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.

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.

[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.

Revelation of ternary complexes between redox partners in cytochrome P450-containing monooxygenase systems by the optical biosensor method.

Formation of binary and ternary complexes in the water-soluble cytochrome P450cam (P450cam)-containing as well as in the membrane P4502B4(2B4)- and the mixed P450scc-containing monooxygenase systems was investigated in real time by the 'resonant mirror' optical biosensor method. It was shown that the inter-protein electron transfer occurs not only during complex formation but also upon random collision--as was the case with the d-Fp/d-b5 pair (2B4 system). Binary complexes may be either facilitative to electron transfer (electron-transfer complexes) or prohibitive to it (non-productive complexes).

Molecular recognition in the p450cam monooxygenase system: direct monitoring of protein-protein interactions by using optical biosensor.

A real-time optical biosensor study on the interactions between putidaredoxin reductase (PdR), putidaredoxin (Pd), and cytochrome P450cam (P450cam) within the P450cam system was conducted. The binary Pd/P450cam and Pd/PdR complexes were revealed and kinetically characterized. The dominant role of electrostatic interactions in formation of productive electron transfer complexes was demonstrated.

Optical biosensor investigation of interactions of biomembrane and water-soluble cytochromes P450 and their redox partners with covalently immobilized phosphatidylethanolamine layers.

A phospholipid-containing biochip was created by covalently immobilizing phospholipids on the optical biosensor's aminosilane cuvette and employed to monitor the interactions of the membrane and water-soluble proteins in cytochrome P450-containing monooxygenase systems with planary layers of dilauroylphosphatidylethanolamine (DLPE) and distearoylphosphatidylethanolamine (DSPE), differing in acyl chain length. It was shown that the full-length membrane proteins-cytochrome P4502B4 (d-2B4), cytochrome b5 (d-b5) and NADPH-cytochrome P450 reductase (d-Fp)-readily incorporated into the phospholipids. The incorporation was largely due to hydrophobic interactions of membranous protein fragments with the phospholipid layer.

Optical biosensor study of ternary complex formation in a cytochrome P4502B4 system.

The optical biosensor method was used for the revelation of ternary complexes, formed by the full-length NADPH-cytochrome P450 reductase (d-Fp) and cytochromes P4502B4 (d-2B4) and b5 (d-b5) in the course of their interactions within the reconstituted d-2B4-containing system. Based on the lack of competition between d-b5 and d-Fp for the binding sites on immobilized 2B4 (3) as well as on the analysis of data obtained in the three proteins' dissociation reactions, the possibility of formation of ternary complexes through the interactions between membranous hydrophobic fragments of proteins was substantiated. All the complexes obtained were productive.

AFM study of membrane proteins, cytochrome P450 2B4, and NADPH-cytochrome P450 reductase and their complex formation.

The application of the AFM technique for visualization of membrane proteins and for measuring their dimensions was demonstrated. The AFM images of the microsomal monooxygenase system components-cytochrome P450 2B4 and NADPH-cytochrome P450 reductase-were obtained by using two types of supports-hydrophobic, highly oriented pyrolytic graphite (HOPG) and hydrophilic mica. It was shown that hemo- and flavoprotein monomers and oligomers can be adsorbed to and visualized on HOPG.

The optical biosensor studies on the role of hydrophobic tails of NADPH-cytochrome P450 reductase and cytochromes P450 2B4 and b5 upon productive complex formation within a monomeric reconstituted system.

The optical biosensor study of interaction between microsomal proteins-NADPH-cytochrome P450 reductase, cytochrome P450 2B4, and cytochrome b5-was carried out in the monomeric reconstituted system in the absence of phospholipids. The formation of individual complexes was kinetically characterized and their association and dissociation rate constants were determined. The association rate constants for the complexes formed were found to be close to the diffusiion limit-(0.

An optical biosensor study of the interaction parameters and role of hydrophobic tails of cytochrome P450 2B4, b5 and NADPH-flavoprotein in complex formation.

The real-time interactions of membrane proteins - cytochrome P450 2B4, NADPH cytochrome P450 reductase and cytochrome b5 - were studied by use of an optical biosensor system. The association and dissociation rate constants for the individual complexes were measured and the affinities of the redox partners for each other were estimated. The association rate constants of these complexes were found to be close to the diffusion limit and their dissociation rate constants were in the order of 1s-1.

Interactions of cytochrome P450 2B4 with NADPH-cytochrome P450 reductase studied by fluorescent probe.

A new method for monitoring the formation of the cytochrome P450 complexes with NADPH-cytochrome P450 reductase (NCPR) is introduced. The method is based on the quenching of fluorescence of NCPR labelled with 7-ethylamino-3-(4'-maleimidilphenyl)-4-methylcoumarin maleimide (CPM). In a monomerized soluble reconstituted system in the absence of phospholipid, cytochrome P450 2B4 and NCPRcpm were shown to form 1:1 complexes with a Kd of 0.

Catalytic activity of cytochrome P4501A2 in reconstituted system with Emulgen 913.

Physicochemical properties and catalytic activity of cytochrome P4501A2 in the reconstituted system with Emulgen 913 were studied. The formation of cytochrome P4501A2 monomers was shown by gel filtration at an Emulgen concentration of 8 g/liter. The catalytic activity of the monomeric monooxygenase system was low.

Comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver. II. Kinetic parameters of reductase and monooxygenase reactions.

