From the Department of Animal Biology and the Mari
Lowe Center for Comparative Oncology, School of Veterinary Medicine,
University of Pennsylvania, and the § Wistar Institute,
Philadelphia, Pennsylvania 19104-6047
Received for publication, January 16, 2001, and in revised form, April 26, 2001
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ABSTRACT |
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Hepatic mitochondria contain an inducible
cytochrome P450, referred to as P450 MT5, which cross-reacts with
antibodies to microsomal cytochrome P450 2E1. In the present study, we
purified, partially sequenced, and determined enzymatic properties of
the rat liver mitochondrial form. The mitochondrial cytochrome P450 2E1
was purified from pyrazole-induced rat livers using a combination of
hydrophobic and ion-exchange chromatography. Mass spectrometry analysis
of tryptic fragments of the purified protein further ascertained its
identity. N-terminal sequencing of the purified protein showed that its
N terminus is identical to that of the microsomal cytochrome P450 2E1.
In reconstitution experiments, the mitochondrial cytochrome P450 2E1
displayed the same catalytic activity as the microsomal counterpart,
although the activity of the mitochondrial enzyme was supported
exclusively by adrenodoxin and adrenodoxin reductase. Mass spectrometry
analysis of tryptic fragments and also immunoblot analysis of proteins
with anti-serine phosphate antibody demonstrated that the mitochondrial
cytochrome P450 2E1 is phosphorylated at a higher level compared with
the microsomal counterpart. A different conformational state of the mitochondrial targeted cytochrome P450 2E1 (P450 MT5) is likely to be
responsible for its observed preference for adrenodoxin and adrenodoxin
reductase electron transfer proteins.
Cytochromes P450
(P450s)1 belong to a
superfamily of heme proteins, which catalyze the oxidation of exogenous
as well as endogenous compounds. P450 proteins are located in different
compartments of the cell. In addition to their localization in the
endoplasmic reticulum, also referred as microsomes, a number of
different forms have been detected in mitochondria (1, 2), Golgi
apparatus (3), and plasma membrane (4, 5). Targeting of P450 proteins to ER and mitochondria require different types of signals. Microsomal P450 is co-translationally inserted in the ER membrane through a
non-cleavable N-terminal hydrophobic signal sequence (6). Mitochondrial
P450 forms, such as P450scc (P450 11A1), P450 11 Recently we demonstrated that, in rat liver and brain,
P450 2E1 has been implicated to have important roles in human health,
as its activity is affected by pathophysiological conditions such as
diabetes, starvation, and obesity (12). Furthermore, it is readily
induced by acute and chronic alcohol ingestion, and the enzyme is known
to actively metabolize alcohol and acetaldehyde. Additionally, the
enzyme efficiently catalyzes the metabolism of a wide spectrum of low
molecular weight hydrophobic compounds, including carbon tetrachloride,
chloroform, and vinylidene chloride in addition to chemical additives
of toxicological and carcinogenic significance (13). Thus, P450 2E1
induction may underlie the increased risks of exposure to such
industrial and environmental chemicals. It is well established that
P450 2E1 causes oxidative stress through the production of reactive
oxygen species in vivo (14) and in vitro (15).
However, the role of mitochondrial targeted P450 2E1 in oxidative
stress remains unknown.
P450 2E1, alternately designated as P450 MT5, has been shown to be
present in rat liver mitochondria as a membrane extrinsic protein with
an apparent molecular mass similar to or slightly smaller (1-2 kDa)
than the 52-kDa size reported for the microsomal form (2). In contrast,
a soluble 40-kDa, putative N-terminal truncated form of P450 2E1 was
recently shown to be localized in mitochondria of cells overexpressing
P450 2E1, lacking the N-terminal 29 amino acid residues (16). A protein
of similar size cross-reacting with antibody to P450 2E1 was also
detected in mitochondria of uninduced rat livers (16). In the present study, we purified P450 2E1 from hepatic mitochondria of
pyrazole-treated rats and characterized its molecular properties and
enzyme activity. Our results show that mitochondrially targeted P450
2E1 has an intact N terminus and that it is phosphorylated at higher
level as compared with the microsomal counterpart. Enzyme
reconstitution experiments show that the activity of the mitochondrial
enzyme is supported preferentially by the Adx+Adr electron transport system, and very poorly by the microsomal CPR system, suggesting a
possible conformational shift, which alters its ability to bind to
different electron transfer proteins.
