Risk factors in heart transplantation. A statistical study of New Zealand cases

The bioactivation of N-nitrosoamines and polycyclic aromatic hydrocarbons (PAH) is mediated by the mixed function oxidase system, which includes dimethylnitrosamine N-demethylase I (DMN-dI), arylhydrocarbon hydroxylase (AHH), cytochrome P-450, cytochrome b5 and NADPH-cytochrome c reductase of liver microsomes. The present study shows the influence of N-nitroso compounds on the activities of the above-mentioned enzymes. Single-dose treatment (20 mg/kg body weight) of male mice with ethylbutylnitrosamine, propylbutylnitrosamine, or dibutylnitrosamine: increased (1) the activity of DMN-dI by 108%, 104%, 51%, respectively; (2) the cytochrome P-450 content by 106%, 72%, 51%, respectively; (3) the activity of AHH by 95%, 106%, 80% respectively; (4) the cytochrome b5 content by 164%, 97%, 94% respectively; and (5) decreased the activity of NADPH-cytochrome c reductase by 55%, 50% and 45%, respectively. Methylpropylnitrosamine decreased the activity of DMN-dI by 44% and the P-450 content by 50%. Diphenylnitrosamine also decreased cytochrome P-450 by 54%, AHH activity by 64% but increased the activity of DMN-dI by 42%, the cytochrome b5 content by 159% and NADPH-cytochrome c reductase activity by 57%. It seems from this study that the activity of AHH is dependent on P-450 content but DMN-dI is not since the compounds that increased or decreased the activity of AHH had parallel effects on P-450 content. Also, the extent to which the altered activities of DMN-dI, P-450, AHH, cytochrome b5 and NADPH-cytochrome c reductase depends on the type of alkyl groups linked to the nitroso group.     

Preparation and properties of partially purified pulmonary cytochrome P-450 from rabbits

Cytochrome P-450 from rabbit pulmonary microsomes was purified approximately 32-fold. The purification method involved solubilization of microsomes using sodium cholate, and recovery of cytochrome P-450 in the precipitate formed between 25 to 42% saturation of the digested microsomes with ammonium sulfate in the absence of glycerol. Further purification was achieved by chromatography on DEAE-cellulose and hydroxylapatite using Emulgen 913 as an eluent. Partially purified preparations containing up to 7.4 nmol of cytochrome P-450 per mg of protein were essentially free of NADPH-cytochrome c reductase activity and cytochromes b5 and P-420. However, epoxide hydrase was found to co-purify with cytochrome P-450. The CO-difference spectrum of dithionite-reduced purified cytochrome showed the expected peak at 450 nm. However, the magnitude of the peak was dependent on added microsomal lipid fraction in the assay medium. Purified pulmonary cytochrome P-450 formed typical types I and II substrate difference spectra with benzphetamine and pyridine, respectively. Sodium dodecyl sulfate-gel electrophoresis of partially purified cytochrome P-450 gave two major bands when stained with Coomassie blue. The faster moving band which contained peroxidase activity had an estimated molecular weight of 49,000 +/- 1,200. The cytochrome P-450 fraction, when combined with solubilized pulmonary microsomal NADPH-cytochrome c reductase and lipid fractions, was active in the O-deethylation of 7-ethoxycoumarin and the N-demethylation of benzphetamine.     

Effects of an immunosuppressive agent, tacrolimus (FK-506), on the activities of cytochrome P-450-linked monooxygenase systems in rat liver microsomes

