Biomedical subjects

A V Lisitsa

Publications and source records attributed to A V Lisitsa.

7 recordsLinked to original sources

PubMed2006

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.

Cytochrome P-450 Enzyme System↗

PubMed2005

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.

Animals↗

PubMed2002

Production of carbon monoxide by cytochrome P450 during iron-dependent lipid peroxidation.

Carbon monoxide (CO) formation was studied in the process of lipid peroxidation in phenobarbital-induced rabbit liver microsomes. The reaction was NADPH-dependent and required Fe(2+), which occurs in microsomes as being protein bound and is not a consequence of heme destruction. Zn-protoporphyrin IX, an inhibitor of the heme oxygenase activity, proved to have no effect on CO production, suggesting that heme oxygenase is not involved into the CO generation reaction. At the same time, the addition of cytochrome P450 typical inhibitors SKF 525A and metyrapone to the reaction mixture had an inhibitory effect on the CO formation rate. Antioxidants such as alpha-tocopherol and desferal inhibited lipid peroxidation in phenobarbital-induced rabbit liver microsomes, and in this case the CO production was not registered. Thus, on the basis of the results presented here it is possible to assert that the process of NADPH, Fe(2+)-dependent carbon monoxide formation in microsomes is a result of lipid peroxidation with cytochrome P450 2B4 participation.

Animals↗

PubMed2001

Cytochrome P450 database.

This paper describes a specialized database dedicated exclusively to the cytochrome P450 superfamily. The system provides the impression of superfamily's nomenclature and describes structure and function of different P450 enzymes. Information on P450-catalyzed reactions, substrate preferences, peculiarities of induction and inhibition is available through the database management system. Also the source genes and appropriate translated proteins can be retrieved together with corresponding literature references. Developed programming solution provides the flexible interface for browsing, searching, grouping and reporting the information. Local version of database manager and required data files are distributed on a compact disk. Besides, there is a network version of the software available on Internet. The network version implies the original mechanism, which is useful for the permanent online extension of the data scope.

Catalysis↗

PubMedDate not supplied

[Computer modeling the cytochrome P450 three-dimensional structure: problems and prospects].

The review is devoted to the description of basic approaches, problems and prospects of computer modelling of 3D structures of cytochromes P450 (P450s). The following questions have been are considered in the review: (1) the role of computer molecular modelling in P450s research; (2) basic stages of homology modelling of protein 3D structures; (3) purposes and tasks of molecular modelling; (4) problems of P450s homology modelling; (5) criteria for successful P450s homology modelling; (6) methods of P450s models refinement, verification of correctness and reliability.

Amino Acid Sequence↗

PubMedDate not supplied

[Study of the mouse liver microsomes by the methods of proteome analysis].

Proteome maps of microsomes and their ghosts (i.e. membranes purified from "ballast" proteins) were obtained using highly purified mouse liver microsomes. Comparative analysis of protein composition of ghosts without and after the induction with phenobarbital (cytochromes P450 inducer) by using 1D- and 2D-electrophoresis and MALDI-TOF-mass-spectrometry revealed more than 30 new proteins, in the course of induction in the 45-60 kDa range (corresponding to the mol. weights of cytochromes P450). In the 17 kDa range (corresponding to the mol. wt. of cytochrome b5) there were 4 additional protein stains about 20 proteins disappeared over the entire electrophoregram). Separation of microsomal ghosts proteins by 1D electrophoresis followed by mass-spectrometric analysis allowed to identify cytochromes P450. The present investigation demonstrates the efficiency of different proteomic methods combination (1D- and 2D-electrophoresis, mass-spectrometry, bioinformatics and determination of the enzyme activities) for cytochromes P450 identification and elucidation of their functioning in different animal tissues and then extrapolating this approach to humans.

Alkylation↗

PubMedDate not supplied

[Nanoelectrochemistry of cytochrome P450s: direct electron transfer and electrocatalysis].

The present study demonstrates the direct electron transfer between cytochrome P450 2B4 (CYP2B4), P450 1A2 (CYP1A2), sterol 14alpha-demethylase (CYP51MT) and screen printed graphite electrodes, modified with gold nanoparticles and didodecyldimethylammonium bromide (DDAB). Electrodetection of heme proteins is possible when 2-200 pmol P450/electrode were adsorbed on the surface of nanostructured electrochemical interfaces. Electron transfer, direct electrochemical reduction and interaction with P450 substrates (oxygen, benzphetamine, lanosterol) and inhibitor ketoconazole were analyzed using cyclic voltammetry (CV), square wave (SWV) or differential pulse (DPV) voltammetry, amperometry.

Animals↗