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S J Pernecky

Publications and source records attributed to S J Pernecky.

15 recordsLinked to original sources
PubMed1999

Cumene hydroperoxide-supported demethylation reactions catalyzed by cytochrome P450 2B4 lacking the NH2-terminal sequence.

Catalytic activities of cytochrome P450 2B4 lacking NH2-terminal amino acids 2-27 (wt Delta2B4) and that of truncated 2B4 containing a Pro to Ser mutation at position 221 were examined in a system supported by cumene hydroperoxide. Demethylation activities of either truncated 2B4 with N-methylaniline, N,N-dimethylaniline, and d-benzphetamine were lower than those of liver microsomal 2B4, whereas the rate of 1-phenylethanol oxidation to acetophenone catalyzed by liver microsomal and truncated 2B4 enzymes was nearly the same. The Km and Vmax values for cumene hydroperoxide in the demethylation of N-methylaniline by wt Delta2B4 were 20% and 28%, respectively, of those obtained for 2B4. The reaction with wt Delta2B4 displayed a lesser dependence on phospholipid than did that with 2B4, and a complex relationship between activity and substrate concentration. The results suggest that the NH2-terminal region contributes to interaction of oxidant, substrate, and phospholipid in cumene hydroperoxide-supported reactions catalyzed by cytochrome P450 2B4.

Aniline Compounds↗
PubMed1998

The detection of cytochrome P450 2E1 and its catalytic activity in rat testis.

Cytochrome P450 2E1 participates in the bioactivation of a wide variety of environmental and occupational pollutants. Such reactions may lead to the production of active carcinogenic metabolites. The presence of P450 2E1 in the testis and prostate has not yet been reported. In the present study, cytochrome P450 2E1 mRNA has been identified in the rat prostate and testis by reverse transcription PCR, southern blotting, and DNA sequencing. P450 2E1 protein from rat testis could be detected with immunoblot analysis, but was not detected in the prostate. The hydroxylation of p-nitrophenol, known to be mediated by P450 2E1, was demonstrated by HPLC measurement of product formation in microsomal fractions from the rat testis, but again not from prostate. Exposure of rats to pyridine resulted in a 2.9-fold increase of p-nitrophenol hydroxylation by testicular microsomes. Diethyldithiocarbamate, a selective mechanism-based inhibitor of P450 2E1, or a P450 2E1 monoclonal antibody, caused marked inhibition of testicular microsomal p-nitrophenol hydroxylase activity. These results indicate that cytochrome P450 2E1 is present in the rat testis, and that it is elevated by the treatment of the animals with pyridine. Thus, the presence and inducibility of cytochrome P450 2E1 in the testis may be of significance in the bioactivation of environmental chemicals to genotoxic metabolites.

Animals↗
PubMed1998

Cytochrome P450 2E1 mRNA in the rat prostate: detection and quantitation by competitive reverse transcription and polymerase chain reaction.

Cytochrome P450 2E1 plays a pivotal role in the metabolic activation of a wide variety of low molecular weight environmental toxicants and procarcinogens. In the present study, expression of the P450 2E1 gene in the rat prostate gland was quantitated by competitive reverse transcription and the polymerase chain reaction. To assess accurately the induction level of P450 2E1 mRNA in the prostate after pyridine treatment of rats, a recombinant standard RNA was generated that is homologous to the sequence of P450 2E1 mRNA except for an internal deletion of 100 bases. The data indicate that P450 2E1 mRNA is present in the prostate of untreated animals and is induced about four-fold by treatment with pyridine. The results suggest that exposure to certain environmental chemicals and procarcinogens may increase P450 2E1 levels in the prostate gland and thus could enhance formation of reactive, carcinogenic metabolites.

Animals↗
PubMed1998

Influence of mutation of the amino-terminal signal anchor sequence of cytochrome P450 2B4 on the enzyme structure and electron transfer processes.