The effects of an immunosuppressive agent, tacrolimus (FK-506), on the activities of cytochrome P-450-linked monooxygenase systems with respect to three cytochrome P-450 isozymes in rat liver microsomes were investigated. FK-506 non-competitively inhibited the aniline p-hydroxylase, p-nitroanisole O-demethylase and lidocaine N-deethylase activities of cytochrome P-450-linked monooxygenase systems, these activities being mainly catalyzed by cytochromes P-450 CYP2E1, CYP2C11 and CYP3A4, respectively, and the Ki values of the activities for FK-506 were determined to be 605, 491 and 97 microM, respectively. The inhibition of cytochrome P-450-linked monooxygenase systems by FK-506 seemed to involve the direct inhibition of cytochromes P-450 because the NADPH-cytochrome c reductase and NADPH-ferricyanide reductase activities of NADPH-cytochrome P-450 reductase were not affected by the presence of 1 mM FK-506 at all. A spectrophotometric study showed that a reverse type I spectral change was induced on the addition of FK-506 to rat liver microsomes, and the Ks value was apparently 125 microM. On the other hand, the EPR spectra of cytochromes P-450 in rat liver microsomes were not affected by 1 mM FK-506. These results suggest direct interaction between FK-506 and cytochrome P-450 apoproteins, except for the heme iron regions of cytochromes P-450, resulting in inhibition of the drug-metabolism activities catalyzed by cytochromes P-450.     

Combined effect of cadmium and nickel on rat hepatic monooxygenases: possible stimulation of certain cytochrome P-450 isozymes

When male rats were given either a single dose of cadmium (3.58 mg CdCl2.6H2O/kg, i.p.) 72 h prior to sacrifice or a single dose of nickel (59.5 mg NiCl2.6H2O/kg, s.c.) 16 h prior to sacrifice, the activities of ethylmorphine N-demethylase, aminopyrine N-demethylase and aniline 4-hydroxylase, and the levels of cytochrome P-450 and microsomal heme were significantly decreased. Cadmium decreased the cytochrome b5 level significantly, whereas it did not alter the NADPH-cytochrome c reductase activity significantly. In contrast, Ni did not alter the cytochrome b5 level significantly but decreased the NADPH-cytochrome c reductase activity significantly. For the combined treatment, animals received the single dose of nickel 56 h after the single dose of cadmium and then they were killed 16 h later. In these animals ethylmorphine N-demethylase, aminopyrine N-demethylase and NADPH-cytochrome c reductase activities and cytochromes P-450 and b5 levels increased significantly as compared to those of controls, whereas aniline 4-hydroxylase activity and microsomal heme level remained unaltered. In concordance with the increase in the enzyme activities, certain P-450 protein bands were observed to be elevated when studied on SDS-polyacrylamide gel electrophoresis. Furthermore, when the monooxygenase activities and SDS-polyacrylamide gel electrophoresis profiles of combined metal-treated animals were compared with those of the animals treated with classic inducers such as phenobarbital (75 mg/kg i.p., 72, 48 and 24 h prior to sacrifice) and 3-methylcholanthrene (20 mg/kg i.p., 72, 48 and 24 h prior to sacrifice), the combination of metals seemed to have tendency to stimulate certain phenobarbital and 3-methylcholanthrene inducible cytochrome P-450 isozymes.     

Some properties of a detergent-solubilized NADPH-cytochrome c(cytochrome P-450) reductase purified by biospecific affinity chromatography

NADPH-cytochrome c (cytochrome P-450) reductase (EC 1.6.2.4) has been purified to homogeneity, as judged by sodium dodecyl sulfate disc gel electrophoresis, from detergent-solubilized rat and pig liver microsomes using an affinity chromatography procedure. Treatment of microsomes with a polyethoxynonylphenyl ether plus either cholate or deoxycholate and subsequent batch-wise DEAE-cellulose chromatography followed by biospecific affinity chromatography on Sepharose 4B-bound N6-(6-aminohexyl)-adenosine 2',5'-bisphosphate (2'5'-ADP-Sepharose 4B) result in a greater than 30% yield of purified reductase from microsomes. The enzyme contains 1 mol each of FAD and FMN and exhibits a molecular weight of 78,000 g mol-1 estimated by comparison with protein standards on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The turnover numbers calculated on the basis of flavin are 1360 min-1 and 1490 min-1 at 25 degrees for the pig and rat liver enzymes, respectively. Titration of these purified preparations aerobically with both NADPH and potassium ferricyanide demonstrated unequivocally that the air-stable, reduced form of NADPH-cytochrome c (P-450) reductase contains 2 electron equivalents, confirming recent results obtained by Masters et al. (Masters, B. S. S., Prough, R. A., and Kamin, H. (1975) Biochemistry 14, 607-613) for the proteolytically solubilized enzyme. In addition, these preparations are capable of reconstituting benzphetamine N-demethylation activity in the presence of partially purified cytochrome P-450 and dilauroylphosphatidylcholine, as measured by formaldehyde formation from benzphetamine.     

