Comparative study of the reaction kinetics of cytochrome P-450 reduction by NADPH-cytochrome P-450 reductase and dithionite

The reactions of NADPH- or dithionite-dependent reduction of cytochrome P-450 were studied using a stopped flow technique. It was found that the kinetic curves for both reactions may be fitted by a sum of the two exponents. The arrhenius plots for the fast phase rate constants are linear for both reactions. On the contrary, the breaks on the corresponding plots for the slow phase rate constants are observed at 22 and 33 degrees C for cytochrome P-450 reduction by dithionite and at 31 degrees C for NADPH-dependent reduction of cytochrome P-450. The coincidence of the values of the rate constants and activation energy (56 +/- 5 kJ/mol) for the fast phase of NADPH-dependent reduction of cytochrome P-450 with values of catalytic constants and activation energy for demethylation of tertiary amines suggests that the first electron transfer process from NADPH-cytochrome P-450 reductase to cytochrome P-450 may be the rate-limiting step. A diverse character of the kinetic parameters for the two cytochrome P-450 reduction reactions is indicative of different nature of biphasity of these processes.     

Metabolization of Porphyrinogenic Agents in Brain: Involvement of the Phase I Drug Metabolizing System. A Comparative Study in Liver and Kidney

(1) We evaluated the involvement of brain mitochondrial and microsomal cytochrome P-450 in the metabolization of known porphyrinogenic agents, with the aim of improving the knowledge on the mechanism leading to porphyric neuropathy. We also compared the response in brain, liver and kidney. To this end, we determined mitochondrial and microsomal cytochrome P-450 levels and the activity of NADPH cytochrome P-450 reductase. (2) Animals were treated with known porphyrinogenic drugs such as volatile anaesthetics, allylisopropylacetamide, veronal, griseofulvin and ethanol or were starved during 24 h. Cytochrome P-450 levels and NADPH cytochrome P-450 reductase activity were measured in mitochondrial and microsomal fractions from the different tissues. (3) Some of the porphyrinogenic agents studied altered mitochondrial cytochrome P-450 brain but not microsomal cytochrome P-450. Oral griseofulvin induced an increase in mitochondrial cytochrome P-450 levels, while chronic Isoflurane produced a reduction on its levels, without alterations on microsomal cytochrome P-450. Allylisopropylacetamide diminished both mitochondrial and microsomal cytochrome P-450 brain levels; a similar pattern was detected in liver. Mitochondria cytochorme P-450 liver levels were only diminished after chronic Isoflurane administration. In kidney only mitochondrial cytochrome P-450 levels were modified by veronal; while in microsomes, only acute anaesthesia with Enflurane diminished cytochrome P-450 content. (4) Taking into account that δ-aminolevulinic acid would be responsible for porphyric neuropathy, we investigated the effect of acute and chronic δ-aminolevulinic acid administration. Acute δ-aminolevulinic acid administration reduced brain and liver cytochrome P-450 levels in both fractions; chronic δ-aminolevulinic acid administration diminished only liver mitochondrial cytochrome P-450. (5) Brain NADPH cytochrome P-450 reductase activity in animals receiving allylisopropylacetamide, dietary griseofulvin and δ-aminolevulinic acid showed a similar profile as that for total cytochrome P-450 levels. The same response was observed for the hepatic enzyme. (6) Results here reported revealed differential tissue responses against the xenobiotics assayed and give evidence on the participation of extrahepatic tissues in porphyrinogenic drug metabolization. These studies have demonstrated the presence of the integral Phase I drug metabolizing system in the brain, thus, total cytochrome P-450 and associated monooxygenases in brain microsomes and mitochondria would be taken into account when considering the xenobiotic metabolizing capability of this organ. Dedicated to the memory of Dr. Susana Afonso     

