Oxidative modification of cytochrome P-450 during its function. II. Study of the mechanism of cytochrome P-450 LM2 inactivation in a soluble reconstructed monooxygenase system

Inactivation of cytochrome P-450 LM2 induced by hydrogen peroxide formed in the active site of the enzyme was studied. Catalase did not protect cytochrome P-450 LM2 from inactivation during its operation in a soluble reconstituted system. The hemoprotein inactivation in this system was found to depend on the ratio of hemo- to flavoproteins. It was demonstrated that cytochrome P-450 LM2 inactivation during catalysis is accompanied by cleavage of the hemoprotein molecule. It is probable that this fact plays a key role in regulation of enzyme decay.     

The induction of cytochrome P-450 and monooxygenase activities in the chick embryo by 2-acetylaminofluorene

Administration of 2-acetylaminofluorence to chick embryos increases the cytochrome P-450 level 3.4 fold but causes no increase in total epoxide hydrase activity or other microsomal electron transport enzymes. The induction response shows some similarity to that elicited by phenabarbitone both in terms of the monooxygenase activities induced and their inhibition characteristics. Induction of a specific cytochrome P-450 subform by this agent may increase its detoxification and in part account for the resistance of avian species to its hepatocarcinogenic effect.     

Inhibition of cytochrome P-450-linked monooxygenase systems by naphthoquinones

Several naphthoquinones, except 2-hydroxy-1,4-naphthoquinone, were found to inhibit microsomal cytochrome P-450-linked monooxygenase activities in rabbit liver and human placenta. In particular, 5-hydroxy-1,4-naphthoquinone inhibited placental estrogen biosynthesis more effectively than it did hepatic drug oxidation reactions. There was little contribution by superoxide radicals to these enzyme inhibitions by naphthoquinones. Spectrophotometric studies revealed that naphthoquinones bind to the cytochrome P-450 component of the monooxygenase complex in both microsomal systems, suggesting that the inhibition is caused by direct interaction of these compounds with the heme.     

Activity of cytoplasmic NADP-dependent dehydrogenase in rat liver during induction of cytochrome P-450 by phenobarbital

Activity of oxidation enzymes of the pentosephosphate way (glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44), cytoplasmic malate dehydrogenase (decarboxylating oxaloacetate) (NADP+) (EC 1.1.1.40) and isocitrate dehydrogenase (NADP+) (EC 1.1.1.42) as well as the content of microsomal cytochromes b5 and P-450 in the rat liver have been studied 24 hours after 1, 2, 3, 4 and 5 intraperitoneal administrations of phenobarbital (4 mg per 100 g of the body weight). It is shown that the cytochrome P-450 content increases after a single administration of phenobarbital and then it gradually grows reaching its maximum after 4 administrations and falls after 5 administrations (though it remains high as compared to the control animals). The content of cytochrome b5 increases only after 4 administrations of phenobarbital and after 5th one it returns to the initial level. The content of microsomal gangliosides calculated per 1 mg of microsomal protein decreases after a single administration of phenobarbital and 5 days later it returns to the initial level. Activity of glucose-6-phosphate dehydrogenase increases after a single administration of phenobarbital, that of malate dehydrogenase--after 3 administrations, 6--phosphogluconate-dehydrogenase--after 4 administrations of the preparation. The 5 administrations of phenobarbital makes activity of all the mentioned dehydrogenases return to the initial level. Activity of isocitrate dehydrogenase under given conditions of the experiment does not change.     

The induction of a specific form of cytochrome P-450 (P-450j) by fasting

In previous work we have demonstrated that liver microsomal N-nitrosodimethylamine demethylase (NDMAd) activity is increased in rats by fasting, and we have postulated that this is due to the induction of a specific form of cytochrome P-450. This communication provides evidence for such a hypothesis. Fasting for 24 and 48 h caused 59 and 116% increases, respectively, in NDMAd activity in male rats, and fasting for 48 h caused a 63% increase in female rats. These increases were accompanied by corresponding increases of cytochrome P-450j (P-450ac) determined by immunoblotting. Fasting for 24 and 48 h also increased the mRNA for P-450j by 153 to 250%, as determined by hybridization with a cDNA probe of this cytochrome. The results suggest that fasting affects the gene expression of P-450j.     