The role of the NH2-terminal hydrophobic patch of cytochrome P4502B4 (CYP2B4) in interactions with NADPH-cytochrome P450 reductase (P450R) and cytochrome b5 (b5) was assessed using a variant lacking the signal anchor sequence (Delta2-27). CD, second-derivative, and fluorescence emission spectra indicated that the structure of the deletion mutant slightly differed from that of the native CYP2B4. Fitting of the initial-velocity patterns for P450R- and b5-directed electron transfer to the ferric CYP2B4 forms to Michaelis-Menten kinetics revealed an approximately 2.3-fold decrease in the affinity of the two electron donors for the engineered enzyme, while the reductive efficiency remained unaffected. Circumstantial analysis suggested that impaired association of the redox proteins with P4502B4(Delta2-27) accounted for this phenomenon. Interestingly, spectral docking of P450R to the truncated pigment was not hampered, while the binding of b5 was blocked. The rates of substrate-triggered aerobic NADPH consumption in systems containing CYP2B4(Delta2-27) and P450R were 16 to 56% those obtained with the unchanged hemoprotein. Decelerated cofactor oxidation did not arise on defective substrate binding or perturbed utilization of the substrate-bound oxy complex. Experiments with b5 as the ultimate electron donor hinted at some damage to second-electron transfer to the truncated enzyme. The results are consistent with the proposal that the NH2-terminal hydrophobic region of CYP2B4 might be of importance in preservation of the catalytic competence of the enzyme.

Amino Acid Sequence↗
PubMed1997

Evidence for a role of a perferryl-oxygen complex, FeO3+, in the N-oxygenation of amines by cytochrome P450 enzymes.

Most cytochrome P450 (P450)-catalyzed reactions are believed to involve an FeO3+ intermediate as the actual oxygenating species. However, studies on the mechanism of steroid aromatization and subsequent model work have provided evidence that a peroxo-iron form (formally FeO2) can be involved directly in some oxidations. The possible involvement of peroxoiron was considered in P450-catalyzed N-oxygenations, because there is precedent for the use of H2O2 and organic peroxides in such reactions in the literature concerning synthetic and flavin reactions. The approach used was to compare P450 reactions involving the normal NADPH/NADPH-P450 reductase/O2 system with those supported by the oxygen surrogates H2O2 (which can directly form FeO2 and subsequently FeO3+) and iodosylbenzene (which can form FeO3 but not FeO2+). Iodosylbenzene was effective in supporting rabbit P450 1A2-catalyzed N,N-dimethyl-2-aminofluorene N-oxygenation, human P450 3A4-catalyzed quinidine N-oxygenation, rat P450 2B1-catalyzed oxidation of N-benzyl-(1-phenyl) cyclobutylamine to the N-hydroxyamine and nitrone, and rat P450 2B1-catalyzed and rabbit P450 2B4-catalyzed N-oxygenation of N,N-dimethylaniline (also N-demethylation). H2O2 also supported most of these reactions. A mutant of P450 2B4 with the substitution of alanine for threonine at position 302 has been shown to have decreased ability to catalyze reactions involving the putative FeO3+ but, presumably because of decreased ability to protonate the FeO2+ complex, to have enhanced activity in oxidative deformylation reactions believed to involve FeO2+. This mutant showed both decreased N,N-dimethylaniline N-demethylation and N-oxygenation activity. Although some contribution of an FeO2+ species to these reactions cannot be ruled out, formation of product in the iodosylbenzene-supported systems cannot be readily explained by an obligatory FeO2 mechanism and the involvement of FeO3+ is concluded to be more likely.

Amines↗
PubMed1996

A role for threonine 302 in the mechanism-based inactivation of P450 2B4 by 2-ethynylnaphthalene.

2-Ethynylnaphthalene (2EN) is a mechanism-based inactivator of P450 2B4 that covalently modifies an amino acid in the peptide Glu273-Met314 with a 2-naphthylacetyl group [Roberts et al. (1994) Biochemistry 33, 3766-3771]. Truncated 2B4 lacking amino acids 2-27, 2B4 (Delta2-27), was expressed in Escherichia coli, purified, and found to catalyze the oxidation of 2EN to 2-naphthylacetic acid (2NA). The metabolism of 2EN resulted in the inactivation and covalent modification of the protein moiety as we have previously reported with P450 2B4 purified from the livers of phenobarbital-induced rabbits. The rate constants of inactivation of the O-deethylation activity of 7-ethoxy-4-trifluoromethylcoumarin (EFC) were 0.15 +/- 0.01 and 0.20 +/- 0.05 min-1 for the protein purified from rabbit liver and 2B4 (Delta2-27), respectively. A protein in which threonine 302 was replaced with alanine, P450 2B4 (Delta2-27, T302A), was inactivated by 2EN with a much slower rate constant (0.05 +/- 0.01 min-1) and formed 1.8-fold more 2NA as compared to P450 2B4 (Delta2-27) over a 10-min incubation. When the formation of 2NA was supported by cumene hydroperoxide, 2B4 (Delta2-27, T302A) formed 30% less product than 2B4 (Delta2-27) over a 5-min incubation. After incubation with [3H]2EN and NADPH, P450 2B4 (Delta2-27) had significant radioactivity associated with the P450 in an NADPH-dependent manner when the incubation mixture was analyzed by SDS-PAGE followed by autoradiography and 10-fold more radioactivity associated with the P450 as compared to P450 2B4 (Delta2-27, T302A) when analyzed by reverse-phase HPLC. Thus, threonine 302 is not required by 2B4 for oxidation of 2EN or EFC but appears to play an important role in the inactivation of P450 2B4 by 2EN and the covalent labeling of the P450 protein by 2EN.

