Characterization of two P-450 isozymes placed in the rat CYP2D subfamily

• Published in: Biochimica et biophysica acta Vol. 1158; no. 3; pp. 227 - 236
• Main Authors: Ohishi, Norihisa, Imaoka, Susumu, Suzuki, Tokuji, Funae, Yoshihiko
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 28.11.1993; Elsevier
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Antibodies - pharmacology; Biological and medical sciences; CYP2D; Cytochrome P-450; Cytochrome P-450 CYP2D6; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P-450 Enzyme System - isolation & purification; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Isoenzymes - chemistry; Lidocaine - metabolism; Male; Microsomes, Liver - enzymology; Mixed Function Oxygenases - antagonists & inhibitors; Mixed Function Oxygenases - chemistry; Mixed Function Oxygenases - isolation & purification; Molecular Sequence Data; Oxidoreductases; rat hepatic microsomes; Rats; Rats, Sprague-Dawley; lidocaine metabolism; rat hepatic microsomes; CYP2D; Cytochrome P-450; Vertebrata; Mammalia; Purification; Hydroxylation; Rat; Isozyme; Cytochrome P450; Liver; Rodentia; Microsome; Physicochemical properties
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/0304-4165(93)90019-5

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 μM and 0.50 nmol/min/nmol of P-450 for P-450 UT-7, and 3.6 μM 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.