P R I O R I T Y P A P E Rin reconstituted membranes Effects of charge and nonbilayer phase propensity of the membrane Pyotr Kisselev1,*, Dieter Schwarz1, Karl-Ludwig Platt2, Wolf-Hagen S
Trang 1P R I O R I T Y P A P E R
in reconstituted membranes
Effects of charge and nonbilayer phase propensity of the membrane
Pyotr Kisselev1,*, Dieter Schwarz1, Karl-Ludwig Platt2, Wolf-Hagen Schunck3and Ivar Roots1
1
Institute of Clinical Pharmacology, University Medical Centrum Charite´, Humboldt University of Berlin, Germany;
2
Institute of Toxicology, University of Mainz, Germany;3Max Delbrueck Centrum for Molecular Medicine, Berlin, Germany
Human cytochrome P4501A1 (CYP1A1) is one of the key
enzymes in the bioactivation of environmental pollutants
such as benzo[a]pyrene (B[a]P) and other polycyclic
aro-matic hydrocarbons To evaluate the effect of membrane
properties and distinct phospholipids on the activity of
human CYP1A1 purified insect cell-expressed human
CYP1A1 and of human NADPH-P450 reductase were
reconstituted into phospholipid vesicle membranes
Con-version rates of up to 36 pmolÆmin)1Æpmol)1CYP1A1 of the
enantiomeric promutagens (–)- and
(+)-trans-7,8-dihy-droxy-7,8-dihydro-B[a]P (7,8-diol) to the genotoxic
diolep-oxides were achieved The highest rates were obtained when
negatively charged lipids such as phosphatidylserine and
phosphatidylinositol and/or nonbilayer phospholipids such
as phosphatidylethanolamine were present in the membrane
together with neutral lipids Both Vmaxand Kmvalues were
changed This suggests a rather complex mechanism of
stimulation which might include altered substrate binding as well as more effective interaction between CYP1A1 and NADPH-P450 reductase Furthermore, the ratio of r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (DE2) to r-7,t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (DE1) formed from (–)-7,8-diol was significantly increased
by the introduction of anionic lipids, but not by that of nonbilayer lipids Thus, charged lipids affect the stereose-lectivity of the epoxidation by leading to the formation of a larger amount of the ultimate mutagen DE2 than of DE1, which is far less carcinogenic These data suggest that membrane properties such as negative charge and nonbi-layer phase propensity are important for the efficiency and selectivity of enzymatic function of human CYP1A1 Keywords: human cytochrome P4501A1; vesicle reconstitu-tion; epoxidareconstitu-tion; benzo[a]pyrene; benzo[a]pyrene-7, 8-diol
Human cytochrome P4501A1 (CYP1A1) is one of the key
enzymes in the bioactivation of environmental pollutants
Benzo[a]pyrene (B[a]P) and other polycyclic aromatic
hy-drocarbons acquire their mutagenic and carcinogenic
prop-erties by its action The first step of activation catalyzed by
CYP1A1 is the formation of
7R,8S-epoxy-7,8-dihydro-B[a]P This is transformed via regioselective hydrolysis by microsomal epoxide hydrolase to (–)-7R,8R-dihydroxy-7,8-dihydrobenzo[a]pyrene ((–)-7,8-diol) and then metabolized
by CYP1A1 to the ultimately genotoxic r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (so-called diolepox-ide-2 or antidiolepoxide, DE2) [1–3] The last reaction appeared to be highly stereoselective as no or much less of the less carcinogenic r-7,t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydo-B[a]P (diolepoxide-1 or syn-diolepoxide, DE1) was produced from (–)-7,8-diol [4,5] Racemic (+/–)-7,8-diol
is mainly converted by human CYP1A1 to the DE2 [6,7] CYP1A1-dependent activity can be reconstituted by mixing the basic components of the monooxygenase system, i.e purified CYP1A1, NADPH-cytochrome P450 reductase, which transfers electrons from NADPH to P450, and dilaurylglycerophosphocholine (Lau2PtdCho) [8–10] How-ever, this micellar system is not appropriate for studying the interactions between the components of the system as some
of its properties are unlike those of the endoplasmic reticulum membrane, the natural environment of the microsomal monooxygenase system This membrane is a bilayer, and it is highly probable that CYP1A1 and NADPH-cytochrome P450 reductase exhibit other impor-tant protein–lipid and protein–protein interactions there Indeed, reconstitution systems using bilayer vesicles yielded higher rates of activity with rabbit liver CYP3A6 and CYP2B4 [11,12] and with human CYP3A4 [13] than micellar
Correspondence to D Schwarz, Charite´, Humboldt University of
Berlin, c/o Max-Delbrueck-Centrum, Robert Roessle Str 10, D-13125
Berlin, Germany.
