Purification of two cytochrome P450 isozymes related to CYP2A and CYP3A gene families from monkey (baboon, Papio papio) liver microsomes

Abstract

Two cytochrome P450 isozymes, FA and FI, were isolated and characterized from liver microsomes of phenobarbital-induced baboons (Pupio papio). The cytochrome FA possesses the same Nterminal amino acid sequence as P450 MK2 from crab-eating monkeys (Macaca irus) and closely resembles the human P450 3A isozymes. This cytochrome was able to oxidize nifedipine and hydroxylate testosterone at the 68 position. The second baboon cytochrome (FI) is closely related to the P450 2A subfamily and has the same N-terminal sequence as human P450 2A7. Like human P450 2A forms, it is highly active as a coumarin 7-hydroxylase. Antibodies against P450 FA and FI crossreact with two human liver proteins of 51 kDa and 49 kDa, respectively. The concentration of the first protein in the human samples, was five-times greater than the second. However, the latter showed marked interindividual variation. In primary cultures of human hepatocytes, rifampicin is a strong inducer of the 51-kDa protein and a moderate inducer of the 49-kDa protein, while phenobarbital has the opposite effect on the two proteins.

Comparison of CYP2A6 catalytic activity on coumarin 7-hydroxylation in human and monkey liver microsomes

“…In addition, CYP2A6 is capable of metabolically activating chemical carcinogens, including N-nitrosodiethylamine, aflatoxin B I, and tobacco-specific nitrosamines (4). 7-hydroxylase activity, was the first such isozyme isolated from monkey liver with a similar N-terminal sequence as human CYP2A7 (8). Later, Ohmori et al (15) isolated a P450 enzyme (referred to as P450 CMLb) from hepatic microsomes of untreated cynomolgus monkeys.…”

Metabolism and Disposition Kinetics of Nicotine

“…A few drugs have been shown to induce CYP2A6 in human primary hepatocyte culture. These include prototypical inducers rifampicin, dexamethasone, and phenobarbital, although there is wide interindividual variability in response (Maurice et al, 1991;Dalet-Beluche et al, 1992;Mattes and Li, 1997;Meunier et al, 2000;Robertson et al, 2000;Rae et al, 2001;Edwards et al, 2003;Madan et al, 2003). Pyrazole is an inducer of CYP2A6 in vitro (Donato et al, 2000).…”

THE OLIVACINE DERIVATIVE S 16020 (9-HYDROXY-5,6-DIMETHYL-N-[2-(DIMETHYLAMINO)ETHYL)-6H-PYRIDO(4,3-B)-CARBAZOLE-1-CARBOXAMIDE) INDUCES CYP1A AND ITS OWN METABOLISM IN HUMAN HEPATOCYTES IN PRIMARY CULTURE

“…Routinely, 20 to 50 g of liver microsomes prepared from cultured hepatocytes were submitted to electrophoresis on an SDS-10% polyacrylamide gel before transferring to nitrocellulose (Millipore Corporation, Bedford, MA). Membranes were incubated with specific anti-P450 antibodies: AK1, a monoclonal antibody directed against rat CYP2C6 cross-reacting with CYP2C9/18/19 (Gerbal-Chaloin et al, 2001); anti-human CYP2B6, kindly provided by P. Beaune (Institut National de la Santé et de la Recherche Médicale, Paris, France) (Gervot et al, 1999); anti-human CYP1A2 and CYP2E1 (Euromedex, Souffelweyersheim, France); and anti-baboon CYP2A and CYP3A cross-reacting with the human orthologous forms prepared in our laboratory (Dalet-Beluche et al, 1992). Blots were developed using coupling antibodies and the enhanced chemiluminescence procedure from Amersham (Amersham Biosciences UK, Ltd.).…”