Treatment of Animals and Isolation of Subcellular
Fractions--
Unless otherwise indicated, chemicals were purchased
from Sigma. Adult male Harlan Sprague-Dawley rats (150-200 g,
Harlan Sprague-Dawley Inc., Indianapolis, IN) were maintained as
described previously (1). Pyrazole, dissolved in saline, was
administered intraperitoneally for 4 days (300 mg/kg body weight
daily). Control rats received saline only. Overnight fasted rats were
sacrificed by CO2 asphyxiation, 16 h after the last
injection. Livers were perfused and washed with ice-cold saline.
Mitochondria were isolated as described (17). Briefly, livers were
homogenized in 10 volumes of sucrose mannitol buffer (70 mM
sucrose, 210 mM mannitol, 2 mM EDTA, 2 mM HEPES, pH 7.4) containing 500 mg/liter of bovine serum
albumin. Resultant mitochondria were washed twice with the homogenization buffer lacking bovine serum albumin, resuspended at a
concentration of 33 mg/ml, and incubated at 4 °C for 2 min with
digitonin (Wako Chemicals, Richmond, VA) at a final concentration of 75 µg/mg of protein. Mitoplasts were then washed three times with the
homogenization buffer and resuspended in 50 mM potassium phosphate (pH 7.3), containing 20% glycerol, 0.1 mM
dithiothreitol, 0.1 mM EDTA, and 0.1 mM
phenylmethylsulfonyl fluoride (buffer A), supplemented with phosphatase
inhibitors (0.5 mM sodium orthovanadate, 0.05 mM sodium molybdate, and 2 mM sodium fluoride).
Microsomes were isolated from the post-mitochondrial supernatant by
centrifugation at 100,000 × g for 1 h at
4 °C.
P450 contents of the mitoplast and microsome membranes were measured in
100 mM potassium phosphate, pH 7.4, containing 20% glycerol, 0.5% sodium cholate, and 0.4% of Triton N-101 by the CO-binding difference spectrum of dithionite-reduced aliquots of each
fraction (18).
Protein content was estimated by the method of Lowry et al.
(19).
Purification of Mitochondrial and Microsomal P450
2E1--
Mitoplasts from pyrazole-treated rat livers were disrupted by
pulse sonication on ice (at setting 5 of a Branson sonifier) for 3 min
and diluted in buffer A at a final concentration of 5 mg of protein/ml.
P450 proteins were solubilized by stirring the suspension with 0.8%
sodium cholate for 1 h at 4 °C, and the soluble proteins were
fractionated with 20% PEG as described previously (20). The
P450-enriched 0-20% PEG fraction was applied to a
The microsomal P450 2E1 was purified from the microsomal membranes of
pyrazole-treated rat livers according to the same procedure, except
that 0.6% sodium cholate was used for solubilization of microsomal
P450 proteins.
Immunoprecipitation--
Liver mitochondria and microsomes (200 µg of each) from control and pyrazole-treated rats were solubilized
in 500 µl of phosphate-buffered saline (10 mM potassium
phosphate, pH 7.4, and 150 mM NaCl) containing 0.2% sodium
cholate and phosphatase inhibitors (0.5 mM sodium orthovanadate, 0.05 mM sodium molybdate, and 2 mM sodium fluoride). The 15,000 × g
supernatant fractions were incubated with anti-P450 2E1 antibody (5 µg of IgG) (Oxford Biomedical Research, Oxford, MI) overnight at
4 °C. 30 µl of protein A-agarose (Life Technologies, Inc.) was
added to each sample and shaken for 3 h at room temperature. Beads
were pelleted by centrifugation and washed four times with phosphate-buffered saline containing 0.05% Tween 20 and phosphatase inhibitors. Immunoprecipitated proteins were eluted from protein A-agarose beads by heating at 95 °C for 5 min in 30 µl of 2×
Laemmli buffer (23) without added 2-mercaptoethanol.
