FIGURE 4.

Cytochrome P450 contents and metabolic activities of mitochondrial and microsomal fractions from stable Neuro-2A cells. A, ferrous-CO versus ferrous difference spectra of mitochondria and microsomes of Neuro-2A cells. The P450 contents of mitochondrial and microsomal isolates were determined by using the dithionite-reduced and CO-bound difference spectra as described under “Materials and Methods.” B, bufuralol 1′-hydroxylation activity of microsomal 2D6 from WT and +SRP 2D6-expressing cells. Enzyme reconstitution and reaction conditions to ensure first-order rate kinetics were as described by Hanna et al. (17). Microsomes (Micro) from −SRP 2D6-expressing cells could not be used because of low CYP content. The values represent the mean of two separate estimates. Inhibition studies were performed by preincubating enzymes for 20 min on ice with 10 μl of 2D6 inhibitory antibody (10 mg of protein/ml, BD Gentest, BD Biosciences) or 10 μm quinidine and 10 μm pargyline at 37 °C for 20 min. C, MPTP oxidation by mitochondria from WT and −SRP 2D6-expressing Neuro-2A cells. Enzyme reconstitution was carried out as described under “Materials and Methods” in the presence of added Adx +Adr. Mitochondria from +SRP-expressing cells were not used because of lower than desirable levels of CYP content. The values represent means of duplicate reactions.