Purification and molecular properties of 2-carboxybenzaldehyde (CBA) reductase from phenobarbital-treated rat liver

1. A rat liver cytosol enzyme, tentatively named CBA reductase, catalyses the conversion of 2-carboxybenzaldehyde (CBA) to 2-hydroxymethyl benzoic acid in the presence of NADH (or NADPH). CBA reductase is useful for exploring the mechanism of in vitro enzyme induction, as the enzyme can be induced by phenobarbital (PB) both in vivo and in vitro.

2. Possible involvement of glutathione (GSH) in gene expression was suggested by a recent study with cultured rat hepatocytes.

3. CBA reductase was purified about 200-fold by a combination of column chromatography and isoelectric focusing in the presence of mercaptoethanol.

4. The ability to form 2-hydroxymethyl benzoic acid was lost when the enzyme was chromatographed on a hydroxylapatite column in the absence of mercaptoethanol: however, it was restored if sulphydryl compounds or bovine serum albumin was added to the eluate from the column.

5. Gel filtration showed the molecular sizes of CBA reductase from the 105***000g supernatant fraction of rat liver extracts and the purified preparation were 64kDa and 49kDa, respectively.

6. The results suggest that sulphydryl substances and some proteins play important roles in preserving the molecular and catalytic properties of CBA reductase.     

Enzymatic reduction of arsenic compounds in mammalian systems: reduction of arsenate to arsenite by human liver arsenate reductase

An arsenate (As(V)) reductase has been partially purified from human liver. Its apparent molecular mass is approximately 72 kDa. The enzyme required a thiol and a heat stable cofactor for activity. The cofactor is less than 3 kDa in size. The thiol requirement can be satisfied by dithiothreitol (DTT). However, the extent of stimulation of reductase activity by glutathione, thioredoxin, or reduced lipoic acid was negligible compared to that of DTT. The heat stable cofactor does not appear to be Cu(2+), Mn(2+), Zn(2+), Mg(2+), or Ca(2+). The enzyme does not reduce monomethylarsonic acid (MMA(V)). The isolation and characterization of this enzyme demonstrates that in humans, the reduction of arsenate to arsenite is enzymatically catalyzed and is not solely the result of chemical reduction by glutathione as has been proposed in the past.     

Purification and characterization of 3-mercaptopyruvic acid S-conjugate reductases

Three kinds of 3-mercaptopyruvic acid S-conjugate reductase (MPR-I, MPR-II and MPR-III) were purified from rat liver cytosol. These enzymes reduced 3-mercaptopyruvic acid S-conjugates derived from cysteine conjugates and some endogenous alpha-keto acids to the corresponding alpha-hydroxy acids in the presence of either NADH (for MPR-I and MPR-II) or NADPH (MPR-III), while simple aldehydes or ketones did not significantly induce substrate activity. The molecular weight of the present enzymes was about 80 kDa composed of two subunits of the same molecular weight. Km values of MPR-I, MPR-II and MPR-III were 0.38, 0.06 and 0.29 mM for S-(4-bromophenyl)-3-thiopyruvic acid, respectively, and 0.15 mM for NADH (MPR-I, MPR-II) and NADPH (MPR-III). Vmax values of MPR-I, MPR-II and MPR-III for this substrate were 5.3, 20 and 13 nmol/min/mg, respectively. The sulphydryl-modifying agents inhibited the enzyme activities of all the three reductases. Based on the properties including substrate selectivity for alpha-keto acids derived from aromatic amino acids, we assumed that MPR-II and aromatic alpha-keto acid reductase are the same enzyme, while enzymes similar to MPR-I and MPR-III have not been reported. From the viewpoints of metabolism of xenobiotics, these enzymes are likely to be important in biotransformation of cysteine conjugates to 3-mercaptolactic acid S-conjugates.     

