Characterization of human liver cytochromes P450 by combining the biochemical and proteomic approaches.
Highly purified human liver microsomes were processed by a combination of the biochemical and proteomic methods. Microsomes were purified from the morphologically normal liver tissue obtained from the resected and discarded masses of surrounding liver upon surgical treatment for hemangioma (control) or hepatic metastases arising from colon cancer (pathology). Proteins of each sample were separated by two-dimensional (2-DE) and one-dimensional electrophoresis (1-DE); selected gel regions were excised, in-gel digested and analyzed by matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry. Analysis of collected fingerprints has revealed a total of 13 microsomal membrane proteins involved in the biotransformation of xenobiotics. These were disulfide isomerase, flavine monooxygenase, NADPH-cytochrome P450 reductase and 10 cytochrome P450 forms, namely: CYPs 1B1, 2A6, 2E1, 2C8, 2C9, 2C10, 2D6, 3A4, 4A11, 4F2. These same samples were characterized by the enzymatic assays using the marker substrates for CYPs 1A, 2B, 3A4, 2C and 2E1. Correlations between mass spectrometric data and enzymatic activities were investigated to demonstrate the manner in which the functional and structural aspects of proteomics meet each other in the field of cytochromes P450.
Petushkova
N A
NA
V.N. Orekchovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Pogodinskaya St., 10, 119121 Moscow, Russia. Natalia.Petushkova@ibmc.msk.ru
Kanaeva
I P
IP
Lisitsa
A V
AV
Sheremetyeva
G F
GF
Zgoda
V G
VG
Samenkova
N F
NF
Karuzina
I I
II
Archakov
A I
AI
D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D008862:Microsomes, Liver / Q000201:enzymology*; D040901:Proteomics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D003577:Cytochrome P-450 Enzyme System