Electron transfer to cytochrome P-450scc limits cholesterol-side-chain-cleavage activity in the human placenta

The aim of this study was to determine whether electron transfer from adrenodoxin reductase and adrenodoxin limits the activity of cytochrome P-450scc in mitochondria from the human placenta. Mitochondria were disrupted by sonication to enable exogenous adrenodoxin and adrenodoxin reductase to deliver electrons to cytochrome P-450scc. After sonication, the rate of pregnenolone synthesis was greatly decreased relative to that by intact mitochondria, due to dilution of endogenous adrenodoxin and adrenodoxin reductase into the incubation medium. The addition of saturating concentrations of bovine or human adrenodoxin and bovine adrenodoxin reductase to the disrupted mitochondria gave an initial rate of pregnenolone synthesis that was 6.3-fold higher than that for intact mitochondria. Similar results were observed when 20alpha-hydroxycholesterol was used as substrate rather than endogenous cholesterol. The turnover number of cytochrome P-450scc in sonicated placental mitochondria supplemented with adrenodoxin and adrenodoxin reductase was comparable to that for the purified enzyme assayed under conditions where electron transfer was not limiting. Addition of exogenous adrenodoxin and adrenodoxin reductase to sonicated mitochondria from the pig corpus luteum and rat adrenal had a much smaller effect on pregnenolone synthesis compared with intact mitochondria, than observed for the placenta. We conclude that in the human placenta, electron transfer to cytochrome P-450scc is limiting, permitting pregnenolone synthesis to proceed at only 16% maximum velocity.     

Steroidogenic electron transport in adrenal cortex mitochondria

The flavoprotein NADPH-adrenodoxin reductase and the iron sulfur protein adrenodoxin function as a short electron transport chain which donates electrons one-at-a-time to adrenal cortex mitochondrial cytochromes P-450. The soluble adrenodoxin acts as a mobile one-electron shuttle, forming a complex first with NADPH-reduced adrenodoxin reductase from which it accepts an electron, then dissociating, and finally reassociating with and donating an electron to the membrane-bound cytochrome P-450 (Fig. 9). Dissociation and reassociation with flavoprotein then allows a second cycle of electron transfers. A complex set of factors govern the sequential protein-protein interactions which comprise this adrenodoxin shuttle mechanism; among these factors, reduction of the iron sulfur center by the flavin weakens the adrenodoxin-adrenodoxin reductase interaction, thus promoting dissociation of this complex to yield free reduced adrenodoxin. Substrate (cholesterol) binding to cytochrome P-450scc both promotes the binding of the free adrenodoxin to the cytochrome, and alters the oxidation-reduction potential of the heme so as to favor reduction by adrenodoxin. The cholesterol binding site on cytochrome P-450scc appears to be in direct communication with the hydrophobic phospholipid milieu in which this substrate is dissolved. Specific effects of both phospholipid headgroups and fatty acyl side-chains regulate the interaction of cholesterol with its binding side. Cardiolipin is an extremely potent positive effector for cholesterol binding, and evidence supports the existence of a specific effector lipid binding site on cytochrome P.450scc to which this phospholipid binds.     