Fax: + 49 30 9406 3329, Tel.: + 49 30 9406 3711,
E-mail: schwarz@mdc-berlin.de
Abbreviations: P450, human cytochrome P4501A1 (CYP1A1);
Lau 2 PtdCho, dilaurylglycerophosphocholine; Ole 2 PtdCho,
diol-eoylglycerophosphocholine; Ole 2 PtdPEtn,
dioleoylglycerophospho-ethanolamine; PtdCho, phosphatidylcholine; PtdEtn,
phosphatidylethanolamine; Ela 2 PtdEtn,
dielaidoylglycerophospho-ethanolamine; PtdSer, phosphatidylserine; PtdIns,
phosphatidylinos-itol; PA, phosphatidic acid; B[a]P, benzo[a]pyrene; DE2, diolepoxide 2
(r-7,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P); DE1,
diolepoxide 1
(r-7,t-8-dihydroxy-c-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P); (+/–)-7,8-diol, (+/–)-trans-7,8-dihydroxy-7,8-dihydro-B[a]P].
*Present address: Institute of Bioorganic Chemistry, Academy
of Sciences of Belarus, Minsk, Belarus.
(Received 21 December 2001, revised 14 February 2002, accepted 20
February 2002)
Trang 2Lau2PtdCho systems There is some evidence that the
membrane charge is an important determinant for
P450-dependent activity [14–16] Furthermore, the presence of
specific lipids of the nonbilayer class was found to be essential
for optimal activity of microsomal and mitochondrial P450s
For instance, rabbit liver CYP2B4 requires
phosphatidy-lethanolamine (PtdEtn) [12], and bovine adrenal CYP11A1
requires cardiolipin, PtdEtn or branched
phosphatidylcho-line (PtdCho) [17–19] for optimal activity as well as for
membrane reconstitution Reconstitution of purified human
CYP1A1 using phospholipid vesicles with CYP1A1 and
P450-reductase incorporated into the membrane has not
been reported to our knowledge There are no data available
which demonstrate how lipids influence the stereo- and/or
regioselectivity of a P450-catalyzed reaction We investigated
whether human CYP1A1 could be reconstituted into vesicle
membranes effectively enough to study the effects of
mem-brane properties and/or distinct phospholipids on CYP1A1
activity We characterized the impact of negatively charged
and nonbilayer lipids on CYP1A1-dependent stereoselective
epoxidation by using both (optically pure) enantiomeric
promutagens
(+)-7S,8S-dihydroxy-7,8-dihydrobenzo[a]py-rene ((+)-7,8-diol) and
(–)-7R,8R-dihydroxy-7,8-dihydro-benzo[a]pyrene ((–)-7,8-diol), as substrates
E X P E R I M E N T A L P R O C E D U R E S
Materials
Ole2PtdCho, Ole2PtdEtn, Ela2PtdEtn, and PtdSer were
bought from Avanti Polar Lipids (Alabaster, AL, USA)
The mixture PtdCho/PtdEtn/PA (2 : 1 : 0.06, w/w/w) and
PtdIns were bought from Lipid Products (Redhill, Surrey,
UK), and the [14C]Ole2PtdCho was from Amersham
Pharmacia Biotech (Freiburg, Germany)
For the preparation of (+)-7,8-diol and (–)-7,8-diol,
racemic 7,8-diol was synthesized [20] and
chromatographi-cally separated into the enantiomers using a chiral stationary
phase [21] Structure, assignment of absolute configuration
and optical purity of the two enantiomers were confirmed by
UV, CD and measurements of the rotation UV spectra were
recorded with a Shimadzu (Japan) UV 2401 PC, CD spectra
were recorded with a Jasco J-720, and specific rotations with
a Perkin-Elmer 241-MC automatic polarimeter The specific
rotations [a20D in acetone were)395 ° and +417 ° for
(–)-7,8-diol and (+)-(–)-7,8-diol, respectively These are nearly
identical with the data reported for these compounds when
optically pure [22] To prove the absolute configuration CD
spectra were recorded for both enantiomers As expected, the
CD curves of the enantiomeric pair were almost mirror
images of each other (not shown)
The tetraols
r-7,t-8,t-9,c-10-tetrahydroxy-7,8,9,10-tetra-hydro-B[a]P (RTTC), r-7,t-8,t-9,t-10-tetrahydroxy-7,8,9,
10-tetrahydro-B[a]P (RTTT),
r-7,t-8,c-9,t-10-tetrahydroxy-7,8,9,10-tetrahydro-B[a]P (RTCT),
r-7,t-8,c-9,c-10-tetra-hydroxy-7,8,9,10-tetrahydro-B[a]P (RTCC) were obtained