Animals↗
PubMed1996

Peroxo-iron and oxenoid-iron species as alternative oxygenating agents in cytochrome P450-catalyzed reactions: switching by threonine-302 to alanine mutagenesis of cytochrome P450 2B4.

Among biological catalysts, cytochrome P450 is unmatched in its multiplicity of isoforms, inducers, substrates, and types of chemical reactions catalyzed. In the present study, evidence is given that this versatility extends to the nature of the active oxidant. Although mechanistic evidence from several laboratories points to a hypervalent iron-oxenoid species in P450-catalyzed oxygenation reactions, Akhtar and colleagues [Akhtar, M., Calder, M. R., Corina, D. L. & Wright, J. N. (1982) Biochem. J. 201, 569-580] proposed that in steroid deformylation effected by P450 aromatase an iron-peroxo species is involved. We have shown more recently that purified liver microsomal P450 cytochromes, including phenobarbital-induced P450 2B4, catalyze the analogous deformylation of a series of xenobiotic aldehydes with olefin formation. The investigation presented here on the effect of site-directed mutagenesis of threonine-302 to alanine on the activities of recombinant P450 2B4 with N-terminal amino acids 2-27 deleted [2B4 (delta2-27)] makes use of evidence from other laboratories that the corresponding mutation in bacterial P450s interferes with the activation of dioxygen to the oxenoid species by blocking proton delivery to the active site. The rates of NADPH oxidation, hydrogen peroxide production, and product formation from four substrates, including formaldehyde from benzphetamine N-demethylation, acetophenone from 1-phenylethanol oxidation, cyclohexanol from cyclohexane hydroxylation, and cyclohexene from cyclohexane carboxaldehyde deformylation, were determined with P450s 2B4, 2B4 (delta2-27), and 2B4 (delta2-27) T302A. Replacement of the threonine residue in the truncated cytochrome gave a 1.6- to 2.5-fold increase in peroxide formation in the presence of a substrate, but resulted in decreased product formation from benzphetamine (9-fold), cyclohexane (4-fold), and 1-phenylethanol (2-fold). In sharp contrast, the deformylation of cyclohexane carboxaldehyde by the T302A mutant was increased about 10-fold. On the basis of these findings and our previous evidence that aldehyde deformylation is supported by added H202, but not by artificial oxidants, we conclude that the iron-peroxy species is the direct oxygen donor. It remains to be established which of the many other oxidative reactions involving P450 utilize this species and the extent to which peroxo-iron and oxenoid-iron function as alternative oxygenating agents with the numerous isoforms of this versatile catalyst.

Animals↗
PubMed1995

Subcellular localization, aggregation state, and catalytic activity of microsomal P450 cytochromes modified in the NH2-terminal region and expressed in Escherichia coli.

This laboratory previously expressed cDNAs encoding rabbit liver cytochrome P450 2E1 (the ethanol-inducible isoform) and the corresponding protein lacking amino acids 3-29, a proposed membrane anchor, in Escherichia coli. Unexpectedly, the shortened protein, like the full-length form, was found to be predominantly located in the bacterial inner membrane rather than the cytosol and to have full catalytic activity. Additional proteins with alterations in the NH2-terminal region of P450 2E1 or P450 2B4 (the phenobarbital-inducible isoform) were similarly expressed, and it was concluded that such modifications can change the cytochrome to an increased cytosolic localization and that the first two hydrophobic segments are not uniquely involved in attachment to the bacterial membrane (Pernecky et al., 1993, Proc. Natl. Acad. Sci. USA 90, 2651-2655). In the present study, three chimeric cytochromes were produced to determine the effect on subcellular localization: 2E1:2B4, in which the first 17 residues of 2E1 (delta 3-29) replaced the corresponding 17 residues in 2B4 (delta 2-27), and BM-3:2B4 and BM-3:2E1, in which the first 19 residues of P450BM-3 replaced the first 17 in 2B4 (delta 2-27) and 2E1 (delta 3-29), respectively. Of the total cytochrome expressed, the localization in the E. coli cytosol was about 60, 70, and 80% for the respective chimeras, with 80% being the highest for any P450 we have examined. A plot of the extent of membrane binding versus hydropathy of the NH2-terminal region showed that the terminal sequence strongly influences the subcellular distribution and that a group of 2E1 proteins and a group of 2B4 proteins each have other regions that characteristically determine the extent of membrane attachment. The role of the NH2-terminal region in the high level of aggregation of purified full-length P450 is indicated by the finding that the multimeric state of 2E1 or 2B4 is unaffected by sodium cholate at concentrations that convert 2E1 (delta 3-29) or 2B4 (delta 2-27) to the monomeric state. In contrast to our earlier experience with P450 2E1, purified P450 2B4 (delta 2-27) has on the average only about half the activity of full-length 2B4 with substrates that undergo oxidative dealkylation or oxygenation at a hydroxyl group.

