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. 2012 Dec 20;288(6):4436–4451. doi: 10.1074/jbc.M112.402123

FIGURE 1.

FIGURE 1.

Oxidation of MPTP by mitochondrial and ER-targeted human CYP2D6. A, MAO activities of mitochondrial preparations from COS-7 cells mock-transfected with Dox-inducible lentiviral vector and mouse brain mitochondria. Metabolism of kynuramine to 4-hydroxyquinoline was carried out as described under “Materials and Methods” using isolated mitochondria. Mitochondria (Mito) were preincubated with or without added inhibitors (deprenyl (Dep) or chlorgyline (Clor), 20 μm each) for 10 min on ice before starting the assay. Means ± S.E. are shown (n = 3). B, immunoblots of whole cell extracts (50 μg each) from COS-7 cells expressing WT CYP2D6 treated with or without Dox (to induce CYP2D6). The blot was also developed with antibody to succinate dehydrogenase (SDH) to assess loading levels. C, ER membrane integration assay; the SRP binding affinities of WT, +SRP, and −SRP proteins were performed as described under “Materials and Methods.” The method essentially tests the extent of integration of labeled nascent proteins into unwashed canine pancreatic ER membrane. T, total translation product used in the assay; M, proteins bound to the ER membrane fraction that was re-isolated and washed. Proteins were analyzed by SDS-PAGE and subjected to autoradiography. Radiometric analysis was performed to determine the percentage of the total translation product that associated with the ER membrane for each construct. D, mitochondrial (Mt) and microsomal (Mc) CYP2D6 levels in WT, −SRP, and +SRP CYP2D6-expressing cells (induced with Dox). A rat liver microsomal (Micro) sample was used as a positive control. Proteins (30 μg each) were subjected to immunoblot analysis as described under “Materials and Methods.” The blot was developed with a monoclonal antibody to human CYP2D6 (1:1000 dilution, v/v) and co-developed with NPR antibody (1:1500 dilution, v/v) to assess levels of microsomal contamination of mitochondrial preparations. E, oxidation of MPTP by mitochondria from Dox-induced COS-7 cells expressing −SRP and +SRP CYP2D6. The oxidation products were quantified by nano-LC-MS-MS analysis as described under “Materials and Methods.” A standard curve of MPP+ was used to calculate moles of MPP+ formed per mg of protein. Inhibition studies were performed by preincubating enzymes for 20 min on ice with 10 μl of CYP2D6 inhibitory antibody or control ascites protein (10 mg/ml, BD Gentest, BD Biosciences) or 10 μm quinidine and 10 μm pargyline and 10 μm deprenyl at 37 °C for 20 min. F, schematic representation of WT2D6 and its mutant constructs. Mutated amino acids are underlined.