7-ethoxycoumarin Deethylation Research Articles

Directed evolution of cytochrome P450 for sterol epoxidation

16,17-Epoxysterol plays an important role in pharmaceutical steroid synthesis. To investigate the potential application of cytochrome P450 for epoxysterol synthesis, an approach to the epoxidation of 16,17-epoxysterol, based on directed evolution of cytochrome P450 BM-3, was developed. This comprised random gene mutagenesis for optimizing the activity of P450 BM-3 for epoxidation of hydrophobic sterol, followed by the 7-ethoxycoumarin de-ethylation assay for general enzyme activity detection and the modified picric acid assay for epoxidation activity screening. By the two-step screening, one mutant from 792 clones showed specific substrate activity of converting progesterone to 16,17-epoxysterol, which validated the possibility to evolve the cytochrome P450 for the synthesis of steroidal epoxides.

Expression and Characterization of Dog Cytochrome P450 2A13 and 2A25 in Baculovirus-Infected Insect Cells

Dog CYP2A13 and CYP2A25 were coexpressed with dog NADPH-cytochrome P450 reductase (OR) in baculovirus-infected Sf9 insect cells. CYP2A13 effectively catalyzed 7-ethoxycoumarin (7EC) deethylation and coumarin hydroxylation with apparent K(m) values of 4.8 and 2.1 microM, respectively, similar to those observed using dog liver microsomes (7.5 and 0.75 microM, respectively). CYP2A25 exhibited much lower affinity toward 7EC, with an apparent K(m) value of 150 microM, which indicates that CYP2A13 plays a more significant role in the metabolism of these CYP2A substrates. Similar to the dog CYP1A2 enzyme, CYP2A13 efficiently catalyzed phenacetin deethylation with a K(m) value of 3.9 microM, which suggests that phenacetin is not a selective probe for dog CYP1A2 activity. Both dog CYP2A13 and CYP2A25 exhibited little or no catalytic activity toward other common cytochrome P450 probe substrates, including bupropion, amodiaquine, diclofenac, S-mephenytoin, bufuralol, dextromethorphan, midazolam, and testosterone. These results provided additional information about the selectivity of these commonly used probe substrates.

Systematic screening of Escherichia coli single-gene knockout mutants for improving recombinant whole-cell biocatalysts

Systematic screening of single-gene knockout collection of Escherichia coli BW25113 (the Keio collection) was performed to select mutants that could enhance the deethylation of 7-ethoxycoumarin catalyzed by CYP154A1. After 96-well plate high-throughput screening followed by test tube assays, four mutants (Delta cpxA, Delta gcvR, Delta glnL, and an unknown-gene-deleted one (Delta uk)) were able to increase the CYP154A1 activity by approximately 1.4-1.7 times compared with that of the control strain. When new mutants were constructed by disrupting individually the cpxA, gcvR, glnL, and uk genes in E. coli BW25113, three of them (Delta cpxA, Delta gcvR, and Delta glnL) showed high levels of CYP154A1 activity. However, the uk-disruptant failed to enhance the CYP154A1 activity, suggesting that the high CYP154A1 activity of the Delta uk mutant in the Keio collection was due to a spontaneous mutation in the chromosome. In-frame deletion mutants of Delta cpxA, Delta gcvR, and Delta glnL also exhibited high enzyme activity, and complementation of these mutations could decrease CYP154A1 activity. These results indicated that the enhancement of the enzyme activity was not caused by polar effects on their neighbor genes. To our knowledge, this is the first report on a genome-wide screening of the genes for deletion to improve the activity of a recombinant whole-cell biocatalyst.