Chemical reactivities of bleomycin

Ferric bleomycin was tested for its ability to catalyze a set of six oxidative reactions characteristic of the heme-containing proteins, cytochrome P-450 and chloroperoxidase. These reactions included peroxyacid decarboxylation and aliphatic hydroxylation as typical cytochrome P-450 chemistries. Peroxyacid-supported oxygen evolution and hydrogen peroxide-mediated chlorination were utilized as characteristic chloroperoxidase reactivities. A typical peroxidative reaction and heteroatom dealkylation, common to both O2 activating enzymes, were also studied. Bleomycin was found to catalyze peroxidation of o-dianisidine. The ferric drug complex was found competent in carrying out N-demethylation of N,N-dimethylaniline when peroxides or peroxyacids or iodosobenzene were used as the oxidants. N-Demethylation was not achieved when N,N-dimethylaniline-N-oxide was substituted as the oxidant under similar conditions. Hydroxylation of cumene and decarboxylation of phenylperacetic acid were not found to be catalyzed by bleomycin. Oxygen evolution from m-chloroperbenzoic acid and chlorination of monochlorodimedone from chloride ion and hydrogen peroxide were found to be catalyzed by bleomycin. Cytochrome P-450cam was also evaluated for O2 evolution, and halogenation activity and was found not to demonstrate such reactivities. The results of this initial survey, along with those of previous studies, appear to indicate that the chemical reactivity of bleomycin can be more closely aligned with the reactivities demonstrated by chloroperoxidase than those of cytochrome P-450.     

Effect of carbon source on the accumulation of cytochrome P-450 and cytochrome c in the yeast Rhodosporidium toruloides

The appearance of cytochrome content in the yeast Rhodosporidium toruloides depended on the substrate supporting growth. The cells of R. toruloides growing on benzoate were found to contain cytochrome P-450. The concentration of cytochrome P-450 was maximal at the beginning of the exponential phase and then remained at a relatively constant level, but rapidly decreased at the beginning of the stationary phase. When benzoate was exhausted from the medium, the cytochrome P-450 level decreased to zero. On the other hand, cytochrome P-450 was not detected when R. toruloides grew on glucose. However, cytochrome P-450 was detected, when R. toruloides was grown on benzoate together with glucose.
The maximal content of cytochrome c in the cells was observed at the beginning of the exponential phase of growth on both substrates and decreased most rapidly during late stationary phase of growth. The content of cytochrome c in R. toruloides was 2–3 times lower during the growth on glucose as compared to the growth on benzoate.     

Evidence for induction of hepatic microsomal cytochrome P-450 by cimetidine: binding and kinetic studies

The interaction of cimetidine with liver microsomes has been examined by spectral and equilibrium partition studies. First, difference spectroscopy has been used to evaluate the proportion of cytochrome P-450 in rat liver microsomes that exhibits an affinity for cimetidine in the pharmacologically relevant, low micromolar range of drug concentration. The value of 0.45 so obtained has confirmed that a substantial proportion of rat liver cytochrome P-450 has a high binding affinity for this drug. Second, a study of the binding of cimetidine to human liver microsomes by difference spectroscopy and partition equilibrium has detected a similar interaction, thus providing direct support for the postulate that the clinically observed impairment of oxidative drug metabolism may be due in part to inhibition of cytochrome P-450 monooxygenase by cimetidine. Hepatic microsomes from cimetidine-pretreated rats have been shown to exhibit elevated cytochrome P-450 specific content but a decreased proportion of sites with high affinity for the drug; this finding has been shown not to be the consequence of cimetidine-mediated, time-dependent, irreversible monooxygenase inhibition. Although cimetidine pretreatment caused enhanced specific activity of 7-ethoxyresorufin O-dealkylation, the specific activities for O-dealkylation of 7-ethoxycoumarin and 4-nitroanisole were decreased, as were those for the N-dealkylation of morphine, ethylmorphine, aminopyrine, and dimethylnitrosamine. Since cimetidine pretreatment was shown to cause no change in the Michaelis constants for oxidation of morphine or 7-ethoxyresorufin, it is argued that these results provide strong presumptive evidence for changes in the relative abundance of isoenzymes catalyzing these various oxidations. Thus, a dual role of cimetidine, acting both as inhibitor and inducer of the cytochrome P-450 system, is proposed to account for the impaired oxidative metabolism of some drugs that occurs during coadministration with this H2-receptor antagonist.     

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.     