from NCI Chemical Carcinogen Repository, Midwest
Research Institute, Kansas City, MI, USA
Preparation of enzymes and lipid vesicles
Human CYP1A1 was heterologously expressed as
C-terminal 6xHis-fusion protein in Spodoptera frugiperda
insect cells using baculovirus [23] Purification was performed with nickel-chelate chromatography,
essential-ly as described for human CYP2D6 [24] but with the modification that a mixture of emulgen 913 (2%) and Na-cholate (0.2%) was used for solubilization CYP1A1 was electrophoretically homogeneous and had a specific P450 content of 11 nmolÆmg)1 protein Human
NAD-PH cytochrome P450 reductase was purified from Spodoptera frugiperda insect cells as described previously [25]
Phospholipid vesicles were prepared essentially as described by Ingelman–Sundberg et al [13] by cholate gel filtration 5 mg of lipid or lipid mixture were dried under nitrogen and resuspended in 1.25 mL of 50 mM
Tris buffer, pH 7.5, containing 100 mM NaCl and 2% sodium cholate 1 nmol P450 and 0.5 nmol human NADPH cytochrome P450 reductase were added to
250 lL of the suspension and incubated for 60 min at
4°C (final cholate concentration: 1%) Cholate was removed by Sephadex G-50 gel filtration The vesicular fractions were collected as void volume eluting from the column and were immediately used for the assays The vesicular fractions were characterized in terms of P450, reductase, and phospholipid content The amount of CYP1A1 was determined by CO difference spectrometry using an extinction coefficient of 91 mM )1Æcm)1[26] Rates
of NADPH-cytochrome c reduction by reductase were measured using an extinction coefficient of 21 mM )1Æcm)1 [27] Lipid was quantitated by measuring the 14 C-radio-activity of the fractions by liquid scintillation counting using [14C]-Ole2PtdCho as marker The final vesicular preparations are characterized as follows: 1 lM CYP1A1, molar reductase/P450 stoichiometry of 0.9, and molar lipid/protein ratio of 1200 The intra- and interday degree
of variation in relative P450, reductase, and lipid content did not exceed 10% in any of the vesicular preparations Finally, standardized amounts of CYP1A1 were used for the enzymatic assays, usually 5 or 10 pmol (vesicular) CYP1A1
Enzyme assays The epoxidation assays were performed as described for the racemic 7,8-diol [7] with the following modifications: incubations contained 50 mMTris/HCl (pH 7.5), 100 mM
NaCl with either (+)- or (–)-7,8-diol, and vesicles with
5 pmol CYP1A1 (for (–)-7,8-diol) and 10 pmol CYP1A1 (for (+)-7,8-diol) in a final volume of 0.5 mL Extraction and HPLC separation of the products were performed essentially as described earlier [7] The rates of DE1 and DE2 formation were estimated from the accumulation of their hydrolysis products, the tetraols, as follows: RTCC + RTCT represent DE1 formation and RTTC + RTTT represent DE2 formation [28]
Kinetic constants were determined by nonlinear analysis
of Michaelis–Menten kinetics using the computer program
ENZFITTER(by J R Leatherbarrow, Elsevier-Biosoft) The data presented are the means and standard deviations of three separate experiments Statistical significance of results between lipid systems was analysed using one-way ANOVA software (GraphPad software, San Diego,
CA, USA)
Trang 3R E S U L T S
Effect of charged lipids on epoxidation rates
We evaluated the kinetics of CYP1A1-dependent
epoxida-tion of 7,8-diol in vesicles prepared from neutral Ole2
Ptd-Cho and mixtures of Ole2PtdCho and anionic lipids
(PtdSer, PtdIns, PA) PtdSer is the most frequently
occur-ring negatively charged microsomal lipid, whereas PtdIns
and PA are minor constituents The mixture PtdCho/
PtdEtn/PA with the two main lipid components of the
microsomal membrane, PtdCho and PtdEtn, in a 2 : 1
ratio, and with a slightly negative charge introduced by PA,
roughly imitates the properties of the microsomal