Antigenotoxic effect of Xanthohumol in rat liver slices

Xanthohumol (XN), the principal prenylated flavonoid in the hop plant, Humulus lupulus L., is suggested to have cancer chemo-preventive activities. Its mechanisms of protection have been proposed to be inhibition of metabolic activation, induction of detoxifying enzymes and antioxidant activity. Our previous study showed that XN efficiently protected human hepatoma HepG2 cells against the genotoxic effects of two pro-carcinogens (2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and benzo(a)pyrene (BaP)) that are dependent on cytochrome P450 (CYP) mediated metabolic activation, and against genotoxic effects of the oxidative damage inducing tert-butyl hydroperoxide (tBOOH). In the present study, we investigated the antigenotoxic effects of XN in precision-cut rat liver slices. Using the comet assay, we detected that at non-cytotoxic concentrations (0.01-10 microM) XN completely prevented IQ and BaP-induced DNA damage. The protective effects of XN against tBOOH-induced DNA damage was less efficient; the maximal 50% reduction of DNA damage was observed at 0.1 microM XN. In rat microsomes, XN (0.001-10 microM) inhibited CYP1A activity (7-ethoxycoumarin (7EC) de-ethylation) in a concentration-dependent manner. Surprisingly, no inhibition of 7EC metabolism by XN was observed in rat liver slices. XN also did not have any influence on mRNA expression of the enzymes CYP1A2 and quinone reductase (QR). These results indicate that inhibition of metabolic activation of pro-carcinogens by CYP1A is not likely to be the mechanism of its antigenotoxic action. In conclusion, XN efficiently protects DNA against genotoxicity of IQ and BaP and against oxidative DNA damage. Although the mechanism of the protective effect of XN is unclear, our results indicate that XN exhibits antigenotoxic effects in fresh liver tissue and provide additional evidence for the cancer preventive potential of XN.

Role of LYS271 and LYS279 Residues in the Interaction of Cytochrome P4501A1 with NADPH–Cytochrome P450 Reductase

It has been proposed that negatively charged amino acids on the surface of reductase and positively charged amino acids on the surface of P450 mediate the binding of both proteins through electrostatic interactions. In this study, we used a site-directed mutagenesis approach to determine a role for two lysine residues (Lys271 and Lys279) of cytochrome P4501A1 in the interaction of P4501A1 with reductase. We prepared two mutants P4501A1Ile271 and P4501A1Ile279 with a mutation of the lysine at positions 271 and 279, respectively. We observed a strong inhibition (>80%) of the 7-ethoxycoumarin and ethoxyresorufin deethylation activity in the reductase-supported system for both mutants. In the cumene hydroperoxide-supported system, P4501A1Ile279 exhibited wild-type activity, but the P4501A1Ile271 mutant activity remained low. The CD spectrum and substrate-binding assay indicated that the secondary structure of P4501A1Ile271 is perturbed. To evaluate further the involvement of these P4501A1 lysine residues in reductase binding, we measured the KM of reductase for wild type and mutants. Both wild type and P4501A1Ile271 reached saturation in the range of reductase concentrations tested with KM values 5.1 and 11.2 pM, respectively. The calculated KM value for P4501A1Ile279 increased 9-fold, 44.4 pM, suggesting that the mutation affected binding of reductase to P4501A1. Stopped-flow spectroscopy was employed to evaluate the effect of mutations on electron transfer from reductase to heme iron. Both wild type and P450Ile279 showed biphasic kinetics with a ∼40% participation of the fast step in the total activity. On the other hand, only single-phase kinetics for iron reduction was observed for P450Ile271, suggesting that the low activity of this mutant can be attributed not only to major structural changes but also to a disturbance in the electron transport.

Assessment of specificity of eight chemical inhibitors using cDNA-expressed cytochromes P450

1. The selectivity of eight chemical inhibitors has been extensively evaluated with 10 cDNA-expressed human cytochrome P450 isoforms (CYP). The results indicate that sulphaphenazole, quinidine and α-naphthoflavone are selective inhibitors of CYP2C9 (IC50 = 5 0.5-0.7 μM), CYP2D6 (0.3-0.4 μM) and CYP1A (0.05-5 μM) respectively on the basis of the IC50, which are much lower than those of other P450 isoforms (> 10-fold). 2. Ketoconazole exhibited potent inhibition of both CYP3A4-catalysed metabolism of phenanthrene, testosterone, diazepam (IC50 = 0.03-0.5 μM) and CYP1A1-catalysed deethylation of 7-ethoxycoumarin (0.33 μM). The selectivity of ketoconazole for other P450s was highly related to the concentration used. 3. Diethyldithiocarbamate, orphenadrine and furafylline were shown separately to be less selective inhibitors of CYP2E1, CYP2B6 and CYP1A isoforms by a broad range of IC50 that overlap those observed with other P450 isoforms. 4. Furafylline, quinidine and α-naphthoflavone activated CYP3A4-catalysed phenanthrene metabolism by 1.7-, 2- and 15-fold respectively. 5. The selectivity of orphenadrine and ketoconazole was further examined by using inhibitory monoclonal antibodies (MAb). Inhibitory MAb specific for the individual P450 isoforms may be of greater value than chemical inhibitors.

