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^Summary of the literature data on P450s involved in steroid, thromboxane, and bile acid synthesis in the rat brain.

^Summary of the literature data on P450s involved in steroid, thromboxane, and bile acid synthesis in the rat brain.

Detection of P450 protein in the brain is not a simple task. The P450 content of the brain is 20-50 pmol/g wet weight or 2-5 pmol/mg brain microsomal protein. This should be compared with the liver level which is 1 nmol/mg microsomal protein. In tissues where it is abundant, P450 can be quantitated in microsomal fractions by its CO difference spectrum (47) or by Western blotting. In the brain, the low levels of the enzyme causes several problems. With a good spectrophotometer, 10 pmol P450/mL is about the lower limit of detection. This means 2 mg brain microsomal protein/mL. In most laboratories, problems of turbidity and interference by other chromophores makes quantitation impossible. Similar limitations occur with Western blotting of brain microsomes. With a very good antibody 0.5 pmol P450/lane can be detected. If there were only one or two forms of P450 in the brain there would be no problem. However, there are multiple forms of P450 in the brain, and in order to detect a P450 which represents 10% of the total brain P450, 1 mg of microsomal protein would have to be loaded onto each lane. The result is usually a very poor signal to noise ratio. Another problem with brain P450 is the difficulty of separating microsomal and mitochondrial fractions. A large part of brain microsomes sediments with the mitochondrial fraction and this reduces the yield of P450 in microsomes. The obvious solution to these technical problems is the use of in situ hybridization of mRNA to identify cells in which the P450 genes are transcribed and immuno-histochemical techniques to localize the proteins. Both of these techniques are also associated with artefacts and must be combined with other methods of detection for definitive evidence for the presence of specific forms of P450. One of the major drawbacks of the in situ hybridization technique is that it cannot detect post transcriptional regulation of genes. As will be discussed later this appears to be a major mechanism for the regulation of brain P450. With immunohistochemical staining, the major problem is the amount of cross reaction with irrelevant proteins. Such staining is called specific when it is completely eliminated by pre-adsorption of the antibody with the antigen. This, of course, is not evidence that the correct protein is recognized in the tissue section. We feel that immunohistochemistry must be accompanied with Western blots, N-terminal sequencing of the protein or catalytic activity. In addition, for each P450 there has to be some reasonable correlation between the amount of immunohistochemical staining and the amount of signal quantitated on Western blots or the amount of catalytic activity. We have chosen to get around these problems of P450 quantitation by solubilizing and partially purifying P450 from a total membrane fraction prepared from brain homogenates. The P450 so obtained contains both microsomal and mitochondrial forms of the enzyme which can be spectrally quantitated, characterized by N-terminal sequencing after SDS PAGE, and by reconstitution of catalytic activities (8). This method has led to the identification of several P450 proteins in the brain (23,7,39-41).

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