mem-brane [29] This lipid mixture has already been used in
studies of P450–lipid interaction (e.g [12]) Figure 1A
represents the effects of the phospholipids on the conversion
of (+)- and (–)-7,8-diol to DE1 and DE2
Table 1 summarizes the results of kinetic analysis
Typical Lineweaver–Burk plots of the kinetic rates of
(–)-7,8-diol epoxidation to DE2 by human CYP1A1 are
shown for selected lipid vesicle systems in Fig 2 The results
demonstrate a clear dependence on the phospholipid charge
and the type of lipid used For instance, Vmaxfor (–)-7,8-diol
oxidation to DE2 was about 33 pmolÆmin)1Æpmol)1 in
vesicles containing PtdSer This rate is very high for a
CYP1A1-catalyzed reaction; it is more than 13 times higher
than the rate obtained with Ole2PtdCho, which is a neutral
lipid The incorporation of PtdIns and PtdCho/PtdEtn/PA
led to a sevenfold and twofold to threefold activation,
respectively Statistical analysis of the data showed that the
differences must be considered very significant (P < 0.05)
apart from the lipid system PtdCho/PtdEtn/PA (P > 0.05)
The activation of the CYP1A1-catalyzed oxidation of the
(+)-7,8-diol was less pronounced
Effects of nonbilayer lipids on the metabolic rates
of epoxidation
We analyzed the effects of a typical member of the
nonbilayer class of phospholipids, namely PtdEtn [30]
Together with PtdCho, it belongs to the main components
of the liver microsomal membrane [29] Comparison of
CYP1A1 activity in vesicles consisting of Ole2PtdCho/
Ole2PtdEtn and Ole2PtdCho/Ela2PtdEtn revealed striking
evidence for the importance of the hexagonal phase forming
tendency of the membrane The two di-18:1-acyl-PtdEtn,
Ole2PtdEtn and Ela2PtdEtn, differ only in their
conforma-tion of the double bond, which is cis in Ole2PtdEtn and
trans in Ela2PtdEtn, whereas both headgroup and chain
length are identical This difference results in a much higher
bilayer-hexagonal phase transition temperature in Ela2
Pt-dEtn (about 65°C) than in Ole2PtdEtn (about 10°C) and
has been used to investigate the impact of the hexagonal
phase forming tendency by Yang and Hwang [31] Data in
Fig 1A and in Table 1 show that the activity of CYP1A1 is
significantly enhanced (P < 0.001, considered extremely
significant), e.g about eightfold for the formation of the
main product DE2 from (–)-7,8-diol in vesicles containing
Ole2PtdEtn, whereas Ela2PtdEtn has almost no activation
potential (P > 0.05, no significant activation) For the
(+)-7,8-diol metabolism the stimulation by the incorporation of
OlePtdEtn was less but also pronounced (fourfold to
fivefold) and statistically significant (P < 0.001) These results clearly demonstrate a strong correlation between the activation of the CYP1A1-catalyzed epoxidation reaction of 7,8-diols and the enhanced nonbilayer phase propensity in membranes containing PtdEtn
Effect of lipids on the stereoselectivity of epoxidation The stereoselectivity of the epoxidation reaction can be demonstrated by the ratio of the formation rates of the two diol-epoxides, DE2 and DE1 These differ only in the conformation of the 9,10-epoxy-group, anti in DE2 and syn
in DE1 The respective data in Fig 1B and Table 1 show
Fig 1 Effect of phospholipids on the total epoxidation (A) and the ratio
of diolepoxide-2 to diolepoxide-1 formation (B) of (–)- and (+)-7,8-diol
by human CYP1A1 in reconstituted vesicles Vesicles consisting of pure Ole 2 PtdCho, or of a lipid mixture of Ole 2 PtdCho and the particular lipid in a weight ratio of 2 : 1 were prepared as described under Experimental procedures PtdCho/PtdEtn/PA is a lipid mixture of egg PtdCho, egg PtdEtn, and phosphatidic acid in a weight ratio of
2 : 1 : 0.06 Rates represent V max values determined by kinetic analysis based on data from three separate experiments Ratios were calculated from these V max values.
