Cytochrome P4502D and -2C enzymes catalyze the oxidative N-demethylation of the parkinsonism-inducing substance 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in rat liver microsomes.
We have examined the oxidative N-demethylation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a parkinsonism-inducing neurotoxin, in liver microsomes from adult Wistar and Dark Agouti (DA) rats. The oxidation of MPTP to 4-phenyl-1,2,3,6-tetrahydropyridine (PTP) in these preparations required NADPH as a cofactor and was significantly inhibited by SKF 525-A (2 mM). MPTP N-demethylation exhibited biphasic kinetics, consistent with two enzymes, a low Km system (Km1, 10.0 +/- 2.2 microM; Vmax1, 0.048 +/- 0.009 nmol/(min.mg of protein)) and a high Km system (Km2, 1180 +/- 91 microM; Vmax2, 4.80 +/- 0.75 nmol/(min.mg of protein)). We thus employed two substrate concentrations, 5 microM and 5 mM, for the low and high Km system, respectively, to assay enzyme activity in subsequent experiments. The oxidation activity was significantly decreased by pretreatment of rats with phenobarbital and beta-naphthoflavone. Furthermore, marked strain (Wistar > DA) and sex (male > female) differences were observed at low (5 microM) and high (5 mM) substrate concentrations, respectively. Reconstitution experiments with cytochrome P450BTL, which belongs to the P4502D subfamily, and P450m1 (P4502C11) demonstrated that MPTP N-demethylase occurs at concentrations of 5 microM and 5 mM. At 5 mM the male-specific P450m1 showed a remarkably high activity, over 400-fold that of P450BTL. Polyclonal antibodies against P450BTL and P450m1 effectively suppressed the activity at the low (5 microM) and the high (5 mM) substrate concentrations, respectively. These results suggest that, in the microsomal preparations used, MPTP N-demethylation is mainly mediated by P4502D enzyme(s) at lower substrate concentrations and by P4502C11 at higher substrate concentrations.
Narimatsu
S
S
Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Tachibana
M
M
Masubuchi
Y
Y
Suzuki
T
T
D015632:1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001189:Aryl Hydrocarbon Hydroxylases; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000494:pharmacology*; D005260:Female; D008658:Inactivation, Metabolic; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology* / Q000378:metabolism* / Q000648:ultrastructure; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*; D010300:Parkinson Disease / Q000209:etiology*; D051381:Rats; D017208:Rats, Wistar; D012737:Sex Factors; D013045:Species Specificity; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000276:immunology / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D003577:Cytochrome P-450 Enzyme System; D015632:1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D039181:Steroid 16-alpha-Hydroxylase; D010089:Oxidoreductases, N-Demethylating