Purification and characterization of a form of P450 from horse liver microsomes.
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.
Komori
M
M
Department of Veterinary Science, University of Osaka Prefecture.
Higami
A
A
Imai
Y
Y
Imaoka
S
S
Funae
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001189:Aryl Hydrocarbon Hydroxylases; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D000072467:Cytochrome P450 Family 2; D006736:Horses / Q000378:metabolism*; D007120:Immunochemistry; D007527:Isoenzymes / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000276:immunology
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C450279:CYP2C11 protein, rat; D000072467:Cytochrome P450 Family 2; D039181:Steroid 16-alpha-Hydroxylase; C022724:testosterone 7-alpha-hydroxylase, hamster