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Proteomic and biochemical analysis of the mouse liver microsomes
I P Kanaeva1, N A Petushkova, A V Lisitsa
1V.N. Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, 119121, Pogodinskaya St., 10, Moscow, Russia.
Abstract:
The efficiency of the proteomic approach for the revelation of proteins, including components of the liver microsomal monooxygenase system (cytochromes b5 and P450) was demonstrated. The liver microsomes and their ghosts (i.e. membranes devoid of "ballast" proteins) were prepared from the control and phenobarbital-treated mice. Microsomes and their ghosts were characterized using the conventional biochemical assay and analysed by one- and two-dimensional electrophoresis (1-DE and 2-DE, respectively) coupled with MALDI-TOF peptide mass fingerprinting procedure. Catalytic activity of cytochromes P450 was measured using specific fluorogenic substrates for CYP1A, CYP2A, CYP2B and CYP2C families. The protein composition of control and phenobarbital-induced ghosts was analysed. The proteomic 2D-based protein separation method enabled us to reveal up to 1005 proteins, the majority of them being soluble. Among the 34 identified proteins, the cytochrome b5-like protein was revealed; however, cytochromes P450 appeared to be undetectable under 2-DE separation conditions. The separation of microsomal ghosts proteins by 1-DE, followed by mass-spectrometric analysis of bands from the 45 to 66 kDa gel range made it possible to identify hydrophobic proteins including cytochromes P450 (CYP2A4 and CYP2A5) and dimethylaniline monooxygenase. The high O-deethylation rate of 7-ethoxycoumarin-a substrate for rodent CYPs 2A and 2B, in particular for CYP2A5-was observed, in agreement with the results of mass-spectrometric identification. Collectively, the data obtained indicate that a combination of enzyme activity assays and various protein separation techniques coupled with mass-spectrometric protein identification allows a more comprehensive insight into the machinery of the cellular detoxifying system.
Insights
Proteomics successfully identified liver proteins, including cytochrome b5, but struggled with hydrophobic cytochromes P450. Combining 1D electrophoresis with mass spectrometry identified these key drug-metabolizing enzymes.
Area of Science:
- Biochemistry and Molecular Biology
- Proteomics
- Drug Metabolism
Background:
- Liver microsomes are crucial for drug metabolism, containing enzymes like cytochromes P450 and b5.
- Phenobarbital treatment induces specific drug-metabolizing enzymes in mice.
- Understanding the microsomal proteome is key to comprehending cellular detoxification pathways.
Purpose of the Study:
- To evaluate the efficacy of proteomic techniques in identifying liver microsomal proteins, particularly cytochromes P450.
- To compare protein profiles of liver microsomes and their ghosts from control and phenobarbital-treated mice.
- To characterize the liver microsomal monooxygenase system using a combination of biochemical and proteomic methods.
Main Methods:
- Preparation and characterization of liver microsomes and microsome-derived ghosts.
- Analysis using one-dimensional (1-DE) and two-dimensional electrophoresis (2-DE).
- Mass spectrometry (MALDI-TOF peptide mass fingerprinting) for protein identification.
- Enzyme activity assays using specific fluorogenic substrates for CYP families.
Main Results:
- 2-DE identified up to 1005 proteins, including cytochrome b5-like proteins, but failed to detect cytochromes P450.
- 1-DE combined with mass spectrometry successfully identified hydrophobic proteins, including cytochromes P450 (CYP2A4, CYP2A5) and dimethylaniline monooxygenase.
- Enzyme activity assays confirmed high activity for CYP2A5, aligning with proteomic findings.
Conclusions:
- Proteomics, especially 2-DE, is effective for soluble liver microsomal proteins but limited for hydrophobic ones like cytochromes P450.
- A combined approach of 1-DE, mass spectrometry, and enzyme assays is essential for a comprehensive analysis of the cellular detoxifying system.
- This integrated strategy provides deeper insights into the machinery of drug metabolism and detoxification.
