Abstract
Hepatic mitoplasts from 3-methylcholanthrene-treated rats contain cytochrome P-450 which can metabolize polycyclic aromatic hydrocarbons like benzo(a)pyrene. Mitochondrial cytochrome P-450 was partially purified and reconstituted in vitro using adrenodoxin and the adrenodoxin reductase electron transfer system and [3H]benzo(a)pyrene as the substrate. A polyclonal antibody to purified microsomal P-450c (a major 3-methylcholanthrene-inducible form) inhibited the activity of mitochondrial enzyme in a concentration-dependent manner and also reacted with a 54-kDa protein on the immunoblots. A monoclonal antibody having exclusive specificity for P-450c, on the other hand, did not inhibit the aryl hydrocarbon hydroxylase activity of the mitochondrial enzyme and showed no detectable cross-reaction with the 54-kDa mitochondrial protein. Similarly, two-dimensional analysis and immunodetection using the polyclonal antibody showed distinct molecular properties of the mitochondrial enzyme different from the similarly induced microsomal P-450c with respect to the isoelectric pH. In vitro translation of free polysomes from 3-methylcholanthrene-induced liver, transport of precursor proteins by isolated mitochondria in vitro, and immunoprecipitation with the polyclonal antibody showed the presence of a 57-kDa putative precursor which is transported and processed into mature 54-kDa species. These results present evidence for the true intramitochondrial location of the P-450c-antibody reactive isoform detected in 3-methylcholanthrene-induced rat liver mitochondria.
Highlights
From the $Department of Animal Biobgy, School of Veterinary Medicine, University of Pennsylvania, Philudelphh Pennsylvania 19104 and the VDepartment of Phurmobgy, University of Wisconsin MedicalCenter
It has been demonstrated that varied polycyclic aromatic hydrocarbons cause distinct alterations in mitochondrial structure andfunction (20,36-38)
P-450 in hepatic mitochondriawhich are capableof activating various polycyclic aromatic hydrocarbons.Recent studies from our laboratory showedthat mitoplast preparations from 3-methylcholanthrene-treatedrat liver contain cytochrome P-450 isoforms which can activate benzo(a)pyrene under in chased with unlabeled methionine in the presence of cycloheximide as described under “Experimental Procedures.”The lysatewas clarified by centrifugation at 150,000 X g for 90 min at 4 “C, and the supernatantdesignated as in vitro translation products(about10‘ cpm/pl) was used for immunoprecipitation and in vitro transport as described under “Experimental Procedures.A”, lane 1, 2 pl of in vitro vitro conditions (18, 19)
Summary
Fisher Scientific Co., Research and Development Biotechnology, 1 Reagent Lane, Fairlawn, NJ 07410. Microsomal cytochrome P-450 was solubilized and fractionated with I n Vitro Transport of Proteins into Mitochondria-Free polysomes. The in uitro transport of translation products was generated in a reaction mixture containing 0.08 M potassium phos- carried out essentially as described before using mitochondria washed phate buffer (pH 7.4), 3.3 mM MgCI,, 0.2 mM EDTA, 2 mM NADP, 3 times with the isolation buffer (33). Mitochondrial or translation products (2-4 X 10' cpm) with 60 pl of the modified microsomalcytochrome P-450 (10-15 pmol), adrenodoxin (0.4 nmol), sucrose-mannitol buffer containing 180-200pgof freshly isolated adrenodoxin reductase (0.045 nmol), or NADPH cytochrome P-450 mitochondria with intact outer membrane. About 5 mg of IgG purified by adsorption to protein A-Sepharose according
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