Cytochrome P-450 2E1 in Rat Liver Peroxisomes: Downregulation by Ischemia/Reperfusion-Induced Oxidative Stress

Cytochrome P-450 containing enzymes, known to be present in the endoplasmic reticulum and mitochondria, catalyze the oxidation of various compounds. In this study we have used highly purified peroxisomes (>95%) to provide evidence by analytical cell fractionation, enzyme activity, Western blot, and immunocytochemical analysis that cytochrome P-450 2E1 (Cyp 2E1) is present in peroxisomes. Similar specific activities of aniline hydroxylase, a Cyp 2E1-dependent enzyme, in purified peroxisomes (0.72 ± 0.03 nmol/min/mg protein) and microsomes (0.58 ± 0.03 nmol/min/mg protein) supports the conclusion that peroxisomes contain significant amount of Cyp 2E1. This peroxisomal Cyp 2E1 was also induced in acetone-treated rat liver. The status of microsomal and peroxisomal Cyp 2E1 was also examined following ischemia/reperfusion-induced oxidative stress. Ischemia alone had no effect; however, reperfusion following ischemia resulted in decrease in Cyp 2E1 both in microsomes and peroxisomes. This demonstration of cytochrome P-450 2E1 in peroxisomes and its downregulation during ischemia/reperfusion describes a new role for this organelle in cytochrome P-450 related cellular metabolism and in oxidative stress induced disease conditions.     

Kinetics of cytochrome P-450 reduction: studies in bovine adrenocortical microsomes

The results of the present study indicate first that in the microsomal preparation, the components of the P-450 reduction system are heterogeneously distributed, comprising dissociable and nondissociable parts. Second, the P-450 reduction curve can be adequately described by a sum of two exponential functions, indicating two concurrent first-order reactions. Third, the two phases can be altered independently. The addition of the substrate increased the extent of the fast phase while it had little or no effect on that of the slow phase. Changes in the interaction of the dissociable and nondissociable components affected the extent of the slow phase while they were without effect on that of the fast phase. Experiments with different steroids indicated that the independence of the two phases is not due to functionally different P-450's and that the cytochrome reduced in both phases is essentially P-450C-21. The results are interpreted as follows: Transformation of P-450 from the low- to the high-spin state controls the total P-450 reduced. The rate and the biphasicity of the reduction are functions of the interaction of P-450 and the reductase.     

Mechanistic studies of lanosterol 14 alpha-methyl demethylase: substrate requirements for the component reactions catalyzed by a single cytochrome P-450 isozyme

Lanosterol 14 alpha-methyl demethylation is a cytochrome P-450-dependent process that proceeds through the oxidative sequence of alcohol, aldehyde followed by decarbonylation with formic acid release. Microsomal metabolism studies shown here indicate that only lanostenols and 32-oxy-lanostenols with unsaturation at either the delta 7 or delta 8 position in the sterol can be demethylated. The 14 alpha-methyl group of either lanostan-3 beta-ol or delta 6 lanostenol is not oxidized to the anticipated C-32 alcohol or aldehyde by the enzyme, nor are the corresponding 32-oxy-lanostanols demethylated when incubated with microsomal preparations. Despite the lack of metabolism, the saturated and delta 6 sterol analogues are effective competitive inhibitors of demethylase activity. Utilizing preferred substrates, comparison of the component reactions of the demethylation sequence shows that both the oxidative function and lyase function are sensitive to common inhibitors and that both activities require NADPH. These findings strongly support the premise that a P-450 isozyme does catalyze each phase of the lanosterol 14 alpha-methyl demethylation sequence. Collectively these results demonstrate the double-bond requirement for both components of the demethylation sequence and suggest that the olefinic electrons at delta 7 or delta 8 but not delta 6 may participate directly during demethylation. This participation may involve stabilizing a transition state intermediate or directing activated oxygen insertion as part of the P-450 monoxygenase mechanism.     

