Characterization of two P-450 isozymes placed in the rat CYP2D subfamily.
Two P-450s with debrisoquine 4-hydroxylation activity, designated P-450 UT-7 and UT-7b, were purified and partially purified, respectively, from hepatic microsomes of untreated male rats. Both purified P-450s with an apparent molecular weight of 49,000, were associated with another protein with an apparent molecular weight of 29,000 which was designated 29 k-protein. The CO-reduced spectra of both P-450 UT-7 and UT-7b showed a peak at 448 nm. The NH2-terminal amino acid sequences of P-450 UT-7 and UT-7b were the same as the amino acid sequences of CYP2D1 and CYP2D2 deduced from the cDNA, respectively, except for the lack of a terminal methionine for P-450 UT-7b. In a reconstituted systems, P-450 UT-7 and UT-7b catalyzed lidocaine 3-hydroxylation and N-deethylation in the presence of the 29 k-protein. The Km and Vmax values for lidocaine 3-hydroxylation were 3.6 microM and 0.50 nmol/min/nmol of P-450 for P-450 UT-7, and 3.6 microM and 0.93 nmol/min/nmol of P-450 for P-450 UT-7b, respectively. Antibody against P-450 UT-7, which also cross-reacted with P-450 UT-7b, inhibited lidocaine 3-hydroxylation in liver microsomes from untreated male rats, but had little effect on lidocaine N-deethylation. These findings suggested that lidocaine 3-hydroxylation in hepatic microsomes from untreated male rats was catalyzed by P-450 UT-7 and/or UT-7b.P-450 UT-7 not containing 29 k-protein was obtained as the non-absorbed fraction from hydroxylapatite HPLC. The activities of debrisoquine 4-hydroxylation as well as lidocaine 3-hydroxylation and N-deethylation in a reconstituted system with P-450 UT-7 without 29 k-protein were one-fifth of those of P-450 UT-7 containing 29 k-protein at the same substrate concentration. These findings suggested that the 29 k-protein was essential to express the maximal metabolic activities. However, the lidocaine metabolic activity in a reconstituted system with P-450 UT-7 containing 29 k-protein and in hepatic microsomes were not inhibited by 29 k-protein antibody.
Ohishi
N
N
Laboratory of Chemistry, Osaka City University Medical School, Japan.
Imaoka
S
S
Suzuki
T
T
Funae
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D019389:Cytochrome P-450 CYP2D6; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000302:isolation & purification; D007527:Isoenzymes / Q000737:chemistry*; D008012:Lidocaine / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley
D000906:Antibodies; D065607:Cytochrome P-450 Enzyme Inhibitors; D007527:Isoenzymes; D003577:Cytochrome P-450 Enzyme System; D008012:Lidocaine; D006899:Mixed Function Oxygenases; D019389:Cytochrome P-450 CYP2D6