Cytochrome P-450d Research Articles

Role of Glu318 at the putative distal site in the catalytic function of cytochrome P450d.

Most microsomal P450s have a conserved "threonine cluster" composed of three Thrs (Thr319, Thr321, Thr322 for P450d) at a putative distal site. An ionic amino acid at 318 is also well conserved as Glu or Asp for most P450s. To understand the role of these conserved polar amino acids at the putative distal site in the catalytic function of microsomal P450, we studied how mutations at this site of P450d influence the activation of molecular oxygen in the reconstituted system. Catalytic activity (0.02 min-1) toward 7-ethoxycoumarin of the Glu318Ala mutant of P450d was just 6% of that (0.33 min-1) of the wild type, while those of Glu318Asp, Thr319Ala, and Thr322Ala were comparable to or even higher than that of the wild type. Consumption rates of O2 and formation rates of H2O2 of those mutants varied in accord with the catalytic activities. Especially, the efficiency (0.5%) of incorporated oxygen atom to the substrate versus produced H2O2 for the Glu318Ala mutant was much lower than that (3.7%) of the wild type, while that (58.8%) for the mutant Glu318Asp was 16-fold higher than that of the wild type. In addition, the autoxidation [Fe(II)---- Fe(III)] rate (0.074 s-1) of the Glu318Ala mutant was much lower than those (0.374-0.803 s-1) of the wild type and other mutants. Thus, we strongly suggest that Glu318 plays an important role in the catalytic function toward 7-ethoxycoumarin of microsomal P450d.

Absorption spectral study of cytochrome P450d-phenyl isocyanide complexes: effects of mutations at the putative distal site on the conformational stability

Interactions of phenyl isocyanide (PheNC) with purified engineered cytochrome P450d wild type and putative distal mutants, Glu318Asp and Glu318Ala, were studied with optical absorption spectra. The wild type and the mutant Glu318Asp were purified as the high-spin state, while the mutant Glu318Ala was purified as the oxygen-bound low-spin form. Thus, it is suggested that Glu318 is important to make the appropriate heme environment of P450d. Spectral dissociation constants (0.19-0.39 mM) of the ligand for the ferric mutants were lower than that (0.74 mM) of the wild type. These dissociation constants were changed by adding a substrate, 7-ethoxycoumarin. The reduced wild type-PheNC complex showed a Soret peak at 451 nm, while the reduced mutant-PheNC complexes showed two peaks at 451 and 423 nm. The 451-nm peak of the complexes decreased with the concomitant increase of a new peak at 433 nm at room temperature. Thus, it was suggested that P450d can take two conformationally different forms from the characteristic spectral features. The Soret spectral conversions which followed the first-order kinetics were analyzed by changing the temperature. The activation energy (69 kcal/mol) for the conversion for the wild type was higher than those (37-50 kcal/mol) for the mutants. The activation energy for the wild type further increased (by 55%) by adding the substrate, while those for the mutants were essentially unchanged by adding the substrate. We discuss the important role of Glu318 at the putative distal site of P450d in the packing or the conformational stability of the putative distal site of the P450d molecule.

Novel stereoselectivity of rat liver cytochrome(s) P450 toward enantiomers of the trans-1,2-dihydrodiol of triphenylene