Effects of morphine sulfate on NADPH-cytochrome c reductase and cytochrome P-450 of mouse liver microsomes

Three hr after i.p. administration of a single dose of 30 mg/kg of morphine to male mice, an increase in specific activity of NADPH-cytochrome c reductase by about 10% and the content of cytochrome P-450 by about 14% of their liver microsomes was observed.Administration of 30 mg/kg of morphine, once daily,during 5 days, caused about 16% and 9% increases in specific activity of c reductase and the content of P-450 respectively. Administration of a single dose of morphine to male and female mice caused no sex-dependent differences in the specific activity of c reductase and the content of P-450. Repeated administration of morphine up to 100 mg/kg to male mice increased the specific activity of microsomal c reductase by about 70%. Repeated administration of morphine up to 55 mg/kg also increased the microsomal content of P-450 by about 22%, but with higher doses of morphine, the content of P-450 declined and finally dropped below control levels. The levels of c-reductase activity and P-450 content returned to normal levels about 2 weeks after termination of morphine administration.     

Effects of erythromycin on hepatic drug-metabolizing enzymes in humans

In rats, erythromycin has been shown to induce microsomal enzymes and to promote its own transformation into a metabolite which forms an inactive complex with reduced cytochrome P-450. To determine whether similar effects also occur in humans, we studied hepatic microsomal enzymes from six untreated patients and six patients treated with erythromycin propionate, 2 g per os daily for 7 days. In the treated patients, NADPH-cytochrome c reductase activity was increased; the total cytochrome P-450 concn was also increased but part of the total cytochrome P-450 was complexed by an erythromycin metabolite. The concn of uncomplexed (active) cytochrome P-450 was not significantly modified and the activity of hexobarbital hydroxylase remained unchanged. We also measured the clearance of antipyrine in six other patients; this clearance was not significantly decreased when measured again on the seventh day of the erythromycin propionate treatment. We conclude that the administration of erythromycin propionate induces microsomal enzymes and results in the formation of an inactive cytochrome P-450-metabolite complex in humans. However, the concn of uncomplexed (active) cytochrome P-450 and tests for in vitro and in vivo drug metabolism were not significantly modified.     

Effects of motorcycle exhaust on cytochrome P-450-dependent monooxygenases and glutathione S-transferase in rat tissues

The effects of motorcycle exhaust (ME) on cytochrome P-450 (P-450)-dependent monooxygenases were determined using rats exposed to the exhaust by either inhalation, intratracheal, or intraperitoneal administration. A 4-wk ME inhalation significantly increased benzo[a]pyrene hydroxylation, 7-ethoxyresorufin O-deethylation, and NADPH-cytochrome c reductase activities in liver, kidney, and lung microsomes. Intratracheal instillation of organic extracts of ME particulate (MEP) caused a dose- and time-dependent significant increase of monooxygenase activity. Intratracheal treatment with 0.1 g MEP extract/kg markedly elevated benzo[a]pyrene hydroxylation and 7-ethoxyresorufin O-deethylation activities in the rat tissues 24 h following treatment. Intraperitoneal treatment with 0.5 g MEP extract/kg/d for 4 d resulted in significant increases of P-450 and cytochrome b5 contents and NADPH-cytochrome c reductase activity in liver microsomes. The intraperitoneal treatment also markedly increased monooxygenases activities toward methoxyresorufin, aniline, benzphetamine, and erythromycin in liver and benzo[a]pyrene and 7-ethoxyresorufin in liver, kidney, and lung. Immunoblotting analyses of microsomal proteins using a mouse monoclonal antibody (Mab) 1-12-3 against rat P-450 1A1 revealed that ME inhalation, MEP intratracheal, or MEP intraperitoneal treatment increased a P-450 1A protein in the hepatic and extrahepatic tissues. Protein blots analyzed using antibodies to P-450 enzymes showed that MEP intraperitoneal treatment caused increases of P-450 2B, 2E, and 3A subfamily proteins in the liver. The ME inhalation, MEP intratracheal, or MEP intraperitoneal treatment resulted in significant increases in glutathione S-transferase activity in liver cytosols. The present study shows that ME and MEP extract contain substances that can induce multiple forms of P-450 and glutathione S-transferase activity in the rat.     