7-ethoxycoumarin deethylation activity in perfused isolated rat brain.

7-ethoxycoumarin (7-EC) deethylation activity was measured in the perfused rat brain in situ. Infusion of 7-EC into a brain through an internal carotid artery resulted in the formation of 7-hydroxycoumarin (7-HC) and its conjugates in the effluent perfusate collected from the superior vena cava. The rate of formation of products was 200 nmol/h/g when 130 microM 7-EC was infused. This value was much higher (more than 100 times) than that determined from the brain microsomal activity ( approximately 1 nmol/h/g), indicating that the activity determined with microsomes was an underestimate. This value was comparable to the activity in the perfused liver (30-50%), suggesting that drug metabolizing enzymes can play important roles within the brain. Pretreatment of rats with P-450 inducers such as phenobarbital and beta-naphthoflavone increased the deethylation activity in the perfused brain, as in the perfused liver. We conclude that the perfused brain is suitable for evaluating drug metabolizing activities under physiological conditions.

Diverse Function of Aromatase and the N-terminal Sequence Deleted Form

The diverse function of human placental aromatase including estradiol 6α-hydroxylase and cocaine N-demethylase activity are described, and the mechanism for the simultaneous metabolism of estradiol to 2-hydroxy- and 6α-hydroxyestradiol at the same active site of aromatase is postulated. Comparison of aromatase activity is also made among the wild type and N-terminal sequence deleted forms of human aromatase which are recombinantly expressed in Escherichia coli . Aromatase cytochrome P450 was reconstituted and incubated with [6α,7α- 3 H 2 ,4- 14 C]estradiol, 7-ethoxycoumarin, and [N-methyl- 3 H 3 ]cocaine. 6α-Hydroxy[7α- 3 H,4- 14 C]estradiol was isolated as the metabolite of estradiol and the 3 H-water release method based on the 6α- 3 H label was established. The initial rate kinetics of the 6α-hydroxylation gave K m of 4.3 μM, V max of 4.02 nmol min −1 mg −1 , and turnover rate of 0.27 min −1 . Testosterone competed dose-dependently with the 6α-hydroxylation and showed the K i of 0.15 μM, suggesting that they occupy the same binding site of aromatase. The deethylation of 7-ethoxycoumarin showed K m of 200 μM, V max of 12.5 nmol min −1 mg −1 and turnover rate of 1.06 min −1 . The N-demethylation of cocaine was analysed by the 3 H-release method, giving K m of 670 μM, V max of 4.76 nmol min −1 mg −1 , and turnover rate of 0.49 min −1 . All activity was dose-responsively suppressed by anti-aromatase P450 monoclonal antibody MAb3-2C2. The N-terminal 38 amino acid residue deleted form of aromatase P450 was expressed in particularly high yield giving a specific activity of 397 ± 83 pmol min −1 mg −1 ( n = 12) of crude membrane-bound particulates with a turnover rate of 2.6 min −1 .

