Identification of three protein targets for reactive metabolites of bromobenzene in rat liver cytosol.

The hepatotoxicity of bromobenzene and many other simple organic chemicals is believed to be associated with covalent binding of chemically reactive metabolites to cellular proteins. Recently, a rat liver microsomal esterase was shown to be targeted by bromobenzene metabolites formed in vitro [Rombach, E. M., and Hanzlik, R. P. (1998) Chem. Res. Toxicol. 11, 178-184]. To identify protein targets for bromobenzene metabolites in cytosol, we incubated liver microsomes and glutathione-depleted liver cytosol from phenobarbital-treated rats with [(14)C]bromobenzene in vitro. In a separate experiment, we intraperitoneally injected a hepatotoxic dose of [(14)C]bromobenzene to phenobarbital-treated rats. The cytosol fractions from both experiments were recovered and analyzed for protein-bound radioactivity. Under the conditions that were used, 2.6 and 3.9 nmolar equiv of bromobenzene/mg of cytosolic protein was bound in vitro and in vivo, respectively. Denaturing polyacrylamide gel electrophoresis of these cytosolic proteins followed by phosphor imaging analysis revealed several radiolabeled protein bands over a broad molecular mass range, the patterns observed in vitro and in vivo being generally similar to each other. Cytosolic proteins labeled in vitro were separated by ion exchange chromatography and electrophoresis, and three major radioactive bands with estimated molecular masses of ca. 14, 25, and 30 kDa were in-gel digested with trypsin, followed by on-line HPLC electrospray ionization mass spectrometry of the resulting peptide mixtures. For the three protein bands, the observed peptide masses were found to match the predicted tryptic fragments of liver fatty acid binding protein, glutathione transferase subunit A1, and carbonic anhydrase isoform III, respectively, with 83, 45, and 59% coverage of the corresponding complete sequences. The possible relationship of the adduction of these proteins to the toxicological outcome is discussed.

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 0.038 microM. Formation of the complex follows the kinetics of reversible second order transition with k(on) = 6.5 10(5) M-1 s-1. Application of high hydrostatic pressure induces dissociation of the complex (delta V degrees = -65 mL/mol). Succinylation of the hemoprotein increases the value of Kd to 0.5 microM primarily by decreasing k(on). In contrast to what was shown for intact 2B4, rising pressure does not take apart succinylated hemoprotein and NCPRcpm molecules, but causes some internal transition in their complex that diminishes the quenching. This transition is characterised by a very large volume change (delta V degrees = -155 mL/mol). The following conclusions were drawn: 1) a molecule of 2B4 contains two distinct contact regions involved in the interactions with NCPR. Only one of these regions is polar and highly hydrated in unbound hemoprotein; 2) interactions of the polar regions of 2B4 and NCPR are necessary to bring CPM-labelled cysteine of NCPR in short distance of the heme of 2B4; and 3) some of the lysine residues located in the proximity of the polar binding regions are apparently involved in the formation of the internal salt bridges in the molecule of 2B4.

High-pressure-induced transitions in microsomal cytochrome P450 2B4 in solution: evidence for conformational inhomogeneity in the oligomers.

Pressure-induced changes in ferric P450 2B4 (LM2) were studied as a function of benzphetamine concentration (0.05 divided by 2 mM) and state of aggregation of the hemoprotein in solution. Application of factor analysis to the spectral changes in the Soret region allowed us to resolve two particular pressure-induced processes in 2B4 oligomers. The first process was identified as the conversion of the low-spin P450 into the P420 state. At 25 degrees C it was followed by decay (bleaching) of about 50% of the newly formed P420. The second process was a pressure-induced high- to low-spin shift. Both transitions were reversible, except the hemoprotein bleaching. The amplitude of the P450-->P420 transition accounted for 67 +/- 5% of the total hemoprotein content. Furthermore, the fraction of the hemoprotein exposed to spin equilibrium was not affected by the P450-->P420 conversion and was estimated to be only about 31 +/- 5% of the total hemoprotein content. After the dissociation of the oligomers by 0.2% Triton N-101, the inhomogeneity vanished: 95% of the monomers were involved in the P450-->P420 transition (delta V degrees = -86 ml/mol) followed by intense bleaching of the hemoprotein. This agrees with our earlier observations on the reduced carbonyl complex of P450 2B4 and suggests some conformational difference between subunits in P450 LM2 oligomers. The parameters of the P450-->P420 conversion (delta V degrees = -32 ml/mol, P1/2 = 1560 bar) show no dependency on the substrate concentration. Analysis of the pressure-induced spin shift versus benzphetamine concentration shows this transition to be caused mainly by changes in the spin equilibrium of both substrate-bound (delta V degrees = -49 ml/mol) and substrate-free (delta V degrees = -21 ml/mol) hemoprotein, whereas the substrate binding step itself has a very weak pressure dependency (delta V degrees = -8 ml/mol).