The demethylation of guaiacol by a new bacterial cytochrome P-450

Spectroscopic studies were carried with a cytochrome P-450 in Moraxella sp., strain GU2, that could grow on guaiacol or 2-ethoxyphenol as the sole source of carbon and energy. The dissociation constant of the guaiacol-cytochrome complex was estimated to 0.15 microM, as determined in vivo or using the cell soluble extract. Cytochrome P-450 could also bind 2-ethoxyphenol, 2-propoxyphenol, and 2-butoxyphenol, and the dissociation constants have been determined in each case. Metyrapone depressed the degradation of guaiacol by whole bacteria, and was bound competitively to guaiacol with a constant of about 0.8 mM. Some catechol was excreted by the bacteria when growing on either guaiacol or 2-ethoxyphenol. Catechol and the other product of guaiacol demethylation, formaldehyde, were further oxidized by the bacteria. All the data available so far are consistent with cytochrome P-450 in Moraxella GU2 as a hydroxylase for the guaiacol side chain, behaving as a nonspecific O-dealkylase with broad specificity for guaiacol and homologous compounds with a longer carbon part in the side chain.     

Presence of cytochrome P-450- and cytochrome P-448-dependent monooxygenase functions in hepatoma cell lines

Cell lines derived from Reuber H-4-II-E hepatoma cells and their hybrids that differ in the expression of liver-specific functions are shown to contain different forms of monooxygenases. According to 1) the specificity toward the substrates benzo(a)pyrene, aldrin and chenodexycholic acid, 2) the kinetics of the epoxidation of aldrin, 3) the response to inducers, such as benz(a)anthracene and dexamethasone, and 4) the $\text{in}$$\text{vitro}$ modifier 7,8-benzoflavone, the monooxygenases predominating in differentiated cell lines belong to the cytochrome P-450-dependent enzyme(s), those in the less differentiated lines belong to the cytochrome P-448-dependent form(s).     

Molecular induction by phenobarbital of a rat hepatic form of cytochrome P-450: expression of a 4-kilobase messenger RNA

A differential screening procedure was employed to isolate a cDNA clone corresponding to a major phenobarbital (PB)-inducible form of rat hepatic cytochrome P-450. The G-C homopolymer-tailing technique was utilized to construct a cDNA library in the PstI site of plasmid pBR322. The library represented PB-induced poly(A+)RNA sequences from hepatic polysomes of 150-g male Sprague-Dawley rats. Hybrid-selection experiments against total PB-inducible RNA were performed with plasmid DNA derived from clones enriched in PB-inducible information. The mRNA molecules that specifically hybridized were subjected to in vitro translation, were immunoprecipitated with antibody raised in rabbits against purified cytochrome P-450b (P. E. Thomas, D. Korzeniowski, D. Ryan, and W. Levin (1979) Arch. Biochem. Biophys. 192, 524-532), and were electrophoresed under sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoretic conditions. One cDNA clone, designated PB-8, contained a 600-bp insert partially coding for a PB-inducible cytochrome P-450 species that comigrated on SDS-gel electrophoresis with highly purified P-450b. A single injection of PB, 15-18 h before sacrifice, increased the level of polysomal poly(A+)RNA complementary to the isolated cDNA clone by approximately 16-fold. Northern blot hybridizations of polysome-derived poly (A+)RNA, electrophoresed in denaturing agarose gels, demonstrated that the size of the mRNA corresponding to the isolated clone was 4 kb. Isolated heteronuclear RNA species demonstrated a time-dependent increase in the synthesis of a similar 4-kb RNA molecule. By genomic blot hybridization to EcoRI-restricted DNA, at least three complementary DNA fragments migrating at 5.1, 3.2, and 2.9 kb were observed with 32P-labeled PB-8 as a probe. These data, together with restriction endonuclease mapping and partial cDNA sequence information of the PB-8 cDNA, suggest that the PB-8 clone represents a previously unreported cDNA clone for a form of cytochrome P-450 inducible by PB.     