农药叶枯灵在大鼠原位灌流肝中的代谢研究

采用反相HPLC、TLC、UV、IR和MS对农药叶枯灵经大鼠原位灌流肝代谢后所形成的代谢产物进行分离和鉴定。结果表明,叶枯灵在大鼠肝脏中进行了广泛的代谢,包括S-氧化作用、水解作用、丙酮酸缩合作用和乙酰化作用,共分离鉴定出5种代谢产物。     

Purification and characterization of rat liver naloxone reductase that is identical to 3alpha-hydroxysteroid dehydrogenase.

1. Rat liver cytosol produced exclusively 6beta-naloxol from naloxone in the presence of either NADPH or NADH at pH 7.4. The amount of 6beta-naloxol formed with NADPH was about four times that with NADH. The enzyme responsible for this reaction, termed naloxone reductase, was purified to a homogeneous protein by various chromatographic techniques. 2. The purified enzyme is a monomeric protein with a molecular weight of 34000 and an isoelectric point of 5.9, and it has a dual co-factor specificity for NADPH and NADH. The enzyme catalysed the reduction of various carbonyl compounds as well as naloxone analogues, and the dehydrogenation of 3alpha-hydroxysteroids and alicyclic alcohols. Indomethacin, quercetin and sulphhydryl reagents potently inhibited the enzyme, but pyrazole and barbital had no effect on the enzyme activity. 3. Identity of naloxone reductase and 3alpha-hydroxysteroid dehydrogenase in rat liver was demonstrated by comparing the elution profiles of the two enzyme activities during purification, the ratios of the two enzyme activities at each purification steps, and thermal stability and susceptibility to inhibitors for the two enzyme activities. 4. Amino acid sequences of five peptides obtained by proteolytic digestion of the purified enzyme were completely identical to the corresponding regions of previously reported 3alpha-hydroxysteroid dehydrogenase.     

Reductases for carbonyl compounds in human liver

Two aldehyde reductases with mol. wt 78,000 and 32,000 and one carbonyl reductase with mol. wt 31,000 were purified to homogeneity from human liver cytosol. The high molecular weight aldehyde reductase exhibited properties similar to alcohol dehydrogenase; it had a single subunit of mol. wt 41,000 and a pI value of 10 to 10.5, and showed preference for NADH over NADPH as cofactor and sensitivity to SH-reagents, pyrazole, o-phenanthroline and isobutyramide. The enzyme reduced aliphatic and aromatic aldehydes, alicyclic ketones and alpha-diketones and an optimal pH of 6.0, and oxidized various alcohols with NAD as a cofactor at an optimal pH of 8.8. The identity of the enzyme with alcohol dehydrogenase was established by starch gel electrophoresis and co-purification of the two enzymes. The other enzymes were NADPH-dependent and monomeric reductases; the aldehyde reductase reduced aldehydes, hexonates and alpha-diketones and was sensitive to barbiturates, diphenylhydantoin and valproate, while the carbonyl reductase showed a broad substrate specificity for aldehydes, ketones and quinones and was inhibited by SH-reagent, quercitrin and benzoic acid. The latter enzyme appeared in three multiforms with different charges which occurred in differing ratios in liver specimens. Comparison of kinetic constants for aldehydes among the enzymes indicated that alcohol dehydrogenase is the best reductase with the highest affinity and Kcat values. The enzyme also catalyzed oxidation and reduction of aromatic aldehydes in the presence of NAD at physiological pH of 7.2. Tissue distribution of the three enzymes and variation of their specific activities in human livers were examined.     

A carbonyl reductase-catalyzing reduction of N3-phenacyluridine in rabbit liver

Carbonyl reductase activity for a novel hypnotic, N3-phenacyluridine, was mainly localized in the cytosol fraction of rabbit liver. The enzyme (N3-phenacyluridine reductase) which catalyzes the reduction of N3-phenacyluridine to N3-alpha-hydroxy-beta-phenethyluridine was purified from the cytosolic fraction of rabbit liver by various chromatographic techniques (DEAE-Sephacel, Red Sepharose CL-6B and hydroxylapatite). N3-Phenacyluridine reductase had a minimum molecular weight of 39kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. This enzyme required reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor and its optimal pH was 7.5. Flavonoids (quercetin and quercitrin) were potent inhibitors of the enzyme, but pyrazole or barbital had little effect. The apparent Km and Vmax values of the enzyme for the reduction of N3-phenacyluridine were 0.32 mM and 8.7 units/mg protein, respectively. A variety of carbonyl compounds, including N3-phenacyluridine, were effectively reduced by the enzyme. However, the enzyme purified from rabbit liver differs in several respects from known carbonyl reductases in rabbit liver.     