Metabolism of 3H-labeled (+)-( S,S)- and (−)-( R,R)-1,2-dihydrodiols of triphenylene by rat liver microsomes and 11 purified isozymes of cytochrome P450 in a reconstituted monooxygenase system has been examined. Although both enantiomers were metabolized at comparable rates, the distribution of metabolites between phenolic dihydrodiols and bay-region 1,2-diol 3,4-epoxide diastereomers varied substantially with the different systems. Treatment of rats with phenobarbital (PB) or 3-methylcholanthrene (MC) caused a slight reduction or less than a twofold increase, respectively, in the rate of total metabolism (per nanomole of cytochrome P450) of the enantiomeric dihydrodiols compared to microsomes from control rats. Among the 11 isozymes of cytochrome P450 tested, only cytochromes P450c (P450IA1) and P450d (P450IA2) had significant catalytic activity. With either enantiomer of triphenylene 1,2-dihydrodiol, both purified cytochrome P450c (P450IA1) and liver microsomes from MC-treated rats formed diol epoxides and phenolic dihydrodiols in approximately equal amounts. Purified cytochrome P450d (P450IA2), however, formed bay-region diol epoxides and phenolic dihydrodiols in an 80:20 ratio. Interestingly, liver microsomes from control or PB-treated rats produced only diol epoxides and little or no phenolic dihydrodiols. The diol epoxide diastereomers differ in that the epoxide oxygen is either cis (diol epoxide-1) or trans (diol epoxide-2) to the benzylic 1-hydroxyl group. With either purified cytochromes P450 (isozymes c or d) or liver microsomes from MC-treated rats, diol epoxide-2 is favored over diol epoxide-1 by at least 4:1 when the (−)-enantiomer is the substrate, while diol epoxide-1 is favored by at least 5:1 when the (+) enantiomer is the substrate. In contrast, with liver microsomes from control or PB-treated rats, formation of diol epoxide-1 relative to diol epoxide-2 was favored by at least 2:1 regardless of the substrate enantiomer metabolized. This is the first instance where the ratio of diol epoxide- 1/diol epoxide-2 metabolites is independent of the dihydrodiol enantiomer metabolized. Experiments with antibodies indicate that a large percentage of the metabolism by microsomes from control and PB-treated rats is catalyzed by cytochrome P450p (P450IIIA1), resulting in the altered stereoselectivity of these microsomes compared to that of the liver microsomes from MC-treated rats.

Remarkable Changes in Catalytic Activity toward Testosterone of Engineered Cytochrome P-450d by Mutations at Putative Distal Site

Abstract The catalytic activity, 6β-hydroxylation, toward testosterone of engineered cytochrome P-450d was three-times increased by Thr319Ala mutation at the putative distal site, While that was abolished Ala315Ser and important role the steroid of while that was abolished by mutations Thr322Ala at the same site, suggesting the of this region in catalytic activity toward cytochrome P-450d.

Carbon monoxide binding studies of engineered cytochrome P-450ds: effects of mutations at putative distal sites in the presence of polycyclic hydrocarbons

The kinetic parameters of CO binding to genetically engineered cytochrome P-450d (P-450d) and two putative distal mutants, Glu318Asp and Thr322Ala, have been evaluated in the presence and absence of polycyclic hydrocarbons. The dissociation constant (Kd) of CO from wild-type P-450d was decreased by half (from 1.8 microM to approximately 0.9 microM) in the presence of phenanthrene or anthracene but was increased to 11 microM in the presence of 1,2:3,4-dibenzanthracene or 7,8-benzoflavone. These changed Kd values were not altered markedly by mutations at the putative distal site. In contrast, the recombination rate constants (kon) of CO to the Glu318Asp mutant in the presence of phenanthrene (15.5 X 10(5) M-1 s-1) and 7,8-benzoflavone (0.75 X 10(5) M-1 s-1) were much larger than those for the wild type. Similar but smaller increases of the kon values were observed for the Thr322Ala mutant. It was suggested that phenanthrene and anthracene distort the Fe-C-O bond and/or affect the access of CO to wild-type P-450d in an opposite way from 1,2:3,4-dibenzanthracene and 7,8-benzoflavone. Glu318 and Thr322 may be located so close to a CO binding channel in ferrous P-450d that mutations of these residues can open the sterically hindered CO channel caused by the hydrocarbons.

Ethanol interferes with regeneration-associated changes in biotransforming enzymes: A potential mechanism underlying ethanol's carcinogenicity?