Phosphorylation of rabbit liver cytochrome P-450 LM2 and its effect on monooxygenase activity

The phosphorylation of rabbit liver microsomal cytochrome P-450 LM2 by catalytic subunit of cyclic AMP-dependent protein kinase (W. Pyerin et al. (1983) Carcinogenesis 4, 573) has now been studied in detail with purified soluble form of cytochrome P-450 as well as with the purified protein incorporated into model membranes. The apparent Km values for P-450 of the phosphorylation reaction in all experimental systems were in a range of 2-8 microM, while the Vmax values were dependent on the state of P-450. Upon phosphorylation, the reconstituted enzyme activities with benzphetamine (N-demethylation) and 7-ethoxycoumarin (O-deethylation) as substrates were reduced to 30-40% of control.     

Protein-protein and lipid-protein interactions in a reconstituted cytochrome P-450 dependent microsomal monooxygenase

NADPH-cytochrome P-450 reductase and cytochrome P-450, both purified from liver microsomes of phenobarbital-treated rabbits, were incorporated into dimyristoylphosphatidylcholine vesicles. The reduction of cytochrome P-450 by NADPH in the reconstituted vesicles proceeded in a biphasic fashion, and 70-80% of the absorbance change was associated with the fast phase. The Arrhenius plot of the apparent first-order rate constant of the fast-phase reduction showed a marked discontinuity around the phase transition temperature of the synthetic phospholipid; an almost 10-fold change in rate constant was associated with this discontinuity. It was, therefore, suggested that the reduction of cytochrome P-450 by reductase in this system was a diffusion-limited reaction controlled by the viscosity of the phospholipid membrane. The Arrhenius plot of overall drug monooxygenase activity catalyzed by the reconstituted vesicles showed a break but in a different way from that observed for the reduction of cytochrome P-450. This break was accompanied only by a change of the slope of the plot but not by a change in reaction rate. This difference in the two Arrhenius plots was attributed to that in the rate-limiting step of the two reactions. NADPH-cytochrome c reductase activity of the reconstituted vesicles, an activity catalyzed by the reductase alone, and cumene hydroperoxide dependent N-methylaniline demethylation activity catalyzed by cytochrome P-450 alone did not show any break in the Arrhenius plots.     

The 14 alpha-demethylation of obtusifoliol by a cytochrome P-450 monooxygenase from higher plants' microsomes

Microsomes isolated from corn embryos (Zea mays) can demethylate the 14 alpha-methyl group of obtusifoliol 2. An enzymatic assay has been developed for obtusifoliol 14 alpha-methyl-demethylase in higher plants. The enzymatic reaction was shown to occur sequentially, converting obtusifoliol 2 to 4 alpha-methyl-5 alpha-ergosta-8,24(28)-dien-3 beta-ol 4 via the trienol 4 alpha-methyl-5 alpha-ergosta-8,14,24(28)-trien-3 beta-ol 3 which was thoroughly identified. This enzymatic reaction is dependent of NADPH and molecular oxygen. It is inhibited by CO, menadione and specific inhibitors of cytochrome P-450, the CO inhibition being partially reversed by light. It is concluded that in Zea mays microsomes, obtusifoliol is demethylated at C-14 by a cytochrome P-450 containing monooxygenase system.     

Induction of the phenobarbital form of cytochrome P-450 by chemically unrelated compounds

The induction of the phenobarbital form of cytochrome P-450 by xenobiotics (phenobarbital, PB, hexachlorobenzene, HCB; hexachlorocyclohexane. HCCH, and aroclor 1016, Ar) was studied. It was demonstrated that administration of these compounds to animals is accompanied by an increase in the total cytochrome P-450, NADPH-cytochrome P-450 reductase, benzphetamine-N-demethylase and aldrin-epoxidase activities. Using monospecific antibodies against the cytochrome P-450 form isolated from PB-induced microsomes (PB-cytochrome P-450), a double immunodiffusion test revealed immunological identity of cytochrome P-450 forms induced by phenobarbital and other xenobiotics. The content of this form determined by rocket immunoelectrophoresis increased markedly and made up to 20-40% of the total cytochrome P-450 content. Antibodies against PB-cytochrome P-450 inhibited by 50-70% the benzphetamine-N-demethylase and aldrin-epoxidase activities, whereas the antibodies to methylcholanthrene-induced cytochrome P-450 were fairly ineffective. It was concluded that the chemically unrelated compounds induce in liver microsomes a cytochrome P-450 form, whose immunological properties and substrate specificity are close to the PB-form of cytochrome P-450.     