Purification and characterization of rat liver naloxone reductase that is identical to 3alpha-hydroxysteroid dehydrogenase

1. Rat liver cytosol produced exclusively 6β-naloxol from naloxone in the presence of either NADPH or NADH at pH 7.4. The amount of 6β-naloxol formed with NADPH was about four times that with NADH. The enzyme responsible for this reaction, termed naloxone reductase, was purified to a homogeneous protein by various chromatographic techniques. 2. The purified enzyme is a monomeric protein with a molecular weight of 34000 and an isoelectric point of 5.9, and it has a dual co-factor specificity for NADPH and NADH. The enzyme catalysed the reduction of various carbonyl compounds as well as naloxone analogues, and the dehydrogenation of 3α-hydroxysteroids and alicyclic alcohols. Indomethacin, quercetin and sulphhydryl reagents potently inhibited the enzyme, but pyrazole and barbital had no effect on the enzyme activity. 3. Identity of naloxone reductase and 3alpha-hydroxysteroid dehydrogenase in rat liver was demonstrated by comparing the elution profiles of the two enzyme activities during purification, the ratios of the two enzyme activities at each purification steps, and thermal stability and susceptibility to inhibitors for the two enzyme activities. 4. Amino acid sequences of five peptides obtained by proteolytic digestion of the purified enzyme were completely identical to the corresponding regions of previously reported 3alpha-hydroxysteroid dehydrogenase.     

骨碎补的化学成分

目的研究骨碎补(Drynaria fortunei)的化学成分。方法采用硅胶柱色谱、SephadexLH-20柱色谱、中低压ODS柱色谱及制备液相等方法进行分离纯化,并根据化合物的理化性质及波谱数据对结构进行鉴定。结果从骨碎补中分离并鉴定9个化合物:柚皮苷(1)、木犀草素-7-O-β-D-葡萄糖醛酸苷(2)、咖啡酸-4-O-β-D-吡喃葡萄糖苷(3)、4-O-β-D-吡喃葡萄糖基香豆酸(4)、对羟基反式肉桂酸(5)、反式桂皮酸(6)、3,4-二羟基苯甲酸(7)、5-羟甲基糠醛(8)、蔗糖(9)。结论化合物2、5、6、8和9为首次从槲蕨属植物中分离得到。     

Characterization of the fluoroacetate detoxication enzymes of rat liver cytosol

Two forms of fluoroacetate-specific defluorinase (FSD) were purified from rat hepatic cytosol. The first form, FSD1 (molecular weight 38 kDa), contained 81% of the total cytosolic fluoroacetate defluorination activity and did not bind to the glutathione-affinity, orange A or mono P columns used in the purification procedures. The second form, FSD2 (molecular weight 27 kDa), contained only 13% of the fluoroacetate defluorination activity, had a pI = 7.8, and exhibited a high glutathione S-transferase (GST)-like activity towards dichloroacetic acid. The FSD1 proteins were identified from peptide mass data and best matched with rat sorbitol dehydrogenase (SDH) (short form), although pure sheep liver SDH enzyme did not possess defluorination activity when subsequently investigated. The FSD2 protein was identified from peptide mass data and best matched with the amino acid sequence of mouse and human Zeta 1 of glutathione S-transferase (GSTZ1) and showed a high GSTZ1 specific activity. This study suggests that the major FSD component (FSD1) represents a new and unique dehalogenating or dehydrogenating enzyme present in rat liver cytosol. The minor FSD component (FSD2) is due to the GSTZ1 present in rat liver cytosol. However, it is not yet clear that FSD1 is indeed SDH and FSD2 is indeed GSTZ1, due to sequence homology being less than 60 and 45%, respectively.     