The effects of chronic ethanol consumption on enzyme systems involved in carcinogen activation and detoxification were studied in a rat model of liver regeneration. In control rats, steady-state messenger RNAs of cytochrome P450j decreased 12 to 24 hr after partial hepatectomy but were fully recovered by 48 to 72 hr. In contrast, messenger RNA levels of cytochrome P450b and P450d did not vary significantly during that period. Steady-state messenger RNA levels for the placental form of glutathione S-transferase decreased within 30 min after partial hepatectomy but fluctuated until levels returned to normal by 48 hr. Preliminary nuclear run-on analyses suggest that the regulation of cytochrome P450j and the placental form of glutathione S-transferase messenger RNA levels involves posttranscriptional control in these animals. In ethanol-fed rats, as in controls, expression of cytochrome P450j and the placental form of glutathione S-transferase decreased transiently after partial hepatectomy. However, compared with control values, messenger RNA levels for cytochrome P450j were greater in ethanol-fed rats at each time point. Similar results were noted for placental glutathione S-transferase levels from 12 to 48 hr after partial hepatectomy. Ethanol feeding had no apparent effect on steady-state messenger RNA levels of cytochrome P450d, P450b or the multidrug-resistant gene. In both ethanol and control rats, only prehepatectomy levels of cytochrome P450 transcripts correlated with levels of the respective P450 isoenzymes. These data indicate that liver regeneration selectively decreases the steady-state messenger RNA expression of certain isoenzymes of cytochrome P450 and glutathione S-transferase.(ABSTRACT TRUNCATED AT 250 WORDS)

Ligand binding studies of engineered cytochrome P-450d wild type, proximal mutants, and distal mutants

Interactions of various axial ligands with cytochrome P-450d wild type, proximal mutants (Lys453Glu, Ile460Ser), and putative distal mutants (Glu318Asp, Thr319Ala, Thr322Ala) expressed in yeast were studied with optical absorption spectroscopy. P-450d wild type and all five mutants were purified essentially as the high-spin form, but the putative distal mutants contained about 5% low-spin form. Bindings of metyrapone and 4-phenylimidazole to the wild type and all mutants formed nitrogen-bound low-spin forms. In contrast, binding of 2-phenylimidazole to the wild type and most of mutants formed oxygen-bond low-spin forms except for the mutant Glu318Asp in which the nitrogen-bound low-spin form was formed. By analogy with the distal structure of P-450cam, it was thus suggested that Glu318 of P-450d, which corresponds with Asp251 of P-450cam, somehow interacts with 2-phenylimidazole over the heme plane. Addition of 1-butanol and acetanilide, a substrate of P-450d, to the wild type and mutants caused the spin change to the low-spin form. The order of dissociation constants of these oxygen ligands to P-450d was wild type greater than proximal mutants greater than putative distal mutants. Spectral analyses showed that the binding of acetanilide is the same as that of another substrate, 7-ethoxycoumarin, in the putative distal mutants but is not the same in the wild type and proximal mutants. From these findings together with other spectral data, it was suggested that the region from Glu318 to Thr322 is located at the distal region of the heme in membrane-bound P-450d as suggested from the X-ray crystal structure of water-soluble P-450cam and amino acid alignments of P-450s.

Probing the role of lysines and arginines in the catalytic function of cytochrome P450d by site-directed mutagenesis. Interaction with NADPH-cytochrome P450 reductase

To identify amino acids of cytochrome P450d (P450d) which participate in the interaction with NADPH-cytochrome P450 reductase, we changed conserved ionic amino acids of P450d to others by site-directed mutagenesis. Turnover numbers (0.032-0.008 min-1) of purified mutants Lys94-Glu, Lys99-Glu, Lys105-Glu, Lys440-Glu, Lys453-Glu, Arg455-Glu, and Lys463-Glu toward 7-ethoxycoumarin were much lower than that (0.380 min-1) of the wild type at 25 degrees C. Reduction rates (less than 0.054 s-1) of the heme of all mutants (0.1 microM) in the presence of NADPH and the reductase (0.3 microM) were much lower than that (5.9 s-1) of the wild type. Furthermore, a turnover number (0.042 min-1) of a microsomal triple mutant (Arg135-Leu + Arg136-Leu + Arg137-Leu) of a conserved Arg cluster was much lower than that (0.674 min-1) of the wild type at 37 degrees C. Thus, we suggest that Lys94, Lys99, Lys105, Lys440, Lys453, Arg455, Lys463, and perhaps the Arg cluster Arg135-Arg136-Arg137 of P450d will participate in the intermolecular electron transfer process by forming ionic bridges between the two proteins and/or by orienting appropriate geometry for electron transfer on the interfacial surface between the two proteins.

Metabolism in Vitro of 3,4,3',4'- and 2,5,2',5'-Tetrachlorobiphenyl by Rat Liver Microsomes and Highly Purified Cytochrome P-450.