Trang 4that charged lipids strongly affect the stereoselectivity of
epoxidation The presence of the anionic lipid PtdSer
increased the formation of DE2 twice as much as that of
DE1 Even the relatively small portion of the negatively
charged PA in the PtdCho/PtdEtn/PA membrane led to a
pronounced enhancement in the product ratio DE2/DE1
In both cases statistical analysis proved the enhancement in
the ratio DE2/DE1 to be very significant (P < 0.05)
Actually, the incorporation of PtdIns into Ole2PtdCho
membrane also leaded to an increase but cannot be
considered statistically significant higher compared to
Ole2PtdCho (P > 0.05) By contrast, we found almost no
influence of either Ole2PtdEtn or Ela2PtdEtn The ratios
determined for both systems are not significant different
from that of Ole2PtdCho (P > 0.05) Thus, the results
demonstrate that the charge of the membrane has an
important influence on the ratio DE2/DE1, whereas the
nonbilayer phase propensity has hardly any effect on the
stereoselective formation of the DEs
D I S C U S S I O N
The results presented show that purified human CYP1A1
can be efficiently reconstituted into phospholipid vesicle
membranes together with P450-reductase The main results
can be summarized as follows: (a) the presence of negatively
charged lipids in the membrane stimulates diol epoxidation
by human CYP1A1 significantly, (b) negatively charged
lipids affect the stereoselectivity of epoxidation by favouring
the formation of DE2 to the detriment of DE1, and (c)
nonbilayer lipids also lead to strong activation probably by
increasing the effective substrate concentration in the
membrane
Thus, we found that in addition to the membrane charge,
the nonbilayer phase propensity of the membrane is an
important determinant for an effective reconstitution of
CYP1A1-dependent epoxidation activity The reason for
the requirement of such a specific and complex membrane structure including charged and nonbilayer lipids for the maximum activity of CYP1A1 is not known It seems that the native function of human CYP1A1 requires a microso-mal membrane containing negatively charged as well as nonbilayer lipids
Kinetic analysis showed, that both kinetic parameters,
Vmaxand Km,were altered, as is clearly demonstrated by the Lineweaver–Burk plots of the rates of (–)-7,8-diol epoxida-tion for the main mutagenic product DE2 (Fig 2) With regard to dependence on membrane charge, this analysis supports the general concept that the negative charge of the membrane not only improves the electron transfer and interaction between reductase and P450 but also affects the active site conformation of P450 This last conclusion is confirmed by the observation that charged lipids also strongly affect the stereoselectivity of the epoxidation reaction, whereas nonbilayer lipids do not Considering all the data, the observed increase in the formation of DE2 is caused, at least partially, by a lipid-induced conformational change This change mediates more favourable active site spatial coordinates responsible for the binding and produc-tive orientation between heme-bound oxygen and the acceptor 9,10-double bond of the (–)-7,8-diol
CYP3A4 is the only other human liver P450, with which similar high metabolic rates could be reached in a vesicle reconstitution system that includes charged lipids [13] However, only Vmaxwas increased whereas Kmremained unchanged This suggests that CYP3A4 activity is stimu-lated by a more effective interaction between P450 and reductase Recently published data for rabbit liver CYP1A2 belonging to the same P450 subfamily also showed that anionic phospholipids (PA, PtdIns, PtdSer) present in the membrane leaded to enhanced enzymatic activity More-over, evidence by structural studies was presented for considerable changes of the overall conformation of CYP1A2 coinciding with the increase of activity [32,33]
Table 1 Lipid dependence of 7,8-diol epoxidation by human CYP1A1: kinetic analysis for (–)-7,8-diol and (+)-7,8-diol The rates of DE1 and DE2 formation were calculated from the accumulation of their hydrolysis products, the tetraols, as follows: RTCC + RTCT represent DE1 formation and RTTC + RTTT represent DE2 formation (28) Data are means ± SD of V max and K m values, determined by fitting experimental data from three separate experiments to Michaelis–Menten kinetics as described under Experimental Procedures DE2/DE1 data represent the ratio of the respective V max values For preparation of lipid vesicles see legend to Fig 1 V max is in pmolÆmin)1Æpmol P450)1, K m is in l M
Lipid mixture
(w/w)
(pmolÆmin)1Æpmol P450)1)
(–)-7,8-diol
(+)-7,8-diol
Trang 5Note, the enzymatic activity of CYP1A2 was measured in
the presence of cumene hydroperoxide in place of reductase
and NADPH proving the conclusion that the observed
effects are indeed related to lipid-induced conformational
changes of CYP1A2 So far we discussed only the effect of
lipids on P450 However, it is also possible that lipids induce
structural changes in the reductase which improve its
interaction with P450 and thereby enhance Vmax In a
previous study, an increase in Vmaxof CYP2B1-dependent
O-dealkylation activity was ascribed to a PtdSer-induced
conformational change of reductase [34]
We also observed a strong stimulation of CYP1A1
epoxidation activity by typical nonbilayer lipids such as