Methylarsenicals and arsinothiols are potent inhibitors of mouse liver thioredoxin reductase.

Thioredoxin reductase (TR, EC 1.6.4.5) was purified 5800-fold from the livers of adult male B6C3F1 mice. The estimated molecular mass of the purified protein was about 57 kDa. The activity of the purified enzyme was monitored by the NADPH-dependent reduction of 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB); this activity was fully inhibited by 1 microM aurothioglucose. Arsenicals and arsinothiols, complexes of As(III)-containing compounds with L-cysteine or glutathione, were tested as inhibitors of the DTNB reductase activity of the purified enzyme. Pentavalent arsenicals were much less potent inhibitors than trivalent arsenicals. Among all the arsenicals, CH(3)As(III) was the most potent inhibitor of TR. CH(3)As(III) was found to be a competitive inhibitor of the reduction of DTNB (K(i) approximately 100 nM) and a noncompetitive inhibitor of the oxidation of NADPH. The inhibition of TR by CH(3)As(III) was time-dependent and could not be reversed by the addition of a dithiol-containing molecule, 2,3-dimercaptosuccinic acid, to the reaction mixture. The inhibition of TR by CH(3)As(III) required the simultaneous presence of NADPH in the reaction mixture. However, unlike other pyridine nucleotide disulfide oxidoreductases, there was no evidence that mouse liver TR was inactivated by exposure to NADPH. Treatment with CH(3)As(III) did not increase the NADPH oxidase activity of the purified enzyme. Thus, CH(3)As(III), a putative intermediate in the pathway for the biomethylation of As, is a potent and irreversible inhibitor of an enzyme involved in the response of the cell to oxidative stress.     

Bromobenzene-mediated alteration in activity and electrophoretic pattern of biliverdin reductase variants in rat kidney

Here we report on the detection of multiple net-charge and molecular mass variants of biliverdin reductase in the rat kidney and describe selective changes in the tissue profile of the variants after bromobenzene treatment (2 mmol/kg, subcutaneously, 24 hr). Using two-dimensional electrophoresis and isoelectric focusing, two major molecular mass species, Mr 30,400 and 30,700, a minor form of Mr 31,400, and five net-charge groups of pI = 6.23, 5.91, 5.77, 5.61, and 5.48 were detected; the net-charge variants with pI = 5.61 and 5.77 were the most abundant forms. The Mr 30,400 form was the main component of two isoelectric focusing bands with pI = 6.23 and 5.91, and the relative amounts of these net-charge variants was severely decreased in the kidneys of bromobenzene-treated rats. The effect of bromobenzene in vivo could not be duplicated by in vitro experiments involving the direct treatment of purified enzyme with bromobenzene, or incubation of the purified preparation with bromobenzene in the presence of a NADPH-dependent microsomal drug-metabolizing system. Bromobenzene treatment did not alter the immunochemical properties of biliverdin reductase variants, as judged by the similarity of isoelectric focusing patterns of preparations on a Western blot using antibody raised against a rat liver total biliverdin reductase preparation. The treatment, however, caused an alteration in the kinetic properties of the enzyme, and the activity with NADH appeared to be selectively decreased. The possible mechanisms involved in the expression of multiple forms of the reductase and the biological significance of the multiplicity, as well as the change in composition caused by bromobenzene, are discussed.     

Purification of aldose reductase from human placenta and stabilization of the inhibitor binding site

Aldose reductase from human placenta was purified to homogeneity by a rapid (2 day) and efficient purification scheme involving Red Sepharose affinity chromatography, chromatofocusing and high performance liquid chromatography on a size-exclusion column. Addition of NADP+ at all steps in the purification of aldose reductase and during storage of the enzyme at -20 degrees stabilized both the enzyme active site and the major site for binding of aldose reductase inhibitors such as sorbinil and tolrestat. Aldose reductase is a monomer with a molecular mass of 38 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, apparent pI 5.9. Placenta aldose reductase exhibited no cross-reactivity with aldehyde reductase from human liver in an ELISA assay. Aldose reductase showed broad specificity for aldehydes, was specific for NADPH, and was activated by sulfate.     