Metabolism of two polychlorinated biphenyls, 3,4,3',4'- and 2,5,2',5'-tetrachlorobiphenyl (TCB), was studied using rat liver microsomes and the four forms of cytochrome P-450 (P-450), P-450b, P-450e, P-450c and P-450d. At first, effects of various inducers of P-450 such as phenobarbital (PB), 3-methylcholanthrene (MC), isosafrole (ISF) and pregnenolone 16 alpha-carbonitrile on the formation of metabolites of these TCBs by liver microsomes were compared. 3,4,3',4'-TCB was significantly metabolized by liver microsomes from MC-treated rats to form two previously reported metabolites, 4-hydroxy-3,5,3',4'-TCB and 5-hydroxy-3,4,3',4'-TCB with a relative ratio of 2.5:1. Incubation with microsomes from untreated or PB-treated rats produced none of the metabolites. On the other hand, 2,5,2',5'-TCB was metabolized to 3-hydroxy-2,5,2',5'-TCB most easily by liver microsomes from PB-treated rats and at a moderate rate by liver microsomes from ISF-treated rats. Activities of microsomes from untreated or MC-treated rats to hydroxylate 2,5,2',5'-TCB were low or undetectable. When these TCB hydroxylase activities were examined with a reconstituted system consisting of each P-450, reduced nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P-450 reductase, dilauroylphosphatidylcholine and NADPH-generating system, only P-450c catalyzed both the 4- and 5-hydroxylations of 3,4,3',4'-TCB at a ratio of 2.2:1. On the contrary, the hydroxylation of 2,5,2',5'-TCB proceeded efficiently with P-450b and P-450e, being more efficient with the former. P-450d did not show any catalytic activity toward 3,4,3',4'-TCB and 2,5,2',5'-TCB.(ABSTRACT TRUNCATED AT 250 WORDS)

Aryl hydrocarbon induction of rat cytochrome P-450d results from increased precursor RNA processing.

We have previously demonstrated that cytochrome P-450d mRNA accumulation is induced at a posttranscriptional level by 3-methylcholanthrene (MCA) in primary cultures of rat hepatocytes grown in serum-free hormonally defined medium. Using dactinomycin chase experiments in this culture system, we found that MCA had no effect on the P-450d mRNA half-life. In addition, induction of P-450d occurred both in the presence and in the absence of protein synthesis inhibitors. An analysis of nuclear precursors showed that the accumulation of the primary transcript of the P-450d gene was induced to the same extent as that of the mature mRNA after MCA treatment and that the pattern of accumulation of precursors differed between treated and control liver cells. Since P-450d induction is thought to be a receptor-mediated event, these data are consistent with a model in which a direct interaction occurs between the receptor-ligand complex and the primary transcript.

Aryl Hydrocarbon Induction of Rat Cytochrome P-450d Results from Increased Precursor RNA Processing

We have previously demonstrated that cytochrome P-450d mRNA accumulation is induced at a posttranscriptional level by 3-methylcholanthrene (MCA) in primary cultures of rat hepatocytes grown in serum-free hormonally defined medium. Using dactinomycin chase experiments in this culture system, we found that MCA had no effect on the P-450d mRNA half-life. In addition, induction of P-450d occurred both in the presence and in the absence of protein synthesis inhibitors. An analysis of nuclear precursors showed that the accumulation of the primary transcript of the P-450d gene was induced to the same extent as that of the mature mRNA after MCA treatment and that the pattern of accumulation of precursors differed between treated and control liver cells. Since P-450d induction is thought to be a receptor-mediated event, these data are consistent with a model in which a direct interaction occurs between the receptor-ligand complex and the primary transcript.

