Abstract
The kinetic parameters of NADPH-dependent cytochrome P450 LM 2 (2B4) reduction and substrate oxidation in the monomeric reconstituted system, consisting of purified NADPH-cytochrome P450 reductase and cytochrome P450 LM 2 monomers, and in phenobarbital-induced rabbit liver microsomes were compared. In the absence of benzphetamine, NADPH-dependent reduction of cytochrome P450 LM 2 was monophasic in the monomeric reconstituted system and biphasic in the microsomes. The presence of the substrate in the monomeric reconstituted system caused the appearance of the fast phase. In this system substrate-free cytochrome P450 LM 2 was entirely low-spin, and the addition of benzphetamine shifted the spin equilibrium to a high state very weakly. No correlation between high-spin content and the proportion of the fast phase of NADPH-dependent LM 2 reduction was found in the system. V max values for the oxidation of type I substrates (benzphetamine, dimethylaniline, aminopyrine) in the monomeric reconstituted system were higher or the same as in the microsomes, whereas K m values for the substrates and NADPH were lower in the microsomes. Maximal activity of the monomeric reconstituted system was observed at a 1:1 NADPH-cytochrome P450 reductase/cytochrome P450 LM 2 ratio. Measurements of benzphetamine oxidation as a function of NADPH-cytochrome P450 reductase/cytochrome P450 LM 2 ratio at a constant total protein concentration allowed the K d of the NADPH-cytochrome P450 reductase/cytochrome P450 LM 2 complex to be estimated as 6.4 ± 0.5 μ m. Complex formation between the NADPH-cytochrome P450 reductase and cytochrome P450 LM 2 monomers was not detected by recording the difference binding spectra of the reductase monomers with LM 2 monomers or by treatment the mixture of the monomers of the proteins with the crosslinking reagent, water-soluble carbodiimide.
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