Proteomic and biochemical analysis of the mouse liver microsomes.
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.
Kanaeva
I P
IP
V.N. Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, 119121, Pogodinskaya St., 10, Moscow, Russia.
Petushkova
N A
NA
Lisitsa
A V
AV
Lokhov
P G
PG
Zgoda
V G
VG
Karuzina
I I
II
Archakov
A I
AI
D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism*; D010449:Peptide Mapping; D010634:Phenobarbital; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification; D040901:Proteomics; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D007527:Isoenzymes; D011506:Proteins; D003577:Cytochrome P-450 Enzyme System; D010634:Phenobarbital