Covalent interaction of 3,3'-dichlorobenzidine with hepatic lipids: Enzymic basis and stability of the adducts

Administration of a single oral dsoe (20 mg/kg) of [U- 14C]3,3'-dichlorobenzidine to rats resulted in the in vivo covalent binding of the compound to hepatic lipids. More than 70% of the lipid-3,3'-dichlorobenzidine adducts were accounted for in microsomes. Loss of the lipid-bound 3,3'-dichlorobenzidine residues from either total liver or endoplasmic reticulum occurred in at least two phases—an initial fast phase and a terminal slow phase. In vitro studies with hepatic microsomes in the presence of antibodies to specific P450 isozymes and chemical inhibitors to determine the enzymes that activate 3, 3'-dichlorobenzidine to the lipid-binding derivative(s) implicated cytochrome P450d. The 3,3'-dichlorobenzidine-bound microsomal lipids were not mutagenic to Salmonella TA98 in the Ames test. The results suggest that adduct formation between 3,3'-dichlorobenzidine and membrane lipids may provide a measure of 3, 3'-dichlorobenzidine activation. It is speculated that covalent interaction of the compound with membrane lipids may modify cellular processes, leading to either enhancement or attenuation of carcinogenesis by the chemical.

Metabolism of the arylamide herbicide propanil: II. Effects of propanil and its derivatives on hepatic microsomal drug-metabolizing enzymes in the rat

Propanil (3,4-dichloropropionanilide) is an arylamide herbicide that has been reported to be contaminated with the cytochrome P450 enzyme inducers 3,3′,4,4′-tetrachloroazobenzene (TCAB) and 3,3′,4,4′-tetrachloroazoxybenzene (TCAOB), which are structural analogs of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). We determined if treatment of rats with TCAB, TCAOB, propanil, 3,4-dichloroaniline, TCDD, or phenobarbital induced the hepatic microsomal metabolism of propanil and 3,4-dichloroaniline. Acylamidase-catalyzed hydrolysis of propanil to 3,4-dichloroaniline was not induced by any of the pretreatments; however, hydroxylation of propanil at the 2′-position was induced by TCDD, TCAB, TCAOB, propanil, and 3,4-dichloroaniline pretreatments. Ring-and N-hydroxylations of 3,4-dichloroaniline were induced by TCDD, TCAB, TCAOB, and 3,4-dichloroaniline pretreatments. Microsomal 7-ethoxyresorufin- O-deethylase (EROD) and 7-benzoxyresorufin- O-dealkylase (BROD) activities and electrophoretic mobility of microsomal proteins suggested that cytochromes P450c and P450d were induced by TCAB and TCAOB pretreatment. EROD, BROD, and 7-pentoxyresorufin- O-dealkylase activities were slightly increased in microsomes from propanil- and 3,4-dichloroaniline-pretreated rats, which suggests that these compounds may be weak inducers of cytochrome P450 isozymes.

The inducibility and catalytic activity of cytochromes P450c (P450IA1) and P450d (P450IA2) in rat tissues

The metabolism of phenacetin is primarily by cytochrome P450-dependent O-deethylation to paracetamol (POD activity). In untreated rats, microsomal POD activity is detectable in both the liver and lung, but not in the small intestine or the kidney. POD activity is highly induced in both hepatic and extrahepatic tissues of the rat following treatment with polycyclic aromatic hydrocarbons such as 3-methylcholanthrene (MC). Only cytochrome P450c (P450IA1) is inducible in rat extrahepatic tissues by MC or isosafrole, whereas in the liver both cytochromes P450c and P450d (P450IA2) are inducible by these compounds. Specific antibodies to cytochromes P450c and P450d were used to study the expression and function of these two related isoenzymes in rat liver and extrahepatic tissues before and after induction with MC. Whereas cytochrome P450d is responsible for all of the high affinity POD activity in hepatic microsomal fractions of both untreated and MC treated rats, this activity is mediated only by P450c in microsomal fractions from extrahepatic tissues following MC treatment. POD activity of microsomal fractions from lung of untreated rats was not mediated by either cytochrome P450c or P450d.

High affinity phenacetin O-deethylase is catalysed specifically by cytochrome P450d (P450IA2) in the liver of the rat

Phenacetin is metabolized primarily by O-deethylation to paracetamol (POD activity), a reaction catalysed by cytochrome P450. The high affinity component of POD activity is inducible in rat liver by treatment of the animals with polycyclic aromatic hydrocarbons. Following treatment with hydrocarbons such as 3-methylcholanthrene (MC) and isosafrole (ISF) both cytochromes P450c (P450IA1) and P450d (P450IA2) are also induced in rat liver. Studies with the reconstituted enzymes have shown that both forms of P450 catalyse phenacetin O-deethylation at rates that exceeded that of the high affinity component of activity of hepatic microsomal preparations from 3-methylcholanthrenetreated rats (at 4μM phenacetin: P450c, 440 ± 40 pmol/nmol/min; P450d, 1030 ± 10 pmol/nmol/min; microsomal fraction, 163 pmol/mg/min). Specific inhibitory antibodies (both monoclonal and monospecific polyclonal) were used to define the specificity of microsomal POD activity. These studies have shown that hepatic high affinity POD activity is exclusively catalysed by cytochrome P450d in both untreated rats and in rats pretreated with MC.