Diverse function of aromatase and the N-terminal sequence deleted form

The diverse function of human placental aromatase including estradiol 6α-hydroxylase and cocaine N-demethylase activity are described, and the mechanism for the simultaneous metabolism of estradiol to 2-hydroxy- and 6α-hydroxyestradiol at the same active site of aromatase is postulated. Comparison of aromatase activity is also made among the wild type and N-terminal sequence deleted forms of human aromatase which are recombinantly expressed in Escherichia coli. Aromatase cytochrome P450 was reconstituted and incubated with [6α,7α-3H2,4-14C]estradiol, 7-ethoxycoumarin, and [N-methyl-3H3]cocaine. 6α-Hydroxy[7α-3H,4-14C]estradiol was isolated as the metabolite of estradiol and the 3H-water release method based on the 6α-3H label was established. The initial rate kinetics of the 6α-hydroxylation gave Km of 4.3 μM, Vmax of 4.02 nmol min−1mg−1, and turnover rate of 0.27 min−1. Testosterone competed dose-dependently with the 6α-hydroxylation and showed the Ki of 0.15 μM, suggesting that they occupy the same binding site of aromatase. The deethylation of 7-ethoxycoumarin showed Km of 200 μM, Vmax of 12.5 nmol min−1mg−1 and turnover rate of 1.06 min−1. The N-demethylation of cocaine was analysed by the 3H-release method, giving Km of 670 μM, Vmax of 4.76 nmol min−1mg−1, and turnover rate of 0.49 min−1. All activity was dose-responsively suppressed by anti-aromatase P450 monoclonal antibody MAb3-2C2. The N-terminal 38 amino acid residue deleted form of aromatase P450 was expressed in particularly high yield giving a specific activity of 397 ± 83 pmol min−1mg−1 (n = 12) of crude membrane-bound particulates with a turnover rate of 2.6 min−1.

Examination of 7-ethoxycoumarin deethylation and ammonia removal activities in 31 hepatocyte cell lines

Aiming the selection of possible candidates for the reactor cells in a hybrid artificial liver, we investigated the activities of 7-ethoxycoumarin deethylation and ammonia removal in 31 hepatocytes cell lines established from the human, rat, mouse, and chicken, compared with freshly isolated rat hepatocytes. Rat liver homogenate and hepatocytes showed 7-ethoxycoumarin deethylation activity of 0.925 and 1.840 nmol/mg protein/min, respectively. Two human hepatoblastoma cell lines, Hep G2 and HuH 6, showed the activity of 0.190 and 0.027 nmol/mg protein/ min, respectively. While the rat hepatocytes efficiently removed added ammonia form the culture medium, no ammonia removal activity was detectable in any cell line with the assay conditions in this study. Ammonia removal activity, possibly catalyzed by the urea cycle, is likely to be harder to restore than the drug metabolizing system in the course of cell immortalization.

Porcine Hepatocytes From Slaughterhouse Organs

Most recent strategies for the development of hybrid artificial liver devices focus on the use of parenchymal liver cells (hepatocytes). For clinical application of these devices, a sufficient cell supply is mandatory. Because human liver tissue is rarely available, isolated porcine hepatocytes from laboratory animals have been suggested for use in bioartificial livers. The authors introduce a modified isolation protocol to yield large scale numbers of viable porcine hepatocytes from slaughterhouse organs. Perfusion and enzymatic digestion of the left medial liver lobe (n = 74) resulted in 1.0 +/- 0.3 x 10(7) viable hepatocytes per gram of tissue, and an overall yield of 1.92 +/- 0.5 x 10(9) viable cells per isolation (viability: 93 +/- 2%). Collagen gel immobilization maintained morphologic integrity and functional activity of hepatocyte cultures over long-term periods. Cell morphology, as assessed by light microscopic evaluation, was maintained for 2 weeks. Stable DNA content (51 +/- 5 micrograms) and low values of alanine aminotransferase release (8 microU/hr/micrograms DNA) indicated structural stability of cultures after a short period of post isolational adaptation. Albumin secretion (4.5 micrograms/hr/micrograms DNA) and persistent Cytochrome P450 IA1 dependent deethylation of 7-ethoxycoumarin (4.5 nmol/hr/micrograms DNA) indicated long-term metabolic activity of cultured hepatocytes. Hepatocytes from livers of slaughtered pigs represent an unlimited resource of viable material for cell culture, and their usefulness as functional units of bioartificial liver support devices should be tested.