Amino Acid Sequence↗
PubMed1994

The NH2-terminal region of rabbit CYP2E1 is not essential for interaction with NADPH-cytochrome P450 reductase.

Recently we reported that electrostatic forces interfere with the formation of the NADPH-cytochrome P450 reductase/cytochrome P450 electron transfer complex and suggested that this complex is formed by the attraction of the complementary hydrophobic patches (Voznesensky, A., and Schenkman, J. (1994) 269 J. Biol. Chem, 15724-1573). In this report we evaluate the role of the NH2-terminal hydrophobic region of CYP2E1 in the interaction with the reductase by comparing ionic strength dependence of the reduction of the full-length and truncated delta 3-29 CYP2E1. Increasing ionic strength stimulates reduction of both full-length and truncated CYP2E1. The neutralization of electrostatic interactions by increasing ionic strength revealed no impairment of the delta 3-29 CYP2E1 reduction compared to that of the full-length CYP2E1, indicating that the NH2-terminal region is not essential for the interaction of the cytochrome with the reductase.

Animals↗
PubMed1993

Expression of truncated forms of liver microsomal P450 cytochromes 2B4 and 2E1 in Escherichia coli: influence of NH2-terminal region on localization in cytosol and membranes.

The currently accepted model for the membrane topology of microsomal cytochrome P450 is that of a largely cytoplasmic domain bound by only one or two transmembrane segments at the NH2 terminus. However, as we have reported previously, P450 2E1 lacking the hydrophobic NH2-terminal signal peptide, like the full-length protein, is located in the inner cell membrane when expressed in Escherichia coli and is active with typical substrates. In the present study, additional variants of alcohol-inducible P450 2E1 as well as truncated forms of phenobarbital-inducible P450 2B4 were similarly expressed to determine the influence of the NH2-terminal region on the membrane-binding properties. After deletion of S1 (the NH2-terminal hydrophobic segment), or both S1 and L1 (the following hydrophilic region, expected to be lumenal or cytosolic), one-third of the resulting P450 2B4 (delta 2-20) and 2B4 (delta 2-27) remained membrane bound. Furthermore, the idea that the first two hydrophobic segments are required for attachment by a hairpin loop is not supported by the finding that after deletion of the S1, L1, and S2 segments about half of the P450 2E1 (delta 3-48) remained membrane bound. Since Na2CO3 treatment of the membrane fraction had no significant effect, the findings are apparently not attributable to a loose attachment or occlusion of the truncated proteins. The replacement of neutral amino acids by positively charged residues in positions 3 and 8 of P450 2E1 (delta 3-29) changed the amount in the cytosol from 35% to 50%, and the deletion of residues 2-20 or 2-27 from P450 2B4, which resulted in positive charges occurring in the NH2-terminal region, changed the amount in the cytosol from 27% to 67%. We conclude that alterations in the NH2-terminal region can change the location of the cytochrome from largely membranous to largely cytosolic and that the first two hydrophobic segments are not uniquely involved in membrane attachment.

Amino Acid Sequence↗
PubMed1992

Cytochrome P450: progress and predictions.

The cytochrome P450 gene superfamily encodes many isoforms that are unusual in the variety of chemical reactions catalyzed and the number of substrates attacked. The latter include physiologically important substances such as steroids, eicosanoids, fatty acids, lipid hydroperoxides, retinoids, and other lipid metabolites, and xenobiotics such as drugs, alcohols, procarcinogens, antioxidants, organic solvents, anesthetics, dyes, pesticides, odorants, and flavorants. Accordingly, it is not surprising that these catalysts have come under intensive study in recent years in fields as diverse as biochemistry and molecular biology, endocrinology, pharmacology, toxicology, anesthesiology, nutrition, pathology, and oncology. In this review, recent advances in our knowledge of the catalytic properties, reaction mechanisms, and regulation of expression and activity of the P450 enzymes are briefly summarized. In addition, the prospects for research in this field are considered, and advances are predicted in four broad areas: improved basic knowledge of enzyme catalysis and regulation; synthesis of fine chemicals, including drug design and screening; removal of undesirable environmental chemicals; and biomedical applications related to steroid, drug, carcinogen, and alcohol metabolism.

