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Initial purification and characterization of hepatic microsomal cytochrome P-450 from BNF-treated perch (Perca
Y S Zhang1, A Goksøyr, T Andersson
1Department of Zoophysiology, University of Göteborg, Sweden.
Abstract:
1. A procedure was developed for isolating and purifying cytochrome P-450 from hepatic microsomes of BNF-treated perch, using modified versions of the methods of Williams and Buhler (1982. Biochim. biophys. Acta 717, 398-404) and Goksøyr (1985. Biochim. biophys. Acta 850, 409-417). 2. Following chromatography on phenyl-Sepharose CL 4B and DEAE-Sepharose CL-6B, the major peaks, fractions b and c, were resolved into five fractions, possibly representing different isoenzymes, by a FPLC with a strong anion exchange column (Mono Q). 3. These fractions have been characterized on the basis of their spectral, electrophoretic and immunological properties. 4. The purified form of cytochrome P-450 in fraction V from perch liver showed a number of similarities to cytochrome P-450c, the major BNF-inducible cytochrome P-450 in cod liver. 5. Therefore we suggest that this purified form of cytochrome P-450 is a BNF-induced form in perch and that it is closely related to the gene subfamily cytochrome P-450 IA1.
Insights
Researchers developed a method to isolate cytochrome P-450 from perch liver microsomes. The purified cytochrome P-450 fraction V is similar to cod liver P-450c, suggesting it is a benzo[a]pyrene (BNF)-induced form related to P-450 IA1.
Area of Science:
- Biochemistry
- Molecular Biology
- Environmental Toxicology
Background:
- Cytochrome P-450 enzymes are crucial for metabolizing xenobiotics.
- Benzo[a]pyrene (BNF) is a known inducer of certain cytochrome P-450 isoforms.
- Understanding BNF-induced P-450 in fish is important for environmental monitoring.
Purpose of the Study:
- To develop a procedure for isolating and purifying cytochrome P-450 from BNF-treated perch liver microsomes.
- To characterize the isolated cytochrome P-450 fractions.
- To compare perch cytochrome P-450 with known isoforms in other fish species.
Main Methods:
- Modified isolation and purification techniques based on established protocols.
- Chromatography using phenyl-Sepharose CL 4B and DEAE-Sepharose CL-6B.
- Fast protein liquid chromatography (FPLC) with a strong anion exchange column (Mono Q) for isoenzyme separation.
- Characterization via spectral, electrophoretic, and immunological analyses.
Main Results:
- Successful isolation and purification of cytochrome P-450 from BNF-treated perch hepatic microsomes.
- Separation of major fractions into five distinct components using FPLC.
- Fraction V exhibited spectral, electrophoretic, and immunological similarities to cytochrome P-450c from cod liver.
- Identified similarities suggest Fraction V is a BNF-induced cytochrome P-450.
Conclusions:
- A robust method for purifying fish cytochrome P-450 was established.
- The purified cytochrome P-450 (Fraction V) in perch is likely BNF-induced.
- This perch cytochrome P-450 is closely related to the cytochrome P-450 IA1 gene subfamily, similar to cod P-450c.