Abstract
The biochemical and molecular characteristics of cytochrome P4501A1 targeted to rat brain mitochondria was studied to determine the generality of the targeting mechanism previously described for mitochondrial cytochrome P450MT2 (P450MT2) from rat liver. In rat brain and C6 glioma cells chronically exposed to beta-naphoflavone (BNF), P450MT2 content reached 50 and 95% of the total cellular pool, respectively. P450MT2 from 10 days of BNF-treated rat brain was purified to over 85% purity using hydrophobic chromatography followed by adrenodoxin affinity binding. Purified brain P450MT2 consisted of two distinct molecular species with NH(2) termini identical to liver mitochondrial forms. These results confirm the specificity of endoprotease-processing sites. The purified P450MT2 showed a preference for adrenodoxin + adrenodoxin reductase electron donor system and exhibited high erythromycin N-demethylation activity. Brain mitoplasts from 10-day BNF-treated rats and also purified P450MT2 exhibited high N-demethylation activities for a number of neuroactive drugs, including trycyclic anti-depressants, anti-convulsants, and opiates. At 10 days of BNF treatment, the mitochondrial metabolism of these neuroactive drugs represented about 85% of the total tissue activity. These results provide new insights on the role of P450MT2 in modulating the pharmacological potencies of different neuroactive drugs in chronically exposed individuals.
Highlights
The biochemical and molecular characteristics of cytochrome P4501A1 targeted to rat brain mitochondria was studied to determine the generality of the targeting mechanism previously described for mitochondrial cytochrome P450MT2 (P450MT2) from rat liver
The mitochondrial targeted P450MT2 exhibited many molecular and biochemical properties distinct from the parent microsomal P4501A1 (12): 1) P450MT2 interacted with Adx with an affinity of 0.6 M Kd, 2) functionally productive interaction of Adx with P450MT2 occurred through the same COOH-terminal basic domain and the same positively charged residues that are known to be involved in interaction with bona fide mitochondrial P450s, such as P450scc and P450c27 (13, 14), 3) P450MT2 bound to erythromycin (Kd of 50 M) as determined by spectral analysis, and 4) P450MT2 exhibited high ERND activity, but vastly reduced CYP1A1 specific marker, EROD activity, in an Adx supported system
Purity of Mitochondrial Preparations—The purity of rat brain mitochondrial preparations was routinely tested by immunoblot analysis for mitochondrial-specific COX I protein and microsome-specific P450 reductase
Summary
The biochemical and molecular characteristics of cytochrome P4501A1 targeted to rat brain mitochondria was studied to determine the generality of the targeting mechanism previously described for mitochondrial cytochrome P450MT2 (P450MT2) from rat liver. Ticulum (hereafter referred to as microsomes) of liver, brain, and other tissues In contrast to this general belief, recent reports from our laboratory showed that the BNF-inducible P4501A1 and phenobarbital-inducible P4502B1 are targeted to mitochondria under both in vitro and in vivo cell transfection conditions. These results are consistent with previous studies from our, as well as, other laboratories showing the presence of P450 proteins cross-reacting with antibodies to the major microsomal forms, in liver and brain mitochondria from inducer treated and untreated rats, and insects (4 – 10). The chimeric nature of the aminoterminal 45-amino acid stretch of P4501A1 was further supported by the ability of this signal to target heterologous proteins such as the cytosolic dihydrofolate reductase and mature
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