Expression of functionally active hyman cytochrome P-450c21 (CYPXXIA2) in Escherichia coli and single-stage purification of it using metal-affinity chromatography

Active human cytochrome P-450c21 was expressed in Escherichia coli and purified to homogeneity. To increase expression, cDNA encoding for the N-terminal fragment of cytochrome P-450c21 was modified. Four histidine codons were added to cDNA encoding for the C-terminus of the protein; thus, recombinant protein could have been rapidly and effectively purified by metal-affinity chromatography. Modified human cytochrome P-450c21 was expressed (40-50 nmoles/l of culture according to spectrophotometry) which was able to bind to bacterial membrane. Modifications of N- and C-terminal regions of cytochrome P-450c21 did not change Km and Vmax for hydroxylation of progesterone and 17 alpha-hydroxyprogesterone in reconstituted system. Recombinant cytochrome P-450c21 was purified to apparent homogeneity from Escherichia coli membrane extract by metal-affinity chromatography. Purified cytochrome P-450c21 migrates as a single 54 kD band on polyacrylamide gel and exhibits type I spectral changes during interaction with progesterone and 17 alpha-hydroxyprogesterone. Activity of purified cytochrome P-450c21 was reconstituted with mouse liver microsomal NADPH-cytochrome P-450-reductase and NADPH-regenerating system. Purified enzyme had Km 12.2 and 3.21 microM and Vmax 192.9 and 198 nmoles/min/nmole of P-450c21 for 17 alpha-hydroxyprogesterone and progesterone, respectively. According to titration spectra, dissociation constants for progesterone and 17 alpha-hydroxy-progesterone were 14.7 and 31.1 microM, respectively.     

The effect of certain vitamin deficiencies on hepatic drug metabolism

There is increasing evidence that the liver microsomal drug metabolizing system is affected by various vitamins such as ascorbic acid, riboflavin, and alpha-tocopherol. In regard to ascorbic acid deficiency there is a decrease in the quantity of hepatic microsomal electron transport components such as cytochrome P-450 and NADPH-cytochrome P-450 reductase, as well as decreases in a variety of drug enzyme reactions such as N-demethylation, O-demethylation, and steroid hydroxylation. In addition, young animals given high supplements of vitamin C have increased quantities of electron transport components and overall drug metabolism activities. Kinetic studies indicate no change in the apparent Km of N-demethylase, O-demethylase or hydroxylase for drug substrates in animals depleted or given high amounts of the vitamin. However, there are qualitative changes in both type I and II substrate-cytochrome P-450 binding. Ascorbic acid is not involved in microsomal lipid peroxidation or in any qualitative or quantitative change in phosphatidylcholine. Replenishing vitamin C-deficient animals with ascorbic acid required 3 to 7 days for the electron transport components and drug metabolism activities to return to normal levels. Induction with phenobarbital and 3-methylcholanthrene is not impaired in the deficient animal since drug metabolism activities are induced to the same extent as normal controls; however, the administration of delta-aminolevulinic acid, a precursor of heme synthesis, to deficient animals caused an increase in the quantity of cytochrome P-450. The effects of riboflavin deficiency on electron transport components and drug metabolism activities have been noted only in adult animals after prolonged periods of deficiency. Decreases in drug metabolism activities occur with both type I (aminopyrine and ethylmorphine) and type II (aniline) substrates. As was found with ascorbic acid deficiency, drug enzyme induction occurred to the same extent with phenobarbital in deficient and normal animals. In addition, it required from 10 to 15 days for the drug metabolism activities to return to normal levels when deficient animals were replenished with riboflavin. The effect of vitamin E on drug metabolism is specific in N-demethylase activities decrease while O-demethylase activities are not affected in the deficient state. This vitamin differs from ascorbic acid and riboflavin in that several laboratories have reported no quantitative decrease in cytochrome P-450, although there are some reports that it and delta-aminolevulinic acid dehydratase are lowered quantity of cytochrome in E-deficient animals. The effect of vitamin E, if any, on the P-450 is unresolved; an important question that requires further clarification. As with ascorbic acid there is no difference in the apparent Km of N-demethylase enzymes for varous substrates and the protective effect of vitamin E does not appear to be one of an antioxidant inhibiting microsomal lipid peroxidation.     