PtdEtn, but the mechanism of activation might be different
from that discussed above for charged lipids The striking
difference in the activation capacities of Ole2PtdEtn and
Ela2PtdEtn suggests that the hexagonal phase propensity is
probably the characteristic of the membrane which best
explains these changes in activity Nonbilayer lipids do not
cause significant alterations in the metabolite profile, i.e
they do not alter the stereoselectivity of epoxidation
Obviously, there is no alteration of the active site
confor-mation of P450 But the striking parallelism of the curves in
the Lineweaver–Burk plots with different lipid components
(i.e for Ole2PtdCho, Ole2PtdCho/Ela2PtdEtn, and Ole2
Ptd-Cho/Ole2PtdEtn) indicates that Kmand Vmaxwere changed
by the same factor The most probable explanation is an
increase in the effective substrate concentration which is
probably due to the redistribution of the substrate between
the aqueous and the membrane phase and is brought about
by a change in the nonbilayer phase propensity of the
membrane
In accordance with this hypothesis, it is now generally assumed that microsomal P450s apart from their N-terminal transmembrane domain have additional attach-ment region(s) associating the protein partially buried into the membrane [35] This would favour lipophilic substrates
by placing the opening of the substrate access channel into the lipid bilayer Thus the pool of substrate molecules accessible for P450 would consist of the substrate molecules
in the membrane phase
The activity of several membrane enzymes, among which are protein kinase C, mitochondrial reductases and ATPases, mitochondrial cytochrome P450 (CYP11A1), and others [18,36,37], is increased by nonbilayer lipids There is some evidence that the latter influence the conformation of membrane-bound proteins by changing membrane properties, e.g introduce curvature stress, that might mediate an optimal conformation of the protein [37–39] Here, we propose an additional mechanism of action for nonbilayer lipids The nonbilayer phase propen-sity of the membrane might lead to an enhancement of the effective substrate concentration in the membrane by redistributing the substrate between the aqueous and membrane phase However, other reasons for an improved substrate accessibility can not be excluded
We showed for the first time that the stimulation of catalytic CYP1A1 activity and its stereoselectivity depend
on the type of lipid present in the membrane Anionic lipids, particularly PtdSer, favour the formation of the ultimate mutagen DE2 to the detriment of that of the far less carcinogenic DE1 It has been reported that lipids affect several catalytic activities of CYP3A4 [40,41] Thus, it would be interesting to know whether other enzymatic activities of human CYP1A1 also depend on the membrane structure and/or lipids
A C K N O W L E D G E M E N T S
This work was supported by grants of the German Research Foundation (DFG) to I R and D S (RO 1287/2-3), and to P K (436 WER 17/8/01), and the Volkswagen-Stiftung to D S (I/75 468).
We are grateful to Dr F J Gonzalez for providing CYP1A1 cDNA and virus for reductase expression (National Cancer Institute, NIH, Bethesda, MD, USA) We thank Dr A Chernogolov for protein purification, Dr D Zirwer for CD measurements, A Sternke for her skilful cell culturing, and Dr H Honeck and R Zummach (all from Max Delbrueck Centrum for Molecular Medicine, Berlin-Buch, Germany) for assistance with HPLC.
R E F E R E N C E S
1 Thakker, D.R., Yagi, H., Lu, A.Y.H., Levin, W., Conney, A.H & Jerina, D.M (1976) Metabolism of benzo[a]pyrene: conversion of (+/–)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene to highly mutagenic 7,8-diol-9,10-epoxides Proc Natl Acad Sci USA 73, 3381–3385.
2 Kapitulnik, J., Wislocki, P.G., Levin, W., Yagi, H., Jerina, D.M & Conney, A.H (1978) Tumorigenicity studies with diol-epoxides of benzo(a)pyrene which indicate that (+/-)-trans-7beta,8alpha-di-hydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene
is an ultimate carcinogen in newborn mice Cancer Res 38, 354– 358.
3 Conney, A.H (1982) Induction of microsomal enzymes by foreign chemicals and cancerogenesis by polycyclic aromatic hydrocar-bons Cancer Res 42, 4875–4917.
Fig 2 Lineweaver–Burk plots of the kinetic analysis of (–)-7,8-diol
epoxidation to diolepoxide-2 by human CYP1A1 for selected lipid vesicle
membranes Vesicle membranes were reconstituted from CYP1A1,
NADPH-P450 reductase, and either Ole 2 PtdCho, Ole 2 PtdCho/
Ole 2 PtdEtn (2 : 1, w/w), Ole 2 PtdCho/Ela 2 PtdEtn (2 : 1, w/w), or
Ole 2 PtdCho/PtdSer (2 : 1, w/w) Note the parallelism of the curves for
Ole 2 PtdCho, Ole 2 PtdCho/Ole 2 PtdEtn, and Ole 2 PtdCho/Ela 2 PtdEtn.
Trang 64 Yang, S.K., McCourt, D.W., Roller, P.P & Gelboin, H.V (1976)
Enzymatic conversion of benzo[a]pyrene leading predominantly to
the diol-epoxide
dihydroxy-t-9,10-oxy-7,8,9,10-tetra-hydrobenzo[a]pyrene through a single enantiomer of
r-7,t-8-dihydroxy-7,8-dihydrobenzo[a]pyrene Proc Natl Acad Sci USA
73, 2594–2598.
5 Gautier, J.C., Lecoeur, S., Cosme, J., Perret, A., Urban, P.,
Beaune, P & Pompon, D (1996) Contribution of human
cyto-chrome P450 to benzo[a]pyrene and
benzo[a]pyrene-7,8-dihydro-diol metabolism, as predicted from heterologous expression in
yeast Pharmacogenetics 6, 489–499.