The kinetic parameters of NADPH-dependent cytochrome P450 LM2 (2B4) reduction and substrate oxidation in the monomeric reconstituted system, consisting of purified NADPH-cytochrome P450 reductase and cytochrome P450 LM2 monomers, and in phenobarbital-induced rabbit liver microsomes were compared. In the absence of benzphetamine, NADPH-dependent reduction of cytochrome P450 LM2 was monophasic in the monomeric reconstituted system and biphasic in the microsomes. The presence of the substrate in the monomeric reconstituted system caused the appearance of the fast phase.

Comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver. I. Properties of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) monomers.

Oligomers and monomers of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) isolated from the liver microsomes of phenobarbital-treated rabbits were examined for physicochemical properties and catalytic activities. As measured using laser correlation spectroscopy the particle sizes of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 oligomers were 14.8 +/- 1.

Cytochrome C (Fe2+) as a competitive inhibitor of NADPH-dependent reduction of cytochrome P450 LM2: locating protein-protein interaction sites in microsomal electron carriers.

The kinetics of NADPH-dependent reduction of cytochrome P450 LM2 in the soluble monomeric reconstituted system in the absence of any substrate is shown to be monophasic. We show that ferrous cytochrome c acts as a competitive inhibitor of the reduction. In the presence of 1 mM benzphetamine an additional extremely fast phase was observed.

Substrate access channel geometry of soluble and membrane-bound cytochromes P450 as studied by interactions with type II substrate analogues.

The problems of alterations in the tertiary structure at the cytochrome P450 active site after isolation from the microsomal membrane and comparative analysis of the structures of the active sites of membrane-bound P450 and soluble P450cam have been studied in terms of using bifunctional compounds (I-IV). These amphiphilic compounds contain a pyridine radical, an aliphatic chain of variable length (n), and diphosphonic acid at the end of the molecule. There exists an optimal length (n) at which the interaction between I-IV and P450 is rather efficient.

Protein-protein interactions in microsomal cytochrome P-450 isozyme LM2 and their effect on substrate binding.

The effects of protein-protein interactions and substrate binding on the structure of the active site of rabbit liver microsomal cytochrome P-450 LM2 have been analyzed by resonance Raman spectroscopy of the monomeric and oligomeric protein in solution. Also H2O2-dependent catalytic activities of the two states have been compared. The two vinyl substituents of the heme exhibit different orientations, as indicated by the frequencies and intensities of their stretching vibrations.

Reduction and catalytic properties of cytochrome P-450 LM2 in reconstituted system containing monomeric carriers.

The membrane microsomal monooxygenase system can be reconstituted in solution from NADPH-specific flavoprotein and cytochrome P-450 which exist in the monomeric state in the presence of Emulgen 913 at molar ratio of the proteins and detergent of 1:1:300. Oxidized and dithionite-reduced monomers of cytochrome P-450 were much less thermostable than its initial aggregates, while thermal stability of NADPH-specific flavoprotein did not depend on its aggregation state. Binding spectra of cytochrome P-450 monomers with benzphetamine were atypical and had an absorbance minimum at 422 nm only.

Reconstitution of liver monooxygenase system in solution from cytochrome P-450 and NADPH-specific flavoprotein monomers.

Microsomal monooxygenase system was reconstituted in the presence of non-ionic detergent Emulgen 913 from cytochrome P-450 and NADPH-specific flavoprotein isolated from phenobarbital-induced rabbit liver microsomes. At Emulgen 913 concentration of 0.05 g/l mixed complex between flavoprotein and cytochrome was formed with 5: 5 protein molar ratio and molecular weight of 700 kD.

Ultrastructure of reconstituted rat liver microsomal membranes and cytochrome b5- or P-450-containing proteoliposomes.

Thin sectioning and freeze-fracture electron microscopy have been used to show that it is possible to obtain topologically closed vesicles by means of reconstitution of rat liver microsomal membrane "ghosts." The reconstitution by 15 hr dialysis resulted in the formation of vesicles with intramembrane particles (IMP) while after 40 hr dialysis no IMP were observed in the membranes. The protein/lipid ratio and functional activity of NADPH- and NADH-linked enzyme systems were similar in both cases.

[Reconstruction of a membrane monooxygenase cytochrome P 450-containing system in the liver using detergents in solution].

Emulgen 913, Triton N-101 and sodium cholate were compared for their reconstituting action on the dimethylaniline N-demethylation system containing cytochrome P-450 and NADPH-cytochrome P-450 reductase. The comparison showed that emulgen 913 is the most efficient detergent. The optimum molar ratio of the proteins and emulgen appeared to be equal to 1:1:600.

[Comparative study of original and reconstituted by self-assembly endoplasmic reticulum membranes].

A method for biomembrane reconstitution from microsomal proteins and lipids solubilized by sodium cholate consisting in a removal of the detergent by its dialysis followed by treatment with 10% albumin has been developed. A comparison of the original and reconstituted membranes showed that the phospholipid, protein and enzymatic composition of the latter is similar or only slightly different from that of the original ghosts. The reconstituted membranes contained 1.

[Reconstitution of liver endoplasmic reticulum membranes from solubilized proteins and lipids by removal of detergent].

A method for membrane reconstitution from cholate-solubilized microsomal proteins and lipids by a removal of the detergent on a column with charcoal has been developed. A comparative study showed that the membranes reconstituted by a dialysis or absorption do not differ from each other in terms of membrane proteins incorporation into lipid vesicles and cytochrome P-450 reconversion into cytochrome P-450. A possibility of biomembrane reconstitution from membrane proteins and lipids solubilized by a non-ionic detergent Triton X-100 was shown.