Characterization of cytochrome P450TYR, a multifunctional haem-thiolate N-hydroxylase involved in the biosynthesis of the cyanogenic glucoside dhurrin

The haem-thiolate N-hydroxylase cytochrome P450TYR involved in the biosynthesis of the tyrosine-derived cyanogenic glucoside dhurrin in Sorghum bicolor had recently been isolated. Reconstitution of enzyme activity by insertion of cytochrome P450TYR and NADPH-cytochrome P450-reductase into L-alpha-dilauroylphosphatidylcholine micelles and using tyrosine as substrate results in the formation of p-hydroxyphenylacetaldehyde oxime. Quantitative substrate binding spectra demonstrate that tyrosine and N-hydroxytyrosine are mutually exclusive substrates that bind to the same active site of cytochrome P450TYR. The multifunctionality of cytochrome P450TYR has been confirmed in reconstitution experiments using recombinant cytochrome P450TYR expressed in Escherichia coli. It was earlier reported that an in vitro microsomal system catalyzing all but the last step in the biosynthetic pathway for cyanogenic glucosides exhibits catalytic facilitation (channelling). This observation is explained by the multifunctionality of cytochrome P450TYR. The cytochrome P450TYR sequence represents the first amino acid sequence of a functionally characterized cytochrome P-450 enzyme from a monocotyledonous plant and the first sequence of an N-hydroxylase with high substrate specificity. Multifunctional N-hydroxylases of the cytochrome P-450 type have not previously been reported in living organisms.     

Crystallization of a covalently-linked complex of adrenodoxin and adrenodoxin reductase

Two forms of mitochondrial adrenodoxin reductase from bovine adrenals and recombinant bovine adrenodoxin and adrenodoxin reductase expressed in Escherichia coli were isolated, purified to homogeneity and biochemically characterized. Recombinant adrenodoxin reductase was expressed as a single polypeptide; its retention time on DEAE-Fractogel coincides with the second form (F2) of the mitochondrial reductase. Two enzyme forms have similar adrenodoxin reductase activities in two types of systems comprising either cytochrome c or cytochrome P-450 (11 beta) as the terminal electron acceptor. Adrenodoxin and each of two reductase forms were cross-linked using 1-ethyl-3-(dimethyl-amino-propyl)carbodiimide. An effective two-step method for the purification of the active heterologous cross-linked complexes is suggested that enables purification of the functional complexes to homogeneity. The cross-linked bimolecular complex of adrenodoxin and adrenodoxin reductase was crystallized for the first time.     

Formation of a novel 20-hydroxylated metabolite of lipoxin A4 by human neutrophil microsomes

Lipoxin A4 (LXA4) is a biologically active compound produced from arachidonic acid via interactions of lipoxygenases. Incubation of LXA4 either with human neutrophils or with the neutrophil microsomes leads to formation of a polar compound on a reverse-phase high-performance liquid chromatography. We have identified the metabolite as 20-hydroxy-LXA4, a novel metabolite of arachidonic acid, on the basis of ultraviolet spectrometry and gas chromatography-mass spectrometry. The LXA4 omega-hydroxylation requires both molecular oxygen and NADPH, and is inhibited by carbon monoxide, by antibodies raised against NADPH-cytochrome P-450 reductase, or competitively by leukotriene B4 (LTB4) and LTB5, substrates of LTB4 omega-hydroxylase. These findings indicate that the formation of 20-hydroxy-LXA4 is catalyzed by a neutrophil cytochrome P-450, the LTB4 omega-hydroxylase.     

