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Keywords:

  • Cytochrome P450 4A;
  • Fatty acid hydroxylase;
  • Prostate;
  • Brain;
  • Polymerase chain reaction;
  • Western blot;
  • Protein sequencing

Abstract: Members of the P450 4A subfamily are key enzymes in the synthesis and degradation of metabolites of arachidonic acid, which are of physiological importance in the brain. In the rat, four members of this subfamily, 4A1, 4A2, 4A3, and 4A8, have been described. In this study, the expression of members of the 4A subfamily in the rat brain has been examined by PCR amplification, by western and northern blotting, and by protein N-terminal sequencing. With PCR all four members of the subfamily were detectable in the liver and kidney. P450 4A1 was found exclusively in the liver and kidney, whereas P450 4A2 was detectable in all the tissues tested, including the lung, seminal vesicles, prostate, cerebral cortex, hypothalamic preoptic area, cerebellum, and brainstem. The tissue distribution of P450 4A3 was similar to that of 4A2 except that it was not detectable in seminal vesicles. A P450 4A8-specific fragment was amplified from the kidney, liver, and prostate and weakly from the cerebral cortex but not from other brain regions. Despite the evidence of their presence by PCR, no members of the 4A family were detectable on northern blots with mRNA from the brain. On western blots a P450 4A-specific antiserum recognized a band in P450 fractions prepared from the brain. The intensity of the signal with 30 pmol of P450 from the brain was similar to that with 10 pmol of liver microsomal P450. The brain P450 was extracted from 1 g of brain, whereas the 10 pmol of liver P450 is the equivalent of 1 mg of liver. This suggests a brain content of 4A P450 that is 0.1% of that in the liver. N-terminal sequencing of the protein bands in the brain P450 fraction revealed the presence of both P450 4A8 and 4A3. These data show the presence in the brain of forms of P450 whose level of mRNA is too low to be detected on northern blots. The specificity of tissue distribution shows that this is not just a nonspecific background level of expression and suggests a role of brain P450 in the synthesis and degradation of arachidonic acid metabolites.