6 Kim, J.H., Stansbury, K.H., Walker, N.J., Trush, M.A.,
Strick-land, P.T & Sutter, T.R (1998) Metabolism of benzo[a]pyrene
and benzo[a]pyrene-7,8-diol by human cytochrome P450 1B1.
Carcinogenesis 19, 1847–1853.
7 Schwarz, D., Kisselev, P., Cascorbi, I., Schunck, W.-H & Roots,
I (2001) Differential metabolism of benzo[a]pyrene and
ben-zo[a]pyrene-7,8-dihydrodiol by human CYP1A1 variants
Carci-nogenesis 22, 453–459.
8 Guo, Z., Gillam, E.M.J., Ohmori, S., Tukey, R.H & Guengerich,
F.P (1994) Expression of modified human cytochrome P450 1A1
in Escherichia coli: effects of 5¢ substitution, stabilization,
purifi-cation, spectral characterization, and catalytic properties Arch.
Biochem Biophys 312, 436–446.
9 Buters, J.T.M., Shou, M., Hardwick, J.P., Korzekwa, K.R &
Gonzalez, F.J (1995) cDNA- directed expression of human
cytochrome P450 CYP1A1 using baculovirus Drug Metab.
Dispos 23, 696–701.
10 Zhang, Z.Y., Fasco, M.J., Huang, L., Guengerich, F.P &
Kaminsky, L.S (1996) Characterization of purified human
recombinant cytochrome P4501A1-Ile462 and –Val462:
assess-ment of a role for the rare allele in carcinogenesis Cancer Res 56,
3926–3933.
11 Ingelman-Sundberg, M & Glaumann, H (1977) Reconstitution
of the liver microsomal hydroxylase system into liposomes FEBS
Lett 78, 72–76.
12 Bo¨sterling, B., Stier, A., Hildebrandt, A.G., Dawson, J.H &
Trudell, J.R (1979) Reconstitution of cytochrome P-450 and
cytochrome P-450 reductase into
phosphatidylcholine-phosphati-dylethanolamine bilayers: characterization of structure and
metabolic activity Mol Pharmacol 16, 332–342.
13 Ingelman-Sundberg, M., Hagbjo¨rk, A.-L., Ueng, Y.-F.,
Yama-zaki, H & Guengerich, F.P (1996) High rates of substrate
hydroxylation by human cytochrome P450 3A4 in reconstituted
membranous vesicles: Influence of membrane charge Biochem.
Biophys Res Commun 221, 318–322.
14 Ingelman-Sundberg, M., Haaparanta, T & Rydstro¨m, J.
(1981) Membrane charge as effector of cytochrome P-450LM2
catalyzed reactions in reconstituted liposomes Biochemistry 20,
4100–4106.
15 Blanck, J., Smettan, G., Ristau, O., Ingelman-Sundberg, M &
Ruckpaul, K (1984) Mechanism of rate control of the
NADPH-dependent reduction of cytochrome P-450 by lipids in
recon-stituted phospholipid vesicles Eur J Biochem 144, 509–513.
16 Imaoka, S., Imai, Y., Shimada, T & Funae, Y (1992) Role of
phospholipids in reconstituted cytochrome P4503A form and
mechanism of their activation of catalytic activity Biochemistry
31, 6063–6069.
17 Lambeth, J.D (1991) Cytochrome P-450SCC: Cardiolipin as an
effector of activity of a mitochondrial cytochrome P-450 J Biol.
Chem 256, 4757–4762.
18 Schwarz, D., Kisselev, P., Pfeil, W., Pisch, S., Bornscheuer, U &
Schmid, R.D (1997) Evidence that nonbilayer phase propensity of
the membrane is important for the side chain cleavage activity of
cytochrome P450SCC (CYP11A1) Biochemistry 36, 14262–
14270.
19 Kisselev, P., Wessel, R., Pisch, S., Bornscheuer, R.D., Schmid,
D & Schwarz, D (1998) Branched phosphatidylcholines stimulate activity of cytochrome P450SCC (CYP11A1) in phospholipid vesicles by enhancing cholesterol binding, membrane incorporation, and protein exchange J Biol Chem.
273, 1380–1386.
20 Platt, K.L & Oesch, F (1983) Efficient synthesis of non-K-region trans-dihydro diols of polycyclic aromatic hydrocarbons from o-quinons and catechols J Org Chem 48, 265–268.
21 Funk, M., Frank, H., Oesch, F & Platt, K.L (1994) Development
of chiral stationary phases for the enantiomeric resolution of dihydrodiols of polycyclic aromatic hydrocarbons by p-donar– acceptor interactions J Chromatogr A 659, 57–68.
