Abstract

The possible relationship between cytochrome P450 (P450) in brain and degenerative diseases of the central nervous system prompted an investigation into the members of the 2D subfamily in rat brain. The mRNA for P450 2D4 was much more abundant than those for 2D1 and 2D5, which are major hepatic forms. 2D2 and 2D3 mRNAs, which are also abundant forms in liver, were not detectable in brain. To evaluate the quantitative significance of 2D4 protein in brain, specific antibodies were raised. The full length 2D4 cDNA was cloned from brain mRNA by reverse transcription-polymerase chain reaction amplification and was translated, in a reticulocyte lysate system, into a protein of approximately 50 kDa. A [35S]methionine-labeled protein of 50 kDa could be immunoprecipitated from in vitro translated 2D4 mRNA. In Western blots, no signals were obtained with brain microsomes. However, with P450 extracted from brain a band of 50 kDa could be detected when 60 pmol or more were loaded in each lane. There was no detectable developmental regulation of the 2D4 mRNA and no change during pregnancy, during lactation, or after treatment with ethanol, conditions under which the P450 content of brain increases. In Dark Agouti rats, which do not express P450 2D1 mRNA in liver, 2D4 mRNA in brain was as abundant as in Wistar rats. We conclude that 2D4 is expressed as a stable protein in the brains of untreated rats, where it represents < 5% of the total P450.