SDS-Polyacrylamide Gel Electrophoresis and Immunoblot
Analysis--
Proteins were resolved by electrophoresis on a 12%
SDS-polyacrylamide gel (23) and visualized by Coomassie Blue staining or transferred to nitrocellulose membrane for immunoblot analysis as
described (24). Polyclonal antibodies against P4502E1, mtTFA, and CPR,
and monoclonal antibody against Ser-phosphate (Sigma) were used. Blots
were developed by chemiluminescence with the Pierce Super Signal Ultra
kit. Imaging and quantitation were performed using a Fluor-S imaging
system (Bio-Rad).
N-terminal Amino Acid Sequencing--
Purified proteins were
subjected to electrophoresis on a 12% SDS-polyacrylamide gel and
transferred to Sequiblot polyvinylidene difluoride membrane
(Bio-Rad). Protein bands were visualized by reversible staining with
xylene cyanol, and the amino acid sequencing was performed by
phenylthiohydantoin-derivatization procedure in a Beckman LH 2600 gas
phase sequencer.
Peptide Analysis by MALDI-TOF Spectrometry--
Purified P450
2E1 proteins were subjected to electrophoresis on a 12%
SDS-polyacrylamide gel and visualized by staining with Coomassie Blue.
Purified P450 2E1 bands were excised, and gel slices were washed three
times with 50% acetonitrile in 25 mM NH4HCO3 (pH 8), soaked in 100% acetonitrile,
and dehydrated in a vacuum centrifuge. Gel fragments were rehydrated
with a minimal volume of trypsin (10 µg/ml) solution and incubated
for 24 h at 37 °C. The resultant peptides were extracted,
concentrated, and resuspended in 10 µl of 0.1% trifluoroacetic
acid/100% acetonitrile (50:50). A fraction of each sample (0.5 µl)
was applied to the MALDI target and mixed with 0.5 µl of
Enzyme Activities--
PNPH activity was measured according to
Reinke and Moyer (25). Reactions with mitoplasts and microsomes were
carried out in 500-µl final volumes in a buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2,
200 µM p-nitrophenol, and 200 µg of proteins as enzyme source. The reaction mixture was pre-incubated with or
without added antibodies or inhibitors at 4 °C for 20 min. Following
a 3-min incubation at 37 °C in a shaking water bath, the reaction
was initiated by adding 1 mM NADPH and the incubation was
continued for 20 min. These incubation conditions cause mitochondrial swelling making the membrane permeable to antibodies. The reaction was
terminated by adding 0.6 N perchloric acid. Insolubles were removed by centrifuging at 10,000 × g for 3 min. Equal
volume of 10 N NaOH was added to the supernatant, and the
reaction product, p-nitrocatechol, was measured at 546 nm.
Reconstitution with purified mitochondrial or microsomal P450 2E1 was
carried out essentially as described for intact organelles, except that
the reactions were carried out in presence of
dilauryl-phosphatidylcholine vesicles as described (26). The final
reaction volume was 200 µl and contained 50 pmol of P450 2E1, 0.2 nmol of Adx and 0.02 nmol of Adr, or 0.1 nmol of CPR and 200 µM p-nitrophenol. Preincubation with
inhibitors and antibodies was carried out as described for intact organelles.
N-Demethylation of DMNA was assayed as described in detail
in a recent study (11). Briefly, reactions were carried out in 50 mM Tris-HCl (pH 7.4), containing 20 mM
MgCl2. The reaction volumes, amounts of proteins and
substrates used were same as those for PNPH activity. After 3 min of
preincubation at 37 °C, the reactions were initiated by the addition
of 1 mM NADPH and continued for 20 min. Reactions were
terminated by adding 0.5 volume of ice-cold trichloroacetic acid (10%
w/v). The insolubles were pelleted by centrifugation at 10,000 × g, and an aliquot of the supernatant was mixed with an equal
volume of Nash reagent. The mixture was further incubated at 55 °C
for 15 min. The final product, formaldehyde, was measured according to
the method of Nash (27). The background values were lowered by using
fresh Nash reagent and reducing the temperature for color development to 55 °C from the 90 °C used in the original method. Typically, all significant N-demethylation activities reported here
correspond to at least >3-fold of background reading.