Dexamethasone-mediated regulation of 3-methylcholanthrene-induced cytochrome P-450d mRNA accumulation in primary rat hepatocyte cultures.

We have previously demonstrated that cytochrome P-450c and P-450d mRNAs can be induced by 3-methylcholanthrene (MCA) in primary cultures of rat hepatocytes grown in serum-free hormonally defined medium (HDM) plus minimal salts (Silver, G., and Krauter, K. S. (1988) J. Biol. Chem. 263; 11802-11807). Such cultures were used to investigate the role of the individual hormonal components present in the medium in the hydrocarbon-mediated induction process. Replacing HDM with minimal salts plus 10% fetal bovine serum (FBS) resulted in a 4-fold reduction in the accumulation of P-450d mRNA in response to MCA. In contrast, no effect was seen on induced levels of P-450c mRNA. Mixing experiments, in which primary cultures of hepatocytes were grown in medium containing HDM plus 10% FBS, indicated that there was no negative acting component present in FBS, but rather there was a positive acting component present in the mixture of hormones in HDM which permitted P-450d induction by MCA. Testing the effects of singly deleting each of the 10 components in HDM on MCA-induced P-450d expression demonstrated that dexamethasone was the only factor which affected the induction of P-450d. Deletion of this component from HDM resulted in a 4-fold decrease in the maximum MCA induced expression of P-450d mRNA. Moreover, supplementation of minimal salts plus 10% FBS with dexamethasone restored full P-450d inducibility by MCA. Deletion of the other components from HDM had no effects on P-450d mRNA accumulation. Although substratum clearly contributed to the quality of primary hepatocyte cultures, we were unable to demonstrate any role of the substratum on MCA induction of P-450d. In vitro nuclear run-on experiments revealed that dexamethasone had little effect on the rate of transcription of the P-450d genes. Therefore, the effect of dexamethasone on induction must be at the posttranscriptional level.

Interspecies features of hepatic cytochromes P450 IA1 and P450 IA2 in rodents

1. Antibodies to mouse liver cytochrome P3-450 (anti-P3-450) and antibodies to rat liver cytochrome P-450d (anti-P-450d-c) both inhibit the O-deethylation of 7-ethoxy-resorufin (ER) in liver microsomes of benzo(a)pyrene-induced (BP) mice but do not inhibit the O-deethylase activity in liver microsomes of BP-induced rats. 2. Anti-P3-450 and anti-P-450d-c inhibit BP hydroxylation in BP-induced mouse liver microsomes by 20%, but they do not inhibit this rection at all in BP-induced rat liver microsomes. 3. Isolated cytochrome P3-450 in a reconstituted monooxygenase system metabolized 7-ER and BP. In contrast, its homologue, cytochrome P-450d, does not metabolize these substrates. The fraction containing cytochrome P1-450 metabolized 7-ER at a low rate and BP at a rate of 3.6 nmol product/min per nmol cytochrome. 4. Western blot analysis with anti-P-450c + d revealed two bands in SDS-PAGE gels containing BP-induced mouse liver microsomes corresponding to cytochrome P1-450, 55.0 kDa, and cytochrome P3-450, 54.5 kDa. There appeared a single band (cytochrome P3-450) in interaction of mouse liver BP-microsomes with anti-P3-450 and anti-P-450d-c.

Complementary DNA sequence of 3-methylcholanthrene-inducible P-450 from the rat lung.