Polybrominated biphenyl induction of cytochrome P450 mixed function oxidase activity in primary rat and human hepatocytes

Polybrominated biphenyl (PBB) is an industrial chemical and environmental contaminant with known incidence of significant human exposure. PBB has been studied in laboratory animals and found to have significant toxicological effects as well as being a potent inducer of hepatic cytochrome P450 mixed function oxidase (MFO) activity. As part of our program to compare the response of laboratory animals and humans to industrial and environmental toxicants, we studied the effect of a major component of commercial PBB mixtures, 2,2′,4,4′,5,5′-hexabromobiphenyl (HBB), on MFO induction in primary cultures of human and rat hepatocytes. MFO induction was evaluated by measuring the deethylation of 7-ethoxycoumarin by intact hepatocytes. Rat hepatocytes were found to be highly susceptible to HBB induction of 7-ethoxycoumarin O-deethylase (ECOD) activity, with significant induction observed at the lowest concentration tested of 10 −8 M. Human hepatocytes were found to have a higher threshold for HBB induction of ECOD activity than rat hepatocytes. The lowest concentration of HBB required for ECOD induction observed for human hepatocytes was 10- to 1000-fold higher (10 −7, 10 −6, 10 −5 and 10 −5 M for the four human samples) than that found in rat hepatocytes. Future mechanistic investigation of this observed difference in sensitivity towards PBB between rat and human hepatocytes may aid the extrapolation of human health risk from toxicological data obtained from laboratory animals.

Chloroethylene mixtures: pharmacokinetic modeling and in vitro metabolism of vinyl chloride, trichloroethylene, and trans-1,2-dichloroethylene in rat.

Environmental and occupational exposures are typically to mixtures of chemicals, although most toxicity information is for individual compounds. Interactions between chemicals may involve pharmacokinetic and/or pharmacodynamic effects resulting in modulation of toxicity. Therefore, physiologically based pharmacokinetic modeling has been used to analyze data describing the metabolism of vinyl chloride (VC) and trichloroethylene (TCE) mixtures in rats. A single saturable pathway was modeled, representing cytochrome P450 2E1. This was partially validated using preexposure to trans-1,2-dichloroethylene (tDCE) which virtually eliminated in vivo metabolism of both VC and TCE at low concentrations. Microsomes from tDCE-exposed animals showed inhibition of metabolism of P450 2E1 substrates (chlorzoxazone, p-nitrophenol, and TCE) and no effect on 7-ethoxycoumarin deethylation. Studies with liver microsomes from VC-exposed animals found that neither suicide inhibition nor induction occurred during 6-hr exposures to high concentrations. Therefore, these effects were not modeled. Modeling of mixtures of VC and TCE was successful only using competitive inhibition, as might be predicted for cytochrome P450 2E1 substrates, and not uncompetitive or noncompetitive inhibition. These results were further confirmed by determining the depletion of glutathione due to VC metabolism. The validation of a detailed model for the inhibition kinetics of metabolism of these two compounds permits better understanding of the implications of coexposures for toxicity. It is notable that competitive inhibition only becomes significant at relatively high concentrations (tens of ppm), while at typical low environmental concentrations (ppb), absorption is perfusion limited and enzyme is in excess so that the chemicals will be metabolized independently.

Induction of rat liver microsomal epoxide hydrolase by its endogenous substrate 16 alpha, 17 alpha-epoxyestra-1,3,5-trien-3-ol.

1. The influence of the endogenous steroid epoxides 16 alpha, 17 alpha-epoxyestra-1,3,5(10)-trien-3-ol (estroxide) and 16 alpha, 17 alpha-expoxiandrost-4-en-3-one (androstene oxide) and their metabolic precursors estra-1,3,5(10), 16-tetraen-3-ol (estratetraenol) and androsta-4, 16-dien-3-one (androstadienone) on the specific activities of hepatic microsomal and soluble epoxide hydrolase, glutathione S-transferase, dihydrodiol dehydrogenase, and 7-ethoxycoumarin deethylase was investigated in the male Sprague-Dawley rat. 2. Both estroxide and estratetraenol induced microsomal epoxide hydrolase activity towards styrene oxide and estroxide itself 2-2.5-fold and glutathione conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) 1.6-fold after intraperitoneal administration of a high dose of compound (300 mg per kg of body weight). 3. In addition, estroxide decreased 7-ethoxycoumarin deethylation down to 20% of the activity observed in the untreated rat, whereas estratetraenol enhanced the activity of soluble epoxide hydrolase towards trans-stilbene oxide by a factor of 1.7. 4. In contrast, neither androstene oxide nor androstadienone showed a significant influence on any of the parameters under investigation. Dihydrodiol dehydrogenase was not significantly changed by any of the treatments.