Characterization of the fluoroacetate detoxication enzymes of rat liver cytosol

Two forms of fluoroacetate-specific defluorinase (FSD) were purified from rat hepatic cytosol. The first form, FSD1 (molecular weight 38?kDa), contained 81% of the total cytosolic fluoroacetate defluorination activity and did not bind to the glutathione-affinity, orange A or mono P columns used in the purification procedures. The second form, FSD2 (molecular weight 27?kDa), contained only 13% of the fluoroacetate defluorination activity, had a pI?=?7.8, and exhibited a high glutathione S-transferase (GST)-like activity towards dichloroacetic acid. The FSD1 proteins were identified from peptide mass data and best matched with rat sorbitol dehydrogenase (SDH) (short form), although pure sheep liver SDH enzyme did not possess defluorination activity when subsequently investigated. The FSD2 protein was identified from peptide mass data and best matched with the amino acid sequence of mouse and human Zeta 1 of glutathione S-transferase (GSTZ1) and showed a high GSTZ1 specific activity. This study suggests that the major FSD component (FSD1) represents a new and unique dehalogenating or dehydrogenating enzyme present in rat liver cytosol. The minor FSD component (FSD2) is due to the GSTZ1 present in rat liver cytosol. However, it is not yet clear that FSD1 is indeed SDH and FSD2 is indeed GSTZ1, due to sequence homology being less than 60 and 45%, respectively.     

A low-molecular-weight cadmium-binding substance in human and rat livers and human blood

A cadmium-binding substance with a molecular weight even lower than that of metallothionein was demonstrated on Sephadex G-75 gel filtration chromatography of the soluble fractions from newborn human and adult rat liver homogenates and adult human hemolysate which were mixed with CdCl2 in vitro. This substance was purified from rat liver extracts by gel filtration and ion-exchange column chromatography and characterized by 6 M guanidine X HCl thin-layer gel filtration chromatography and N-terminal and total amino acid analyses. The results showed that the isolated low-molecular-weight cadmium-binding substance was a cadmium-reduced glutathione complex, whose molecular weight was found to be approximately 1400 by Sephadex G-15 gel filtration.     

Multiple forms of biliverdin reductase: age-related change in pattern of expression in rat liver and brain

Biliverdin reductase is the dual nucleotide-dependent cytosolic enzyme that converts biliverdin to the bile pigment, bilirubin, and displays extensive microheterogeneity in rat organs. The enzyme is unique in having two pH optima. The present study reports on the tissue-dependent pattern of developmental expression of the reductase in rat liver and brain. When analyzed by Western immunoblotting, two closely migrating immunoreactive proteins were detected in the liver cytosol during the first 2-3 weeks after birth; the protein with greater mobility was not detected in the liver of adult aged animals (6 months old) and was present at low levels in rats during the first week of life. The faster migrating protein was not detected in the brain cytosol at any stage of development. Furthermore, in the brain the total amount of enzyme protein increased as the animal matured, whereas in the liver the enzyme protein level decreased with age. When the purified enzyme was analyzed, age-related changes in the variant composition of the enzyme in the liver were noted. Although in both adult and newborn animals (14 days old) the purified enzyme, when subjected to isoelectric focusing, separates into five net charge forms (pl 6.23, 5.91, 5.76, 5.61, and 5.48), the relative abundance of the variants notably differed in the two preparations. In addition, when the purified preparations were subjected to two-dimensional electrophoresis, although both purified preparations separate into three molecular weight forms (Mr 30,400, 30,700, and 31,400) one species (Mr 31,400, pl = 5.77), which was very prominently expressed in the newborn, was essentially absent in the adult. Biliverdin reductase activity of the liver cytosol with both NADPH (pH 8.7) and NADH (pH 6.7) exhibited developmental changes, with the activity increasing after birth, reaching a peak on day 14, and decreasing to low levels in the adult. The existence of a close correlation between development of biliverdin reductase activity in the brain and liver and that of heme oxygenase in these organs is noted. The suggestion is made that the reductase is not a passive component of the heme degradation pathway; rather, its activity could become limiting in the elimination of heme degradation products.     

