| Budget Amount *help |
¥5,800,000 (Direct Cost: ¥5,800,000)
Fiscal Year 1985: ¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1984: ¥3,900,000 (Direct Cost: ¥3,900,000)
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| Research Abstract |
1. Four constitutive forms of cytochrome P-450 were purified from the liver microsomes of untreated rats and characterized. They were three male-specific forms (M-1, M-2, and M-3) and one female-specific form (F-1), and were immunologically distinct from phenobarbital-inducible forms (PB-1 and PB-2) and 3-methylcholanthrene-inducible forms (MC-1 and MC-2).
2. The quantitation of those constitutive and drug-inducible forms of cytochrome P-450 in liver microsomes was attempted using various immunochemical methods. Although the four constitutive forms of cytochrome P-450 showed some degrees of immunological crossreactions, the scavenging of the antibodies with heterologous antigens enabled us to determine the major forms (M-1 and F-1) by quantitative immunoprecipitation. The quantitative immunoprecipitation method was, however, not suited for minor P-450 species in small samples.
3. To establish suitable microassay methods for individual forms of cytochrome P-450, radial diffusion method, two-dimentional immunoelectrophoresis, immunoblot assays after SDS-polyacrylamide gel electrophoresis, and ELISA were also examined and compared. ELISA was modified to make it fit for detergent-solubilized microsomal samples, and the improved procedure was found to be satisfactory.
4. Preparation of monoclonal antibodies to various forms of cytochrome P-450 was also carried out, and good monoclonal antibodies to P-450(M-1) and P-450(F-1) were obtained.
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