Rotation and membrane topology of genetically expressed methylcholanthrene-inducible cytochrome P-450IA1 lacking the N-terminal hydrophobic segment in yeast microsomes.

A modified rat liver cytochrome P-450IA1, lacking amino acids 2-30, a proposed membrane anchor for cytochrome P-450, was expressed genetically in yeast microsomal membranes. This truncated cytochrome is practically active in the deethylation of 7-ethoxycoumarin. A full-length cytochrome P-450IA1 was also expressed in yeast microsomes. Rotational diffusion of P-450IA1 was examined by observing the flash-induced absorption anisotropy r(t) of the P-450.CO complex. The anisotropy decayed to a time-independent value within a 2-ms time range. Since the decay curve has the characteristics of a slow rotation of membrane-embedded cytochrome, the theoretical analysis of r(t) was performed based on a "rotation-about-membrane normal" model. 41% of the shortened P-450IA1 was rotating with the rotational relaxation time phi of 1020 microseconds, whereas 27% of the full-length P-450IA1 was mobile with phi = 1101 microseconds. The high salt treatment did not remove the shortened cytochrome from the membrane and also did not drastically weaken the interactions of the cytochrome with the membrane, as judged from the slow rotation characteristics (phi = 830 microseconds). These results demonstrate that the N-terminal shortened P-450IA1 is incorporated properly into the yeast microsomal membrane and that the N-terminal hydrophobic segment is not solely responsible for attachment to the membrane, providing evidence that additional segments of P-450IA1 are involved in the membrane binding.

Fasciola-Hepatica: Liver Microsomal Membrane Functions in Host Rat

Changes in different microsomal membrane functions were measured in the liver of rats 3, 6, or 9 weeks following an oral infection with 20 metacercariae of Fasciola hepatica. The parasitic pathology noted at autopsy was accompanied by increased levels in both plasma aspartate aminotransferase (EC 2.6.1.1) and microsomal γ-glutamyltransferase (EC 2.3.2.2). Heme oxygenase activity of microsomes was significantly decreased by Weeks 3 and 6 postinfection and this decrease correlates with those of total microsomal cytochrome P450 and certain P450-dependent monooxygenase activities, namely, benzphetamine demethylation, ethoxycoumarin deethylation, and benzopyrene hydroxylation. Microsomal epoxide hydrolase (EC 3.3.2.3) was only altered 6 weeks after the infection. During the early stages of the parasitism, there were decreases in both microsomal calcium uptake and calcium ATPphosphohydrolase activity (EC 2.6.1.1), whereas membrane fluidity, estimated by the order parameter S, was lower in the infected rats than that in the controls. These alterations could be related to the already described increase in liver cytosolic calcium or lipid peroxidation which occurs in experimental fascioliasis.

Beta-naphthoflavone induction of a cytochrome P-450 arachidonic acid epoxygenase in chick embryo liver distinct from the aryl hydrocarbon hydroxylase and from phenobarbital-induced arachidonate epoxygenase.