Binding of reactive metabolites of CCl4 to specific microsomal proteins

The covalent binding of [14C]carbon tetrachloride to microsomal proteins in rat liver microsomes under anaerobic conditions was investigated by SDS-polyacrylamide slab gel electrophoresis and fluorography. Most of the labeled proteins were observed in the molecular weight range of 52-61 kDa, indicating that cytochrome P-450 forms (EC 1.14.14.1) were labeled. Protein bands at the position of the NADPH-cytochrome P-450 reductase (78 kDa) (EC 1.6.2.4) and NADH-cytochrome b5 reductase (33 kDa) (EC 1.6.2.2) also showed radioactivity. The fluorographic pattern of the protein labeling was cytochrome P-450-dependent, as was demonstrated by CO and metyrapone inhibition as well as by pretreatment of rats with inducing drugs such as 3-methylcholanthrene, benzo(a)pyrene, phenobarbitone and Aroclor 1254. Immuno-precipitation with a purified anti-P-450 immunoglobulin against cytochrome P-450 PB-B (52 kDa) of rat liver indicated that this protein contained about 10-20% of the total bound radioactivity in an average ratio of 0.8 mol [14C]CCl4-metabolite/mol cytochrome P-450 PB-B.     

Heterologous expression of CYP2K1 and identification of the expressed protein (BV-CYP2K1) as lauric acid (omega-1)-hydroxylase and aflatoxin B1 exo-epoxidase.

LMC2 is the most abundant constitutively expressed hepatic cytochrome P450 found in sexually immature rainbow trout (Onchorynchus mykiss) and is also the isozyme that activates the carcinogen aflatoxin B1 (AFB1). This P450 has been cloned, sequenced, and designated as CYP2K1. The present report describes the heterologous expression of enzymatically active CYP2K1 (BV-CYP2K1) in baculovirus Spodoptera frugiperda (Sf9) insect cells and its catalytic and immunoreactivity characterization in comparison with that of the previously purified LMC2 P450. Homogenates of Sf9 cells expressing the CYP2K1 enzyme and LMC2 both catalyzed the hydroxylation of lauric acid and the epoxidation of AFB1 in the presence of rat NADPH-cytochrome P450 reductase. Both LMC2 and BV-CYP2K1 catalyzed the oxidation of lauric acid primarily at the (omega-1) position plus small amounts at the (omega-2) position. Formation of AFB1 epoxide was shown indirectly by the appearance of an AFB1 epoxide-glutathione conjugate when P450 incubation mixtures contained AFB1, glutathione (GSH) together with mouse liver cytosol or purified rat GSH-transferase. When the AFB1 epoxide-GSH conjugate produced by BV-CYP2K1 and purified LMC2 was analyzed by HPLC using a chiral column, it had a retention time identical to that produced by CYP3A4, a human P450 known to form exclusively the AFB1 exo-epoxide. These results, therefore, confirm that the cDNA-expressed CYP2K1 protein is catalytically and immunologically identical to purified trout LMC2 and that these two enzymes produce primarily the highly carcinogenic stereoisomeric exo-epoxide form of AFB1.     

In vitro and in vivo studies of the effect of vitamin E on microsomal cytochrome P450 in rat liver

