| Abstrakt: |
Proteomic approaches have been used for detection and identification of cytochromes P450 from highly-purified membrane preparations of human liver. These included the protein separation by 2D- and/or 1D-electrophoresis and molecular scanning of a SDS-PAGE gel fragment in the range of 45-66 kD (this area corresponds molecular weights of cytochromes P450). The analysis of protein content was statistically evaluated by means of original 1D-ZOOMER software package which allowed to carry out processing of mass spectra mixture instead of individual mass spectra used by standard techniques. In the range of 45-66 kDa we identified 13 microsomal membrane proteins including 11 cytochromes P450, namely CYPs 1A2, 1B1, 2A6, 2E1, 2C8, 2C9, 2C10, 2D6, 3A4, 4A11, 4F2. The microsomal samples were characterized by the enzymatic assays using the marker substrates for CYP1A, 2B, 3A4, 2C and 2E1. The 7-methoxy- and 7-ethoxyresorufin-O-dealkylase activities (i.e. the marker activities for cytochromes P450 1A1/1A2, respectively) and the erythromycin-N-demethylase activity (i.e. the marker activity for cytochrome P450 3A4) are lowered in pathology compared to these activities in norm. At the same time the benzyloxyresorufin-O-debenzylase activity (which characterizes the total activity of CYP2B and CYP2C), the activities of CYP2E1 (methanol), 7-pentoxyresorufin-O-dealkylation (CYP2B), 7-ethoxy- and 7-methoxycoumarin-O-dealkylases (CYP2B1) did not change. On the basis of the results obtained efficiency of a combination proteomic and biochemical analyses for inventory cytochromes P450 and revealing of their level expression is shown, and opportunities of mass spectrometry for a quantitative estimation of proteins are discussed. |