Cytochrome P-450-mediated arachidonic acid metabolism in chick embryo liver microsomes was increased by both Ah receptor-dependent (2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and beta-naphthoflavone) and independent (phenobarbital) P-450 inducers. Arachidonic acid epoxides and monohydroxyeicosatetraenoic acids were increased 9-12-fold. omega-1-OH arachidonic acid was also significantly increased by TCDD and beta-naphthoflavone while omega-OH arachidonic acid, the main metabolite in uninduced livers, was decreased by all three agents. The P-450s catalyzing the enhanced arachidonate metabolism in beta-naphthoflavone- and phenobarbital-treated liver were investigated in reconstituted systems containing wholly or partially purified P-450s. beta-Naphthoflavone induced formation of a 55-kDa P-450 selective for arachidonate metabolism and for epoxygenation in particular. This P-450 was purified (beta NFAA). It was found to be distinct from a 54.5-kDa beta-naphthoflavone-induced P-450 catalyzing aryl hydrocarbon hydroxylase and 7-ethoxyresorufin deethylase (designated NF1). Mean turnover numbers for arachidonate epoxygenase, aryl hydrocarbon hydroxylase, and 7-ethoxyresorufin deethylase were 11.2, 0.56, and 0.04, respectively, for reconstituted beta NFAA and 0.33, 11.8, and 2.4 for NF1. beta NFAA and NF1 also differed in chromatography elution characteristics and N-terminal amino acid sequences. Both were low spin, with carbon monoxide binding peaks at 448 nm. The phenobarbital-induced arachidonate epoxygenation was catalyzed by P-450 fractions containing the main 48- and 49-kDa phenobarbital-induced P-450s; fractions in which the 49-kDa P-450 predominated were the most active. Turnover numbers for arachidonic acid epoxygenation were not correlated with those for aminopyrine demethylation or 7-ethoxycoumarin deethylation for P-450s from phenobarbital-treated livers or with aryl hydrocarbon hydroxylase, 7-ethoxyresorufin deethylase, or 7-ethoxycoumarin deethylase for P-450s from beta-naphthoflavone-treated livers. Also, different P-450s catalyzed the epoxygenation and the omega-hydroxylation of arachidonic acid in both beta-naphthoflavone- and phenobarbital-treated livers. The findings support a physiologic role for P-450-induced arachidonate metabolism and provide a basis for a possible link between TCDD's induction of P-450 and alterations of cellular homeostasis.

The Aqueous Two-Phase System Polyethylene Glycol/Dextran as a Reaction Medium for the Microsomal Monooxygenase System

An aqueous two-phase system of dextran and polyethylene glycol was investigated as a reaction medium for pig liver microsomes in order to find out if the partition of the microsomes, of the substrate 7-ethoxycoumarin and of the product 7-hydroxycoumarin favoured any biotechnological perspectives. Cytochrome-P-450 and NADPH-cytochrome P-450 reductase concentrations and the monooxygenase 7-ethoxycoumarin deethylation activity were measured under a variety of the system parameters. Microsomes totally partition into the bottom phase whereas the concentration ratio of substrate to product is higher in the microsome free top phase. An unfavourable effect is the specific partial deactivation of the cytochrome P-450 by polyethylene glycol.

Chemical modification of rat liver microsomal cytochrome P-450: Study of enzymic properties and membrane topology

Isolated rat liver cytochrome P-450IIB1 was alkylated and acetylated at primary amino groups, and the position of the modified amino acids in the protein was identified. Alkylation of up to nine amino groups did not disturb the interaction of reconstituted P-450 and NADPH-cytochrome- P-450 reductase in a way that hydroxylation of benzphetamine was altered, whereas deethylation of 7-ethoxycoumarin was gradually reduced in parallel with impaired 7-ethoxycoumarin binding. Acetylation of four lysine residues completely inhibited binding and metabolism of 7-ethoxycoumarin but not of benzphetamine. These results suggest the presence of different substrate binding sites on P-450. Exhaustive proteolysis of modified P-450 in proteoliposomes liberated all but the N-terminal modified peptide and 85 to 90% of the cytochrome's mass from intact proteoliposomes. These findings further support our previously proposed model of P-450 topology (Vergères, G., Winterhalter, K.H. and Richter, C. (1989) Biochemistry 28, 3650–3655), in which P-450 is anchored to the membrane with the N-terminal peptide only, the N-terminal methionine facing the lumenal interior.

A Sensitive and Accurate Assay for 7-Ethoxycoumarin Deethylase Activity Determination Using Column-Switching High-Performance Liquid Chromatography

To meet the need for the determination of the low activities of cytochrome P450-catalyzed 7-ethoxycoumarin deethylation (ECDE) in most extra-hepatic tissues and cultured cells, a reliable, sensitive, and accurate assay, which uses a modified column-switching high-performance liquid chromatographic system and is essentially free of sample pretreatment, has been developed. With this system, the recovery of the 7-hydroxycoumarin product added to the biological tissue-containing incubation medium was 87%. With ECDE activity as low as 1 pmol/mg of protein/min, replicate measurements with variation coefficients lower than 5% were easily obtainable. ECDE activities in several extrahepatic tissues and cultured cells were determined with the technique to illustrate its usefulness.