Animals↗
PubMed1991

Purification and characterization of cytochrome P450 2E2 from hepatic microsomes of neonatal rabbits.

The alcohol-inducible P450 2E subfamily in the rabbit has two known members that differ in only 16 amino acid residues scattered throughout the polypeptide chain. P450 2E1 has been thoroughly characterized, and is known to have diverse inducers and substrates. Little is known, however, about the properties of P450 2E2, since efforts to isolate this isozyme from adult rabbits have been unsuccessful. In the present study, 2E2 was purified to electrophoretic homogeneity from liver microsomes of neonatal rabbits with the use of 4-methylpyrazole as a stabilizing agent. The purified cytochrome was identified as 2E2 by NH2-terminal amino acid sequence analysis as well as by immunoblot analysis with three different antibodies to 2E1. Purified 2E2, in contrast to 2E1, is predominantly low-spin in the presence of 20% glycerol, but is in a mixed high- and low-spin state as the concentration of glycerol is decreased. The catalytic properties of purified 2E1 and 2E2 were compared in the reconstituted system with a variety of substrates, including alcohols, ethers, nitrosamines, and aromatic compounds. Differences between the two enzymes in catalytic activity and in the interaction with cytochrome b5 were observed with some but not all of the substrates tested. Purified 2E1 and 2E2 both consume molecular oxygen relatively rapidly during NADPH oxidation in the absence of an added substrate, and stoichiometric determinations indicated that only about 20% of the O2 was reduced to H2O2, with the remainder apparently undergoing four-electron reduction to water.

Amino Acid Sequence↗
PubMed1990

Expression of rabbit cytochrome P-450IIE2 in yeast and stabilization of the enzyme by 4-methylpyrazole.

A rabbit cytochrome P-450IIE2 full-length cDNA was cloned into a yeast episomal plasmid (YEp13) between the copper-responsive yeast metallothionein gene promoter (CUP1) and the iso-1-cytochrome c gene terminator (CYC1), and the cytochrome P-450 was expressed in Saccharomyces cerevisiae. The microsomal fraction prepared from copper-treated cells exhibited a ferrous carbonyl difference spectrum with an absorption maximum at 451 nm and contained approximately 0.07 nmol of P-450IIE2 per mg of protein. The P-450IIE2 protein expressed in yeast microsomes was catalytically competent as judged by the NADPH-dependent deethylation of N-nitrosodiethylamine and by the oxidation of butanol. Cholate solubilization and polyethylene glycol fractionation of yeast microsomal P-450IIE2 yielded a preparation with a markedly lower specific content than that of intact microsomes, but, when 4-methylpyrazole was included during solubilization, the holoenzyme was completely stabilized.

Cholic Acid↗
PubMedDate not supplied

Induction of P-450 cytochromes 2E2, 1A1, and 1A2 by imidazole in neonatal rabbits.

Cytochrome P-450 2E1 is induced in adult rabbits by treatment with alcohol, imidazole, and a variety of other agents, as shown earlier in this laboratory, but it is not known whether the highly homologous P-450 2E2 is similarly induced. In this study, the effects of imidazole on 2E2 expression were examined in neonatal rabbits, in which 2E1 is not detectable. Treatment of the animals with imidazole on days 8 through 11 after birth caused a 3-fold increase in the content of total P-450 in liver microsomes. In contrast, the microsomal content of cytochrome b5 and NADPH-P450 reductase was not changed. Immunoblot analysis revealed a significant increase in the level of P-450 2E2 (3-fold) as well as 1A1 (> 10-fold) and 1A2 (> 2-fold) in hepatic microsomes from imidazole-treated neonatal rabbits. The rates of microsomal N-demethylation of N-nitrosodimethylamine and O-deethylation of 7-ethoxyresorufin were similarly increased from 1.3 and 0.03 nmol/min/mg protein, respectively, to 5.6 and 0.24 nmol/min/mg protein, respectively, by imidazole treatment. Blot analysis indicated that the levels of 2E2, 1A1, and 1A2 mRNAs are not increased by imidazole treatment and that 2E1 mRNA is not detectable in either untreated or imidazole-treated neonates. The induction of P-450 2E2 was confirmed by NH2-terminal amino acid sequence analysis of immunopurified 2E protein from hepatic microsomes of imidazole-treated neonatal rabbits.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