Acetone-inducible cytochrome P-450: purification, catalytic activity, and interaction with cytochrome b5

A procedure was developed for the purification of an acetone-inducible form of cytochrome P-450 (P-450ac) to electrophoretical homogeneity from liver microsomes of acetone-treated rats. The P-450ac preparation containing 16.0 to 16.5 nmol P-450/mg protein moved as a single protein band with an estimated molecular weight of 52,000 upon gel electrophoresis in the presence of sodium dodecyl sulfate. The ferric P-450ac showed an absorption maximum at 394 nm at 25 degrees C, suggesting that it exists mainly in the high-spin form. It also existed in the low-spin form, especially at lower temperatures, as indicated by the absorption maximum in the 412-nm region. Upon reconstitution with NADPH: cytochrome P-450 reductase and phospholipid, P-450ac efficiently catalyzed both the demethylation and denitrosation of N-nitrosodimethylamine (NDMA) showing Vmax values of 23.8 and 2.3 nmol min-1 nmol P-450-1, respectively. The catalytic activity of P-450ac was greatly affected by cytochrome b5 which decreased the Km values of these reactions by a factor of 10 and increased the Vmax values. Cytochrome b5 appeared to interact with P-450 at a molar ratio of 1:1 and an intact cytochrome b5 structure was required for such interaction. Among the substrates studied, the demethylation of NDMA was affected the most by cytochrome b5 and showed the highest rate. P-450ac also catalyzed the oxygenation of N-nitrosomethylethylamine and aniline and the activity was enhanced slightly by cytochrome b5. Cytochrome b5 did not enhance the P-450ac-catalyzed metabolism of other drug substrates such as benzphetamine, aminopyrine, and ethylmorphine. P-450ac appeared to be similar in property to the previously studied rat P-450et (ethanol-inducible), rat P-450j (isoniazid-inducible), and rabbit P-450LM3a (ethanol-inducible). These P-450 species represent a new class of P-450 isozymes that are important in the metabolism of many endobiotics and xenobiotics.     

Phospholipid interactions with cytochrome P-450 in reconstituted vesicles Preference for negatively-charged phosphatidic acid

Cytochrome $\text{P-450}$ LM2 was reconstituted by the cholate-dialysis method into vesicles containing a mixture of either phosphatidylcholine or phosphatidylethanolamine with up to 50 mol% of phosphatidic acid. Phase transition curves in the presence or absence of cytochrome $\text{P-450}$ were obtained from electron paramagnetic resonance experiments by measuring the partitioning of 2,2,6,6-tetramethylpiperidine-1-oxyl. Protein-free phospholipid vesicles exhibit a phase separation into domains of gel phase enriched in phosphatidic acid in a surrounding fluid matrix containing mainly phosphatidylcholine. The phase transition of the phosphatidic acid domains disappeared following incorporation of cytochrome $\text{P-450}$ into the bilayers. In contrast, in vesicles containing mixtures of egg-phosphatidic acid and dimyristoyl phosphatidylcholine, the phase transition of the domains enriched in dimyristoyl phosphatidylcholine was less sharp than in the corresponding vesicles containing cytochrome $\text{P-450}$. The results of both of these experiments could be explained by a redistribution of the mol fraction of the two phospholipids in the gel phase due to preferential binding of the egg-phosphatidic acid to the cytochrome $\text{P-450}$. For comparison, incorporation of cytochrome $\text{P-450}$ into uncharged vesicles of dimyristoyl phosphatidylcholine and egg-phosphatidylethanolamine did not alter the     

An anti-peptide antibody targeted to a specific region of rat cytochrome P-450IA2 inhibits enzyme activity.

An anti-peptide antibody has been produced which binds to and specifically inhibits the activity of cytochrome P-450IA2 in rat hepatic microsomes. This was achieved by raising an antibody against a synthetic peptide (Ser-Glu-Asn-Tyr-Lys-Asp-Asn), the sequence of which occurs in cytochrome P-450IA2 at positions 290-296. The selection of this region of cytochrome P-450IA2 was based on several criteria, including prediction of surface and loop areas, identification of variable regions between cytochromes P-450IA2 and P-450IA1, and consideration of a site on cytochrome P-450IA1 where chemical modification has been shown to cause substantial enzyme inactivation. The specificity of antibody binding was determined by enzyme-linked immunosorbent assay and by immunoblotting using hepatic microsomal preparations and purified cytochrome P-450 isoenzymes. This showed that the antibody binds specifically to rat and mouse cytochrome P-450IA2 and to no other cytochrome P-450, as was predicted from the amino acid sequences of the peptide and the cytochromes P-450. The effect of the antibody upon enzyme activity was studied in hepatic microsomes from rats treated with 3-methylcholanthrene. The antibody was shown to inhibit specifically the activity of reactions catalysed by cytochrome P-450IA2 (phenacetin O-de-ethylase and 2-acetylaminofluorene activation), but had no effect on aryl hydrocarbon hydroxylase activity, which is catalysed by cytochrome P-450IA1, or on aflatoxin B1 activation.     