Administration of a diet supplemented with 0.06% vitamin E acetate to male rats over a 6-week period doubled hepatic microsomal stores of alpha-tocopherol over those in control (vitamin E adequate) rat liver. Total cytochrome P450 content and NADPH-cytochrome P450 reductase activity were significantly elevated in hepatic microsomes from vitamin E-supplemented rats to 111% and 123% of respective control values. Androstenedione 16 alpha-hydroxylase activity was increased in these fractions (2.57 +/- 0.31 nmol product/min/mg protein vs 1.81 +/- 0.38 in controls) whereas activities of the 6 beta-, 7 alpha- and 16 beta-hydroxylase pathways were unchanged. Immunoquantitation of the microsomal 16 alpha-hydroxylase, P450 IIC11, indicated a corresponding increase in the hepatic content of the enzyme. In view of the established antioxidant role of tocopherols, the effects of dietary vitamin E manipulation on the concentration of protein sulphydryl groups and the susceptibility of microsomes to ferric sulphate-ADP-NADPH-mediated lipid peroxidation were also assessed. Dietary supplementation did not influence microsomal protein sulphydryl content (68 +/- 10 nmol glutathione equivalents/mg protein) but decreased the extent of lipid peroxidation produced by the ferric sulphate-ADP-NADPH system in vitro. Further in vitro experiments demonstrated that vitamin E acetate (2 microM) protected protein sulphydryl groups and lipids against peroxidation in control microsomes and partially reduced the associated losses of P450-mediated steroid hydroxylase activities. Western immunoquantitation of P450 IIC11 revealed that exogenous vitamin E acetate protected completely against peroxidation-induced apoprotein loss. These studies establish that the in vitro protective effects of vitamin E acetate against sulphydryl and lipid peroxidation extend to protection of the P450 apoprotein but that enzyme activity is only partially protected. This finding suggests that peroxidation-dependent loss of P450 in vitro is mediated by haem degradation from the P450 holoenzyme and is not directly related to lipid/sulphydryl oxidation. In contrast, the in vivo effects of dietary vitamin E on drug metabolizing enzymes are regulatory in nature and are unrelated to effects on lipid peroxidation.     

The enzymology of doxorubicin quinone reduction in tumour tissue.

We have reported previously that enzymes present in the Sp 107 rat mammary carcinoma catalyse doxorubicin quinone reduction (QR) to 7-deoxyaglycone metabolites in vivo [Willmott and Cummings, Biochem Pharmacol 36: 521-526, 1987]. In order to provide insights into the role of QR in the antitumour mechanism of action of doxorubicin, we have attempted in this work to identify the enzyme(s) responsible. NAD(P)H: (quinone acceptor) oxidoreductase (DT-diaphorase) was the major quinone reductase in the tumour accounting for approximately 70% of all the activity measured in microsomes and cytosols (microsomal activity, 28.4 +/- 4.6 nmol/min/mg; cytosolic activity, 94.3 +/- 11.9 nmol/min/mg). Its presence was confirmed by western blot analysis. Low levels of NADH cytochrome b5 reductase (15.6 +/- 6.3 nmol/min/mg) and NADPH cytochrome P450 reductase (14.5 +/- 4.0 nmol/min/mg) were detectable in microsomes. The presence of the latter was confirmed by western blot analysis. Pretreatment of tumours with doxorubicin (48 hr) at a therapeutic dose decreased the level of activity of all the reductases studied by at least 2-fold (P < 0.01, Student's t-test). Doxorubicin was shown not to be a substrate for purified rat Walker 256 tumour DT-diaphorase with either NADH or NADPH as co-factor and utilizing up to 20,000 units of enzyme/incubation but was confirmed to be a substrate for purified rat liver cytochrome P450 reductase. 7-Deoxyaglycone metabolite formation by purified cytochrome P450 reductase had an absolute requirement for NADPH as co-factor, was inhibited by molecular oxygen and dicoumarol (IC50 approx. 50 microM), and modulated by specific reductase antiserum. Reductive deglycoslation of doxorubicin to 7-deoxyaglycones was localized to the microsomal fraction of the Sp 107 tumour, with negligible activity being found in cytosols (NADH, NADPH and hypoxanthine as co-factors) and mitochondria (NADH and NADPH). The tumour microsomal enzyme had an absolute co-factor requirement for NADPH, was inhibited by oxygen and dicoumarol, and modulated by cytochrome P450 reductase antiserum. These data indicate strongly that NADPH cytochrome P450 reductase is the principal enzyme responsible for catalysing doxorubicin QR in the Sp 107 tumour.