Circular Dichroism Spectroscopy--
CD spectra of mitochondrial
and microsomal P450 2E1 were measured on a Jasco J720 instrument at
room temperature as described previously (9). The proteins were
concentrated to 0.2 mg/ml and dialyzed against buffer C. Background
spectra were obtained using buffer C alone and digitally subtracted
from the test spectra. Mean residue ellipticity ([ Synthetic Peptides--
Peptides were synthesized using an ABI
model 431A synthesizer and purified by high performance liquid
chromatography on a reverse phase column. Peptides used in this study
are listed in Table I.
Extent of Induction of Mitochondrial P450 2E1 by Pyrazole--
The
rat liver mitochondrial preparations were routinely checked for purity
by marker enzyme assays as described previously (11, 26, 28). The
digitonin-stripped mitochondria used in this study contained
approximately 95% of mitochondrial-specific marker enzyme activity,
cytochrome c oxidase, but less than 1% microsome-specific,
rotenone-insensitive NADPH cytochrome c reductase activity.
The rate of induction of mitochondrial P450 2E1 by pyrazole treatment
was studied by immunoblot analysis of mitoplast preparations from
control untreated and pyrazole-treated rat livers. As shown in Fig.
1, pyrazole treatment induced the level
of antibody-reactive 52-kDa putative P450 2E1 protein by 3-fold,
whereas the level of the protein in the microsomes was induced by
2-fold. Based on the recovery of 7.5 mg of mitochondrial proteins and 9 mg of microsomal proteins/g of liver, the mitochondrial content in the pyrazole-treated liver corresponds to 30-40% of the total tissue pool
of P450 2E1. In order to ascertain the purity of membrane isolates, the
immunoblot was co-developed with antibodies to the mitochondria-specific protein mtTFA, and the microsome-specific marker
protein CPR. Results show that only the 28-kDa mtTFA is detected in the
mitochondrial fractions with no detectable 78-kDa CPR protein,
indicating the purity of mitochondrial preparation. The microsomal
fractions, on the other hand, contained only the 78-kDa CPR protein
with no detectable 28-kDa mitochondria-specific mtTFA protein.
The effects of pyrazole treatment on P450 2E1-dependent
PNPH and DMNA N-demethylation (25, 29) were measured to gain
further insight on the extent of induction (Fig.
2, A and B,
respectively). Results show that, in both mitochondrial and microsomal
fractions, pyrazole treatment resulted in 2-3 fold increased
N-demethylation of DMNA and PNPH activities. Furthermore,
both the activities were inhibited 83-97% by P450-specific inhibitor
SKF-525A and CO (latter results not shown). Thus, the extent of
increase in enzyme activity with both substrates is consistent with the
increased antibody reactive protein in pyrazole-treated fractions.
Results also show that the activities with both pyrazole-treated and
untreated livers were effectively inhibited by Adx antibody, but
minimally with antibody to CPR, further demonstrating the purity of
mitochondrial preparations. As expected, the activity of the microsomal
fraction was specifically inhibited by antibody to CPR.
Purification of Mitochondrial P450 2E1--
In order to determine
if the mitochondrial targeted P450 2E1 is N-terminally truncated or
not, we purified the protein from both mitochondria and microsomes from
pyrazole-treated rat livers using the conventional purification
procedures (20). As shown in Table II,
the cholate extract of mitoplasts from pyrazole-treated livers
contained 0.6 nmol of P450/mg of protein. The 20% PEG precipitate contained 83% of the input P450. The protein fraction eluted with 0.06% Emulgen 911 from the
Fig. 3A shows the
electrophoretic pattern of protein fractions at various stages of
purification of mitochondrial P450 2E1. Proteins in the size range of
50 and 55 kDa appeared to be enriched in the PEG fraction. One protein
of approximately 52 kDa was further enriched in the
The electrophoretic patterns of protein fractions at various stages of
microsomal P450 2E1 purification are presented in Fig. 3B.
As shown above for the purification of the mitochondrial protein, fraction 1 from DEAE-Sephacel column, showing a P450 content of approximately 10 nmol/mg protein (data not shown) exhibited the highest
purity (75-80%) and was used for further characterization and
comparison with the mitochondrial P450 2E1.