Enzymatic Mechanisms Involved in Phenanthrene Degradation by the White Rot Fungus Pleurotus ostreatus

The enzymatic mechanisms involved in the degradation of phenanthrene by the white rot fungus Pleurotus ostreatus were examined. Phase I metabolism (cytochrome P-450 monooxygenase and epoxide hydrolase) and phase II conjugation (glutathione S-transferase, aryl sulfotransferase, UDP-glucuronosyltransferase, and UDP-glucosyltransferase) enzyme activities were determined for mycelial extracts of P. ostreatus. Cytochrome P-450 was detected in both cytosolic and microsomal fractions at 0.16 and 0.38 nmol min(sup-1) mg of protein(sup1), respectively. Both fractions oxidized [9,10-(sup14)C]phenanthrene to phenanthrene trans-9,10-dihydrodiol. The cytochrome P-450 inhibitors 1-aminobenzotriazole (0.1 mM), SKF-525A (proadifen, 0.1 mM), and carbon monoxide inhibited the cytosolic and microsomal P-450s differently. Cytosolic and microsomal epoxide hydrolase activities, with phenanthrene 9,10-oxide as the substrate, were similar, with specific activities of 0.50 and 0.41 nmol min(sup-1) mg of protein(sup-1), respectively. The epoxide hydrolase inhibitor cyclohexene oxide (5 mM) significantly inhibited the formation of phenanthrene trans-9,10-dihydrodiol in both fractions. The phase II enzyme 1-chloro-2,4-dinitrobenzene glutathione S-transferase was detected in the cytosolic fraction (4.16 nmol min(sup-1) mg of protein(sup-1)), whereas aryl adenosine-3(prm1)-phosphate-5(prm1)-phosphosulfate sulfotransferase (aryl PAPS sulfotransferase) UDP-glucuronosyltransferase, and UDP-glucosyltransferase had microsomal activities of 2.14, 4.25, and 4.21 nmol min(sup-1) mg of protein(sup-1), respectively, with low activity in the cytosolic fraction. However, when P. ostreatus culture broth incubated with phenanthrene was screened for phase II metabolites, no sulfate, glutathione, glucoside, or glucuronide conjugates of phenanthrene metabolites were detected. These experiments indicate the involvement of cytochrome P-450 monooxygenase and epoxide hydrolase in the initial phase I oxidation of phenanthrene to form phenanthrene trans-9,10-dihydrodiol. Laccase and manganese-independent peroxidase were not involved in the initial oxidation of phenanthrene. Although P. ostreatus had phase II xenobiotic metabolizing enzymes, conjugation reactions were not important for the elimination of hydroxylated phenanthrene.     

Detection of the enzymatic activity of cytochrome P-450 enzymes by high-performance liquid chromatography

The reactions catalysed by the various cytochrome P-450 enzymes are reviewed with respect to the analysis of products by high-performance liquid chromatography (HPLC). Especially biotransformation reactions of purified cytochrome P-450 enzymes in a reconstituted system and in microsomes mainly of rat liver origin are considered. Emphasis is put on the specificity of product formation due to the individual isozymes of cytochrome P-450. It is shown that the presence of eight cytochrome P-450 isozymes can be monitored and determined by specific product formation after HPLC analysis, which is an important parameter in toxicological studies.     