Molecular cloning and expression in Saccharomyces cerevisiae of tobacco NADPH-cytochrome P450 oxidoreductase cDNA

We obtained information on the full length tobacco NADPH-cytochrome P450 oxidoreductase (P450 reductase) by a combination of the cDNA clone pCTR1 and the genomic DNA clone pGTR1. The deduced primary structure consisting of 713 amino acid residues contained sequences corresponding to FMN, FAD, and NADPH-binding regions. Based on this information, we prepared the full-length cDNA pFTR of tobacco P450 reductase by RT-PCR and expressed it in the yeast Saccharomyces cerevisiae. The transformed yeast cells carrying pFTR produced the corresponding mRNA and protein, and had increased cytochrome c reductase activity in the microsomes. An in vitro reconstitution system of the yeast microsomal fractions expressed tobacco P450 reductase and rat P450 1A1 showed an increased 7-ethoxycoumarin O-deethylase activity. These results indicated that tobacco P450 reductase expressed in the yeast microsomes coupled with rat P450 1A1 resulting in an increased monooxygenase activity.     

Enzymology of mitomycin C metabolic activation in tumour tissue. Characterization of a novel mitochondrial reductase

In this study, the enzymology of mitomycin C (MMC) bioactivation in two murine colon adenocarcinomas, MAC 16 and MAC 26, was examined. Subcellular quinone reductase assessment via cytochrome c reduction confirmed a number of active enzymes. MAC 16 exhibited 22-fold greater levels of cytosolic DT-diaphorase than MAC 26, while microsomal NADPH:cytochrome P-450 reductase levels were similar in both tumour types. Metabolism of MMC by subcellular fractions isolated from both MAC 16 and MAC 26 was quantitated by monitoring the formation of the principle metabolite 2,7-diaminomitosene (2,7-DM) via high-performance liquid chromatography (HPLC). In MAC 16 only, activity displaying the properties of cytosolic DT-diaphorase and microsomal NADPH:cytochrome P-450 reductase was detected and confirmed, using the enzyme inhibitors dicoumarol and cytochrome P-450 reductase antiserum, respectively. The highest level of MMC metabolism was associated with the mitochondrial fraction from both tumours and was the sole enzyme activity detected in MAC 26. The greatest mitochondrial drug metabolism was achieved in the presence of NADPH as cofactor and hypoxia (MAC 16-specific activity, 3.67 +/- 0.58 nmol/30 min/mg; MAC 26 specific-activity, 3.87 +/- 0.71 nmol/30 min/mg) and was unaffected by the addition of the inhibitors dicoumarol and cytochrome P-450 reductase antiserum. NADH-dependent mitochondrial activity was only observed in MAC 16 at approximately 4-fold less than that seen with NADPH. MAC 26 homogenate incubations displayed enhanced metabolism under hypoxia, presumably due to the presence of the identified mitochondrial enzyme. MAC 16 homogenates showed no increase in metabolism under hypoxia, suggesting that other enzyme(s) may be predominant. These data indicate the presence of a novel mitochondrial one-electron reductase capable of metabolising MMC in MAC 16 and MAC 26.     

Antioxidant effect of cupric complex on NADPH-dependent lipid peroxidation in rat liver microsomes

The antioxidant effect of His-Cu (Histidine and cupric ion) on NADPH-dependent lipid peroxidation was proportional to the inhibitory effect on cytochrome c reduction i the presence of microsomes and ADP-Fe (ADP and ferric ion). The inhibiton of cytochrome c reduction by His-Cu caused no inhibition of NADPH oxidation. ADP-Fe stimulated NADPH oxidation in the absence of cytochrome c, and inhibited cytochrome c reduction. His-Cu inhibited NADPH-oxidation stimulated by ADP-Fe, but the antioxidant effect of His-Cu was independent of the effect on cytochrome b5 reduction. These results suggest that the antioxidant effect of His-Cu depends on the inhibitory effect on the electron transport from NADPH-cytochrome c reductase to ADP-Fe, but not on the inhibitory effect on the activity of the reductase.     

