Multiple forms of human P450 expressed in Saccharomyces cerevisiae. Systematic characterization and comparison with those of the rat.
We systematically characterized the levels and substrate specificity of P450s from humans and rats to extrapolate drug metabolism data from experimental animals to humans. Human P450s (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2D6, 2E1, and 3A4) were expressed in Saccharomyces cerevisiae and purified. Rat P450s were purified from hepatic microsomes of rats. We investigated the catalytic activities of purified P450s in a reconstituted system. Human CYP2B6 and rat CYP2B1 had high lidocaine N-deethylation activity. Human and rat CYP2D forms had high debrisoquine 4-hydroxylation activity. Human CYP3A4 and rat CYP3A2 had high testosterone 2 beta- and 6 beta-hydroxylation activities in a modified reconstituted system with a lipid mixture. The hydroxylation site of testosterone by CYP2B6 (16 alpha- and 16 beta-positions) agreed with that by rat CYP2B1. Human CYP2E1 had the highest lauric acid (omega-1)-hydroxylation activity and also had catalytic properties similar to those of rat CYP2E1. Human CYP2A and 2C forms had catalytic properties in testosterone metabolism different from those of rats. Antibodies raised against purified P450s were used to measure the levels of hepatic P450s. The level of CYP3A4 was the highest in human hepatic microsomes, comprising 30-40% of the total P450. CYP2C9 comprised 10-20% of the total. The levels of CYP1A2, 2A6, 2C8, 2D6, and 2E1 were moderate (5-15% of total P450). CYP2B6 content was very low. The information of this study is useful for drug metabolism and toxicological studies.
Imaoka
S
S
Laboratory of Chemistry, Osaka City University Medical School, Japan.
Yamada
T
T
Hiroi
T
T
Hayashi
K
K
Sakaki
T
T
Yabusaki
Y
Y
Funae
Y
Y
D000818:Animals; D000906:Antibodies / Q000276:immunology; D001483:Base Sequence; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D015870:Gene Expression; D006801:Humans; D007118:Immunoassay; D015151:Immunoblotting; D007527:Isoenzymes / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013045:Species Specificity; D013379:Substrate Specificity
D000906:Antibodies; D007527:Isoenzymes; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System