Metabolism of 17 alpha-ethynylestradiol in humans

17 alpha-Ethynylestradiol is extensively sulfated but the sulfate is thought to primarily be a storage form of this estrogen. 2-Hydroxylation is clearly the major oxidative reaction, and the 2-hydroxy derivative is further transformed by methylation and glucuronidation prior to urinary and fecal excretion. Alterations in the rate of 2-hydroxylation can have major effects on the pharmacokinetics and effectiveness of 17 alpha-ethynylestradiol as a contraceptive. The major human catalyst of the 2-hydroxylation reaction is liver microsomal cytochrome P-450 IIIA4. Lesser amounts of this enzyme are found in other tissues such as the intestine and may contribute to overall clearance of the orally administered contraceptive. In individuals with very low amounts of this enzyme other forms of cytochrome P-450 may make some contribution. Levels of cytochrome P-450 IIIA4 vary widely among individuals and can explain the variation in rates of 17 alpha-ethynylestradiol 2-hydroxylation. The known inducibility of the enzyme by barbiturates and rifampicin explains their effects in enhancing 17 alpha-ethynylestradiol clearance and reducing the effectiveness of the drug. Mechanism-based inactivation of cytochrome P-450 IIIA4 can be seen with 17 alpha-ethynylestradiol and other 17 alpha-acetylenic steroids, and the progestogen gestodene appears to be unusually active in this regard. Other unknown factors may also modulate levels of cytochrome P-450 IIIA4 and its ability to catalyze 17 alpha-ethynylestradiol 2-hydroxylation.     

Heterogeneity of cytochrome P-450 in rat testis microsomes.

Cytochrome P-450 appears to be a component of the steroid-coverting enzymes, 17alpha-hydroxylase and 17,20-lyase, which catalyze sequential steps in sex hormone synthesis. Further evidence indicates that the steroid substrates of these enzymes bind to cytochrome P-450 during catalysis. The present report deals with the problem of whether a single form of cytochrome P-450 mediates both enzyme reactions or whether two enzymes are involved. Both activities are competitively inhibited by a number of the same inhibitors. Because K1 values of competitive inhibitors are dissociated constants, and thus a property of the cytochrome, different magnitudes of K1, determined for the same inhibitor with each enzyme, are consistent with the participation of more than one form of cytochrome P-450. Differences in the K1 values were found to be statistically significant and varied from 3- to 10-fold. Two competitive inhibitors retarded velocities with one reaction but not the other. In addition, the enzyme activities were markedly different in their sensitivity to carbon monoxide inhibition. The conclusion based on these two lines of evidence is that separate enzymes and different forms of cytochrome P-450 are involved in each reaction.     

Endogenous promutagen activation in the yeast Saccharomyces cerevisiae: factors influencing aflatoxin B1 mutagenicity

The formation of convertants, revertants and other types of mitotic segregants was induced in Saccharomyces cerevisiae D7 upon incubation with aflatoxin B1 (AFB1). The most distinct effects were observed for gene conversion to tryptophan prototrophy. The fact that different cytochrome P-450 inhibitors (ellipticine, penconazole and propiconazole as yeast-specific P-450 inhibitors) abolished the AFB1-induced mutagenicity indicates that activation of the promutagen AFB1 depends on the cytochrome P-450-catalyzed electron-transfer reactions. This hypothesis is further supported by the observation that the cytochrome P-450 content of yeast cells harvested at different phases during growth is directly correlated with their sensitivity for AFB1-induced tryptophan conversion.     

Purification and characterization of hepatic microsomal prostaglandin omega-hydroxylase cytochrome P-450 from pregnant rabbits

Prostaglandin omega-hydroxylase, designated as cytochrome P-450 LPG omega (P-450 LPG omega), has been purified, to a specific content of 15 nmol of cytochrome P-450/mg of protein, from liver microsomes of pregnant rabbits. The purified P-450 LPG omega was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and to have an apparent molecular weight of 52,000. The enzyme showed a maximum at 450 nm in the carbon monoxide (CO)-difference spectrum for its reduced form. This cytochrome P-450 efficiently catalyzed the omega-hydroxylation of prostaglandin E1 (PGE1), prostaglandin E2 (PGE2), prostaglandin D2 (PGD2), prostaglandin F2 alpha (PGF 2 alpha), prostaglandin A1 (PGA1), and prostaglandin A2 (PGA2), as well as the omega- and (omega-1)-hydroxylation of myristate and palmitate, in a reconstituted system containing cytochrome P-450, NADPH-cytochrome P-450 reductase, phospholipid, and cytochrome b5. Various monovalent and divalent cations further stimulated these reactions in the presence of cytochrome b5. In addition, the reactions were also markedly enhanced by various organic solvents, such as ethanol and acetone. This cytochrome P-450 showed no detectable activity toward several xenobiotics tested. P-450 LPG omega was very similar or identical to the pulmonary prostaglandin omega-hydroxylase (P-450p-2) (Yamamoto, S., Kusunose, E., Ogita, K., Kaku, M., Ichihara, K., & Kusunose, M. (1984) J. Biochem. 96, 593-603) in its molecular weight, absorption spectra, catalytic activity, peptide mapping pattern, and N-terminal amino acid sequence. However, P-450 LPG omega was more unstable than P-450p-2 on storage. In sharp contrast to P-450p-2, P-450 LPG omega was not induced by progesterone.     