Glutathione-dependent factors and inhibition of rat liver microsomal lipid peroxidation

The effects of reduced glutathione (GSH) and glutathione disulfide (GSSG) on lipid peroxidation were investigated in rat liver microsomes containing deficient or adequate amounts of alpha-tocopherol (alpha-TH). Rates of formation of thiobarbituric acid reactive substances (TBARS) as well as rates of consumption of alpha-TH and O2 were decreased by GSH and were more pronounced in the NADPH-dependent assay system than in the ascorbate-dependent system. The GSH-dependent inhibition of lipid peroxidation was potentiated by GSSG in the NADPH-dependent assay system, but it had no effect in the nonenzymatic system. Diphenyliodonium chloride, an inhibitor of NADPH cytochrome P-450 reductase, completely prevented lipid peroxidation in the NADPH-dependent assay system whereas it had no effect on the ascorbate-dependent system. This is further evidenced by the fact that purified rat liver microsomal NADPH cytochrome P-450 reductase (EC 1.6.2.4) was inhibited approximately 24% and 52% by 5 mM GSH and 5 mM GSH + 2.5 mM GSSG, respectively. Glutathione disulfide alone had no effect on reductase activity. Similarly, other disulfides such as cystine, cystamine and lipoic acid were without effect on reductase activity. These results clearly delineate different mechanisms underlying the combined effects of GSH and GSSG on microsomal lipid peroxidation in rat liver. One mechanism involves recycling of microsomal alpha-TH by GSH during oxidative stress via a labile protein, ostensibly associated with "free radical reductase" activity. A second glutathione-dependent mechanism appears to be mediated through the inhibition of NADPH cytochrome P-450 reductase. The enhanced inhibition by GSH + GSSG of microsomal lipid peroxidation in the NADPH-dependent assay system suggests suppression of the initiation phase at the level of NADPH cytochrome P-450 reductase which is independent of microsomal alpha-TH.     

Cloning and expression of cDNA for soluble form of rat heme oxygenase-1

Heme oxygenase catalyzes the oxidation of heme to biliverdin and carbon monoxide. The gene encoding the truncated soluble rat heme oxygenase-1 (Metl-Pro267) was cloned. The enzyme protein was expressed in E. coli JM109 and purified to homogeneity. The molecular weight of the recombinant enzyme was 30 kDa as assessed by SDS-polyacrylamide gel electrophoresis. From a 3-L culture, about 90 mg of the purified enzyme was routinely obtained. The dependency of the heme oxygenase reaction catalyzed by the soluble enzyme on the NADPH-cytochrome P-450 reductase concentrations and the effect of catalase on the reaction were examined to compare with the purified membrane-bound form of heme oxygenase-1 (Yoshida and Kikuchi, 1978b). The activity of the soluble enzyme was inhibited at high concentrations of NADPH-cytochrome P-450 reductase and the inhibition was not alleviated by addition of catalase unlike the membrane-bound form. The ferric iron of the heme-heme oxygenase complex was in a typical high spin state at acidic to neutral pH (pH 6.5-7.0) but conversion to low spin state was observed at basic pH (pH 9-10). The heme bound to heme oxygenase was converted to biliverdin at a stoichiometric ratio of unity in the presence of NADPH-cytochrome P-450 reductase system. During the heme degradation of the heme-heme oxygenase complex under atmospheric oxygen, several intermediates, that is, oxygenated heme and verdoheme, were spectrally discriminated.