Cytochrome P-450MC was induced in pulmonary microsomes of 3-methylcholanthrene-treated rats and also at low level in that of isosafrole-treated rats. Cytochrome P-450d was not detected in the lungs of 3-methylcholanthrene- or isosafrole-treated rats by the method of Western blot analysis with a polyclonal antibody raised against cytochrome P-450c which is equally effective against P-450d, nor by the method of Northern hybridization probed with pcP450mc3 (P-450d probe). Complementary DNA of P-450MC was isolated from rat pulmonary cDNA library and the nucleotide sequence of pulmonary cDNA was compared with that of hepatic P-450c cDNA reported by Yabusaki et al. There was no gross change in nucleotide sequences of cDNA between pulmonary P-450MC and hepatic P-450c.

Induction of rat liver microsomal and nuclear cytochrome p-450 by dietary 2-acetylaminofluorene and butylated hydroxytoluene

The influence of dietary 2-acetylaminofluorene (AAF) on the cytochrome P-450 content of rat liver microsomal and nuclear fractions was immunochemically probed with monoclonal and polyclonal antibodies to cytochromes P-450c and P-450d. Cytochrome P-450d but not P-450c was immunodetected in microsomes, nuclear envelopes, and nuclei from untreated rats. The levels of both cytochromes P-450c and P-450d were elevated after a diet of either 0.1% AAF for 1 week or 0.05% AAF for 3 weeks. However, the level of cytochrome P-450c relative to P-450d was lower after the more prolonged AAF feeding. Supplementation of AAF-containing diets with 0.3% butylated hydroxytoluene (BHT), which affords protection against AAF hepatocarcinogenesis in high-fat fed rats, protected and/or induced total (spectral) nuclear envelope cytochrome P-450 content. Immunochemical studies of liver fractions showed that BHT enhanced the AAF-dependent induction of cytochrome P-450c, but not of P-450d. This was a concerted effect of AAF + BHT since dietary BHT by itself did not affect the levels of cytochrome P-450c or P-450d as compared to control rats. Since 1- to 3-week dietary AAF had little effect on total (spectral analyses) microsomal cytochrome P-450 but markedly reduced total P-450 in nuclear envelopes, the coordinated induction of specific cytochrome P-450s in the different fractions suggests selective induction and depression of different forms of cytochrome P-450 and provides additional evidence for independent regulation of the drug-metabolizing system in nuclear envelope and microsomes. In addition, these results suggest that regulation of cytochrome P-450 may play a crucial role in the nutritional modulation of AAF hepatocarcinogenesis.

Site-directed mutageneses of rat liver cytochrome P-450d: catalytic activities toward benzphetamine and 7-ethoxycoumarin.

Catalytic activities toward benzphetamine and 7-ethoxycoumarin of 11 distal mutants, 9 proximal mutants, and 3 aromatic mutants of rat liver cytochrome P-450d were studied. A distal mutant Thr319Ala was not catalytically active toward benzphetamine, while this mutant retained activity toward 7-ethoxycoumarin. Distal mutants Gly316Glu, Thr319Ala, and Thr322Ala displayed higher activities (kcat/Km) toward 7-ethoxycoumarin that were 2.4-4.7-fold higher than that of the wild-type enzyme. Although kcat/Km values of four multiple distal mutants toward benzphetamine were less than half that of the wild type, activities of these mutants toward 7-ethoxycoumarin were almost the same as or higher than the wild-type activity toward this substrate. The distal double mutant Glu318Asp, Phe325Tyr showed 6-fold higher activity than the wild-type P-450d toward 7-ethoxycoumarin. Activities of the proximal mutants Lys453Glu and Arg455Gly toward both substrates were much lower (less than one-seventh) than the corresponding wild-type activities. Catalytic activities of three aromatic mutants, Phe425Leu, Pro427Leu, and Phe430Leu, toward benzphetamine were less than 7% of that of the wild type, while the activities of these aromatic mutants toward 7-ethoxycoumarin were more than 2.5 times higher than the wild-type activity toward this substrate. From these findings, in conjunction with a molecular model for P-450d, we suggest that (1) the relative importance to catalysis of various distal helix amino acids differs depending on the substrate and that these differences are associated with the size, shape, and flexibility of the substrate and (2) the proximal residue Lys453 appears to play a critical role in the catalytic activity of P-450d, perhaps by participating in forming an intermolecular electron-transfer complex.