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. The maximum rate of the 7-ethoxyresorufin O-deethylation reaction, 150 pmol resorufin min-1 nmol-1 cytochrome P4501A2, was observed at an Emulgen concentration of 0.1 g/liter when cytochrome P4501A2 pentamers predominated. The effects of Emulgen on cytochrome P4501A2 cumene hydroperoxide-dependent peroxidase activity, on its affinity to substrate and to cytochrome b5, and on the rate constants of dithionite-dependent reduction were insignificant. Study of the NADPH-dependent reduction of cytochrome P4501A2 in the reconstituted system showed that the rate constant and reduction level of cytochrome P4501A2 were always higher when the reaction was initiated by NADPH than when it was initiated by NADPH-cytochrome P450 reductase. This indicated that the reduction reaction initiated by the reductase was limited by a step in the cytochrome P4501A2 and reductase interaction. Correlation between 7-ethoxyresorufin O-deethylase activity and reduction level of cytochrome P4501A2 was found.

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. In this system substrate-free cytochrome P450 LM2 was entirely low-spin, and the addition of benzphetamine shifted the spin equilibrium to a high state very weakly. No correlation between high-spin content and the proportion of the fast phase of NADPH-dependent LM2 reduction was found in the system. Vmax values for the oxidation of type I substrates (benzphetamine, dimethylaniline, aminopyrine) in the monomeric reconstituted system were higher or the same as in the microsomes, whereas Km values for the substrates and NADPH were lower in the microsomes. Maximal activity of the monomeric reconstituted system was observed at a 1:1 NADPH-cytochrome P450 reductase/cytochrome P450 LM2 ratio. Measurements of benzphetamine oxidation as a function of NADPH-cytochrome P450 reductase/cytochrome P450 LM2 ratio at a constant total protein concentration allowed the Kd of the NADPH-cytochrome P450 reductase/cytochrome P450 LM2 complex to be estimated as 6.4 +/- 0.5 microM. Complex formation between the NADPH-cytochrome P450 reductase and cytochrome P450 LM2 monomers was not detected by recording the difference binding spectra of the reductase monomers with LM2 monomers or by treatment the mixture of the monomers of the proteins with the crosslinking reagent, water-soluble carbodiimide.

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 +/- 1.7 and 19.2 +/- 1.4 nm, respectively. Twenty-four-hour incubation with Emulgen 913 at 4 degrees C at a molar ratio of 1:100 led to the monomerization of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 oligomers, the particle sizes diminishing to 6.1 +/- 1.3 and 5.2 +/- 0.4 nm, respectively. The thermal stability of NADPH-cytochrome P450 reductase monomers was the same as that of oligomers, whereas cytochrome P450 LM2 monomers were less thermostable than oligomers and cytochrome P450 in microsomes. Similar to cytochrome P450 LM2 oligomers and the microsomal hemoprotein, cytochrome P450 LM2 monomers formed complexes with type I and II substrates, but with Kd values higher than those of microsomes and cytochrome P450 LM2 oligomers. Kinetic parameters (Vmax and Km) of H2O2- and cumene hydroperoxide-dependent oxidation of benzphetamine and aniline in the presence of cytochrome P450 LM2 oligomers, monomers, and microsomes were determined. Peroxidase activities of the oligomers and monomers were the same, but were lower than those of microsomes. Thus the substitution of protein-protein interactions in cytochrome P450 LM2 oligomers with protein-detergent interactions in the monomers did not influence the catalytic properties of the hemoprotein.

Some features of nucleo-cytoplasmic RNA transport from isolated nuclei.

Messenger RNA is released preferentially from isolated rat liver nuclei in the presence of the ATP-generating system and cytosol. The release is suppressed by spermidine, while cytoplasmic RNase inhibitor was ineffective and PCMB like some other thiol-blocking agents inhibitory. Cytoplasmic SOD added to the system strongly suppressed RNA release. A similar effect could be obtained by anaerobiosis due to addition of SMP. In both cases the inhibition is reversed by cyanide. In contrast to normal liver where the generation of superoxide radicals takes place almost exclusively in microsomes and is coupled with the oxidation of NADPH, in mouse ascites hepatoma 22a the generation of superoxide radicals occurs mainly in the nuclear envelope and is coupled wih the oxidation of both NADPH and NADH and inhibited by cyanide.