Isolation of partially purified P450 2D18 and characterization of activity toward the tricyclic antidepressants imipramine and desipramine.
Previous reports have shown that rat brain microsomes are capable of metabolizing tricyclic antidepressants such as imipramine. Subsequent studies have shown that the protein products of several clones isolated from rat brain cDNA libraries are capable of metabolizing imipramine to both its active metabolite, desipramine, and its inactive hydroxylated metabolites. We report here the overexpression and partial purification of P450 2D18 using the baculovirus expression system and the incorporation of a C-terminal [His]4 tag. P450 2D18 was partially purified to a specific content of 4.8 nmol/mg protein and shown to be electrophoretically pure. The apparent KM values for P450 2D18 toward imipramine and desipramine were 374 and 314 microM, respectively. While apparent KM values were similar, P450 2D18 was shown to have a fivefold increased Vmax (2.2 nmol/min/nmol P450) for imipramine compared to desipramine (0.44 nmol/min/nmol P450), suggesting a primary involvement in the activation of imipramine to desipramine. We also examined the effect of the CYP2D6 inhibitor quinidine, the CYP3A inhibitor ketoconazole, and the dopamine reuptake inhibitor GBR-12935 for their ability to inhibit P450 2D18-mediated metabolism of imipramine. These results, when compared with previous studies using rat brain microsomes, suggest that P450 2D18 may play an important role in the conversion of imipramine to its active metabolite desipramine in the rat brain.
Thompson
C M
CM
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas, 77225, USA.
Kawashima
H
H
Strobel
H W
HW
D001189:Aryl Hydrocarbon Hydroxylases; D016367:Baculoviridae / Q000235:genetics; D002384:Catalysis / Q000187:drug effects; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000302:isolation & purification*; D003891:Desipramine / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007099:Imipramine / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification; D007654:Ketoconazole / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry
D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D010879:Piperazines; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; C044630:1-(2 (diphenylmethoxy)ethyl)-4-(3-phenylpropyl)piperazine; D001189:Aryl Hydrocarbon Hydroxylases; C115670:Cyp2d4 protein, rat; D007099:Imipramine; D007654:Ketoconazole; D003891:Desipramine