethylation, and testosterone 16 alpha-hydroxylation.
• In the horse liver microsomes, additional reactions, including aniline p-hydroxylation and testosterone 6 beta-hydroxylation, were detected.

Homology and Inhibition

• The N-terminal (beginning portion of the protein molecule) amino acid sequence of P450(h-1) was found to be highly homologous, meaning very similar, to that of the rat P450 2C11 enzyme.
• Using an anti-P450(h-1) antibody, which binds to and inhibits the activity of P450(h-1), the researchers conducted a Western blot analysis. This test showed that the anti-P450(h-1) antibody most strongly recognized P450 2C13 among ten rat P450s from eight different subfamilies involved in hepatic (liver) drug metabolism.
• This suggests that P450(h-1) belongs to the P450 2C subfamily.
• The anti-P450(h-1) antibody also inhibited the testosterone 16 alpha-hydroxylase activity in horse liver microsomes, indicating that P450(h-1) contributes to this specific reaction in horse liver cells.