Oxidative stress limits vitamin D metabolism by bovine proximal tubule cells in vitro

When bovine proximal tubule cells are placed in primary culture, they are subject to elevated oxidative stress which acts to limit the expression of mitochondrial vitamin D3 1 alpha- and 24-hydroxylase activities. This increased oxidative stress was demonstrated by increased production of cell and mitochondrial membrane lipid hyperperoxides (LOOH). This increased production was prevented by the addition of the antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Cell and mitochondrial membrane LOOH increased from 1 to 2 pmol/mg protein on the day of plating to 70-90 pmol/mg protein after 6 days in culture. Pretreatment of cultures with BHA and BHT resulted in membrane LOOH of 15-20 pmol/mg protein after 6 days. Mitochondrial LOOH production was greater than total cell LOOH after 6 days. The increase in cellular oxidative stress was paralleled by decreases in both 1 alpha- and 24-hydroxylase activities toward 25-OH D3. Mitochondrial hydroxylase activities were inversely proportional to the increase in mitochondrial membrane LOOH production. Mitochondrial cytochrome P-450 content, determined spectrophotometrically, was decreased over time in culture. Mitochondrial cytochrome P-450 content determined by a specific polyclonal antibody in an enzyme-linked immunosorbant assay also decreased over time in culture. Specificity of polyclonal antibodies, raised against rat liver microsomal cytochrome P-450 RLM5, was demonstrated by the immunosequestration of both 1 alpha- and 24-hydroxylase activities from a partially purified preparation of renal mitochondrial cytochrome P-450. BHA showed the loss of 1 alpha- and 24-hydroxylase activities and mitochondrial P-450 content measured by all criteria. These experiments indicate that oxidative stress-mediated changes in hydroxylase activities are mediated directly by changes in hydroxylase content and not at distal sites. A partially purified preparation of bovine proximal tubule mitochondrial cytochrome P-450, with purified renal ferredoxin, ferredoxin reductase, and NADPH, expressed both 1 alpha- and 24-hydroxylase activities toward 25-OH D3. LOOH, derived from mitochondrial membranes of 5-day-old cultures, when added to this mixture, caused a dose-dependent decrease in both activities. These experiments suggested that an increase in mitochondrial LOOH production resulted in a loss of 1 alpha- and 24-hydroxylase activities. 1 alpha-Hydroxylase was more sensitive to the effects of LOOH treatment than 24-hydroxylase. At a ratio of LOOH:P-450 of 5:1 (molar), all 1 alpha-hydroxylase activity was lost but 50% of the 24-hydroxylase activity remained.(ABSTRACT TRUNCATED AT 400 WORDS)     

Musk xylene is a novel specific inducer of cytochrome P-450IA2.

The effect of musk xylene on contents of both cytochrome P-450IA1 and cytochrome P-450IA2 in rat liver was investigated using Western blotting analysis. Rats were treated i.p. for five consecutive days with either 50, 100 or 200 mg musk xylene/kg body weight. Musk xylene increased both total cytochrome P-450 and cytochrome b5 contents in rat liver microsomes. Musk xylene induced cytochrome P-450IA2 (384 pmol/mg protein) strongly and preferentially and the ratio of cytochrome P450IA2/P-450IA1 was about 12 at the lowest dose tested. Musk xylene also induced the cytochrome P-450IA1 dose-dependently, but these extents were very small (32-174 pmol/mg protein). These results suggest that musk xylene may be a more specific inducer for cytochrome P-450IA2 than any other inducers reported.