22 Yagi, H., Akagi, H., Thakker, D.R., Mah, H.D., Koreeda, M & Jerina, D.M (1977) Absolute stereochemistry of the highly mutagenic 7,8-diol 9,10-epoxides derived from the potent carci-nogen trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene J Am Chem Soc 99, 2358–2359.
23 Chernogolov, A., Schwarz, D., Schunck, W.H & Roots, I (2000) Purification and characterization of baculovirus-expressed CYP1A1.1, CYP1A1.2, and CYP1A1.4 13th International Sym-posium on Microsomes and Drug Oxidation 5, 111.
24 Modi, S., Paine, M.J., Sutcliffe, M.J., Lian, L.-Y., Primrose, W.U., Wolf, C.R & Roberts, G.C.K (1996) A model for human cyto-chrome P450 2D6 based on homology modeling and NMR stu-dies of substrate binding Biochemistry 35, 4540–4550.
25 Tamura, S., Korzekwa, K.R., Kimura, S., Gelboin, H.V & Gonzalez, F.J (1992) Baculovirus- mediated expression and functional characterization of human NADPH-P450 oxidor-eductase Arch Biochem Biophys 293, 219–223.
26 Omura, T & Sato, R (1964) The carbon monoxide-binding pig-ment of liver microsomes J Biol Chem 239, 2370–2378.
27 Yasukochi, Y & Masters, B.S (1976) Some properties of detergent-solubilized NADPH-cytochrome c (cytochrome P450) reductase purified by biospecific affinity chromatography J Biol Chem 251, 5337–5344.
28 Shou, M., Korzekwa, K.R., Crespi, C.L., Gonzalez, F.J & Gel-boin, H.V (1994) The role of 12 cDNA-expressed human, rodent, and rabbit cytochromes P450 in the metabolism of benzo[a]pyrene and benzo[a]pyrene-trans-7,8-dihydrodiol Mol Carcinogenesis 10, 159–168.
29 Depierre, J.W & Dallner, G (1975) Structural aspects of the membrane of the endoplasmic reticulum Biochim Biophys Acta
415, 411–472.
30 Cullis, P.R & De Kruijff, B (1978) The polymorphic phase behaviour of phosphatidylethanolamines of natural and synthetic origin Biochim Biophys Acta 513, 31–42.
31 Yang, F.Y & Hwang, F (1996) Effect of non-bilayer lipids on the activity of membrane enzymes Chem Phys Lipids 81, 197–202.
32 Ahn, T., Guengerich, F.P & Yun, C.-H (1998) Membrane insertion of cytochrome P450 1A2 promoted by anionic phos-pholipids Biochemistry 37, 12860–12866.
33 Yun, C.-H., Song, M & Kim, H (1997) Conformational change
of cytochrome P450 1A2 induced by phospholipid and detergents.
J Biol Chem 272, 19725–19730.
34 Balvers, W.G., Boersma, M.G., Vervoort, J., Ouwehand, A & Rietjens, I.M (1993) A specific interaction between NADPH-cytochrome reductase and phosphatidylserine and phosphatidyli-nositol Eur J Biochem 218, 1021–1029.
35 Williams, P.A., Cosme, J., Sridhar, V., Johnson, E.F & McRee, D.E (2000) Microsomal cytochrome P450 2C5: comparison to microbial P450s and unique features J Inorg Biochem 81, 183– 190.
36 Stubbs, C.D & Slater, S.J (1996) The effects of non-lamellar forming lipids on membrane protein–lipid interactions Chem Phys Lipids 81, 185–195.
Trang 737 Epand, R.M (1996) The properties and biological roles of
non-lamellar forming lipids Chem Phys Lipids 81, 101–264.
38 Gruner, S.M., Cullis, P.R., Hope, M.J & Tilcock, C.P.S (1985)
Lipid polymorphism: the molecular basis of nonbilayer phases.
Annu Rev Biophys Chem 14, 211–238.
39 Hui, S.W (1987) Non-bilayer-forming lipids: Why are they
nec-essary in biomembranes? Comments Mol Cell Biophys 4/5, 233–
248.
40 Eliasson, E., Mkrtchian, S., Halpert, J & Ingelman-Sundberg, M (1994) Substrate-regulated, cAMP-dependent phosphorylation, denaturation, and degradation of glucocorticoid-inducible rat liver cytochrome P450 3A1 J Biol Chem 269, 18378–18383.
41 Shet, M.S., Fisher, C.W., Holmans, P.L & Estabrook, R.W (1993) Human cytochrome P450 3A4: enzymatic properties of a purified recombinant fusion protein containing NADPH-P450 reductase Proc Natl Acad Sci USA 90, 11748–11752.