The immunoblots of mitochondrial and microsomal proteins at different
stages of purification are presented at the bottom of Fig. 3
(A and B), respectively. The results show that
DEAE fractions representing different levels of purity indeed
cross-react with monoclonal antibody to P450 2E1.
As seen in Fig. 3C, N-terminal amino acid sequencing of the
rat liver mitochondrial P450 2E1 yielded a sequence identical to that
of the rat liver microsomal P450 E1.
Phosphorylation of Mitochondrial P450 2E1--
In a recent study
we showed that PKA-mediated Ser128 phosphorylation of P450
2B1 is a critical factor, which modulates the rate of protein targeting
to ER and mitochondrial membranes (9). We also showed that P450 2B1
purified from phenobarbital-induced mitochondria (termed as P450 MT4)
was phosphorylated at a higher level compared with the microsomal
counterpart (9). Since members of family 2 P450 are known to be
phosphorylated, it was decided to see if mitochondrial targeted P450
2E1 (designated as P450 MT5) is also phosphorylated using two different
approaches. In the first approach, proteins solubilized from
mitochondria and microsomes of livers from control and pyrazole-treated
rats or P450 2E1 purified from these organelles were immunoprecipitated with P450 2E1 antibody, and the immunoprecipitated proteins were probed
with antibody to Ser-phosphate by immunoblot analysis. As shown in Fig.
4, only P450 2E1 immunoprecipitated from
mitochondria or purified from mitochondrial membrane cross-reacted with
anti-Ser-phosphate antibody. The similarly treated microsomal protein
fractions showed very low to negligible cross-reactivity. It is also
seen that the antibody cross-reacted with a single protein band
with apparent molecular mass of 52 kDa with purified mitochondrial P450
2E1, whereas the antibody cross-reacted with two closely migrating bands of 52 and 51 kDa with immunoprecipitates from total mitochondrial proteins. Although the nature of the faster migrating (~51 kDa) band
remains unclear, it may represent a contaminant or degradation product
of P450 2E1 generated during immunoprecipitation.
In the second approach, MALDI-TOF analysis was carried out to evaluate
the phosphorylation status of purified microsomal and mitochondrial
P450 2E1. P450 2E1 sequence (Swiss-Prot no. P05182) was loaded into the
GPMAW software (PerSeptive Biosystems) for theoretical digestion
analysis. The PKA target site on P450 2E1 is part of the tryptic
fragment FS (129)LSILR. With Ser129-phosphorylated P450 2E1
(30), one expects a mass of 914.50 for this fragment and with
unphosphorylated protein, a mass of 835.50. Fig.
5A shows the spectra of the
digested peptide mass fingerprint of P450 purified from rat liver
mitochondria and microsomes. The data base search using MSFit
identified both of these proteins as P450 2E1 with 48-58% matched
masses. Chromatograms with both mitochondrial and microsomal P450 2E1
also showed some fragments (marked with arrows) that did not
match either with P450 2E1 or with any of the known proteins from the
data base. These fragments may therefore be derived from unknown
contaminating proteins. It is also seen that the mass spectra with both
mitochondrial and microsomal P450 2E1 showed the presence of peaks with
molecular mass of 835.50, suggesting the presence of unphosphorylated
species. The mass spectrum with mitochondrial P4502E1 also showed a
small, yet detectable peak with a mass of 914.55. Based on the relative heights of peaks with masses 835.50 and 914.55, approximately 20-25%
of the mitochondrial P450 2E1 is phosphorylated. An enlargement of the
area of the chromatogram corresponding to masses 914 to 918, presented
in Fig. 5B, further demonstrates the presence of the peptide
with molecular mass of 914.55 in the mitochondrial P450 2E1, but
negligible amount in the microsomal protein digest. Thus, the results
of MALDI-TOF analysis together with the immunoblots in Fig. 4 show
that, under steady state conditions, the mitochondrial imported protein
is phosphorylated at higher level than the microsomal P450 2E1.