Tetraphenylporphyrin-Sn4+ induction of cytochrome P-450

TPP-Sn4+ was administered intraperitoneally (25 mg/kg body weight). The study was performed for 1-30 days. A day after administration the increase in the hemoprotein level 1.4 times was observed, as well as an increase in the level of p-hydroxylation of aniline. On 7-14 days the greatest increase in cytochrome P-450 content was observed. To clarity the mechanism of TPP-Sn4+ effect on cytochrome P-450, we studied its effect on the activity of heme oxygenase and LP rate. This compound is an inhibitor of heme oxygenase activity and reduces the rate of LP in the microsomes which regulates porphyrin metabolism in the organism.     

Effect of monooxygenase reactions catalyzed by cytochrome P-450 on the microsomal membrane

Hydroxylation of dimethylaniline in rabbit liver microsomes is accompanied by inactivation of cytochrome P-450 and the formation of products inhibiting the catalytic activity of non-inactivated cytochrome P-450. Other enzymes and electron carriers of microsomal membrane (cytochrome b5, NADH-ferricyanide reductase, NADPH-cytochrome c and NADPH-cytochrome P-450 reductases) as well as glucose-6-phosphatase were not inactivated in the course of the monooxygenase reactions. Phospholipids and microsomal membrane proteins were also unaffected thereby. Consequently, the changes in the microsomal membrane during cytochrome P-450 dependent monooxygenase system functioning are confined to the inactivation of cytochrome P-450.     

Studies on the stimulation of cytochrome P-450-dependent monooxygenase activity by dilauroylphosphatidylcholine

Steady-state kinetic and deuterium isotope effect studies have been conducted to determine the influence of the phospholipid dilauroylphosphatidylcholine on the catalytic activity of a reconstituted monooxygenase system composed of cytochrome P-450 and NADPH-cytochrome c (P-450) reductase. Addition of this lipid up to a concentration equivalent to its CMC resulted in an increase in V for benzphetamine N-demethylation. Above the CMC, no further change in V was observed. In contrast, the K_m was not affected throughout the entire lipid concentration range. Furthermore, the deuterium isotope effect for 7-ethoxycoumarin O-deethylation was not affected by the lipid concentration indicating that the contribution of the carbon-hydrogen bond cleavage step to V was also not affected. These data are consistent with the mass-action model proposed earlier (G. T. Miwa, S. B. West, M. T. Huang, and A. Y. H. Lu, (1979), J. Biol. Chem.254, 5695–5700) for cytochrome P-450 and NADPH-cytochrome c (P-450) reductase association during catalysis. The lipid concentration, below its CMC, appears to decrease the apparent dissociation constant for the cytochrome P-450-reductase complex thus causing an increase in the steady-state concentration of this catalytically active complex.     

Comparative characteristics of isolated forms of cytochrome P-450 induced by phenobarbital and perfluorodecalin

Two forms of cytochrome P-450 were isolated from liver microsomes of perfluorodecalin-induced rats and purified to homogeneity. A comparison of these forms with cytochromes P-450b and P-450e obtained from phenobarbital-induced rat liver microsomes revealed their similarity in a number of properties, e.g., chromatographic behaviour on 1.8-diaminooctyl-Sepharose 4B and DEAE-Sephacel columns, molecular mass determined by SDS polyacrylamide gel electrophoresis, spectral properties, immunoreactivity, peptide mapping, catalytic activity. These findings suggest that in rat liver microsomes perfluorodecalin and phenobarbital which differ in their chemical structure induce identical forms of cytochrome P-450.     

A human cytochrome P-450 characterized by inhibition studies as the sparteine-debrisoquine monooxygenase

The present study compares the debrisoquine monooxygenase and the sparteine monooxygenase activities of human liver microsomes. In the presence of 14 competitive inhibitors, apparent inhibition constants (Ki) as determined by these two activities ranged over four orders of magnitude with a correlation coefficient 0.99. These in vitro results represent the strongest evidence to date that the debrisoquine monooxygenase and the sparteine monooxygenase are identical and involve a single isozyme of cytochrome P-450.