Fig. 5C shows the alignment of P4502E1-specific peptide
fragments from the mass spectra in Fig. 5A. It is seen that
the peptide fragments recovered from the purified mitochondrial protein
digest accounted for approximately 45% of the total 450 2E1 sequence encompassing amino acid sequence 64-486 of the protein. In the case of
microsomal P4502E1, the recovery of peptides was less efficient and
corresponded to approximately 36% of the protein. The N-terminal
as well as middle regions of the protein poorly represented in the mass
spectra in Fig. 5A yield short peptides of less that 8 amino
acid residues, which are not efficiently recovered by the present
extraction procedure. The differential recovery of peptides as
indicated in Fig. 5C may also be responsible for the
difference in the mass spectrometry patterns between the mitochondrial
and microsomal P450 2E1 (see Fig. 5A). Nevertheless, results
of MALDI-TOF analysis, along with the N-terminal sequence data and also
the 52-kDa apparent mass of the purified protein estimated by
SDS-polyacrylamide gel analysis in Fig. 3A, provide evidence
that the mitochondrial P450 2E1 contains an unprocessed intact N terminus.
Reconstitution of Enzyme Activity with Purified P450 2E1--
The
PNPH and N-demethylase of DMNA activities of the purified
mitochondrial P450 2E1 were reconstituted with CPR or Adx+Adr systems,
and compared with those of enzyme purified from the microsomal fraction. Mitochondrial P450 2E1 in the presence of Adx+Adr yielded the
same range of activity as the microsomal P450 2E1 in the presence of
CPR: 1.23 and 1.61 nmol/min/nmol of P450, respectively, for PNPH and
15.4 and 13.5 nmol/min/nmol of P450, respectively, for N-demethylation of DMNA (Figs.
6 and 7).
Notably, the mitochondrial enzyme yielded very low to negligible
activity with the CPR system, whereas the microsomal enzyme was nearly
completely inactive with the Adx+Adr system. The activities with both
enzymes were inhibited by P450 inhibitors SKF 525-A and CO and also by
the P450 2E1-specific inhibitor, 4-methylpyrazole. Furthermore, the
activities were highly dependent on the addition of electron transport
proteins and also NADPH (results not shown). Finally, a polyclonal
antibody specific for P4502E1 inhibited the PNPH activity of both the
Adx+Adr-supported mitochondrial and CPR-supported microsomal P450 2E1
enzymes by over 90%. These results further confirm that the PNPH
activity is catalyzed by mitochondrial and microsomal P450 2E1, rather than any possible contaminating proteins.
We have previously shown that Adx interacts with P450 MT2
(N-terminal truncated P4501A1), through its conserved C-terminal acidic
domain 2, which spans amino acid sequence 70-85 of human Adx (see
Table I) (28). This is the same domain that was shown to interact with
the constitutively expressed mitochondrial P450 forms, such as P450scc,
P450c27, etc. (31, 32). As shown for P450 MT2, Adx+Adr-supported PNPH
activity of the mitochondrial P450 2E1 was inhibited by nearly 100% by
a 30 M excess of Adx-C peptide (Fig. 7A).
However, the Mut Adx-C peptide in which negatively charged residues are
substituted by neutral residues (see Table I) failed to inhibit
significantly the Adx+Adr-supported PNPH activity. On the other hand,
both peptides had no effect on CPR-supported PNPH activity of the
microsomal P450 2E1, showing their specificity. These results suggest
that a functionally productive interaction of Adx with mitochondrial
P450 2E1 occurs through the same C-terminal acidic domain as that
involved in interaction with other mitochondrial P450 forms and P450 MT2.
Recently, we mapped the region of P450 MT2 interfacing with both Adx
and CPR to amino acid sequence 266-279 (putative helix G, sequence of
P450 MT2 peptide in Table I) of the protein, which contains 5 positively charged residues (33). We also showed that
Lys267 and Lys271 were critical for binding to
Adx, while Lys268 and Arg275 were important for
binding to CPR (33). Results of reconstitution in Fig. 7 (A
and B) show that P450 MT2 peptide also inhibited both the
Adx+Adr-supported PNPH activity of mitochondrial P450 2E1 and
CPR-supported PNPH activity of microsomal P450 2E1 by over 95%.
Although not shown, P450 MT2 peptide also inhibited both the Adx+Adr-
and CPR-supported DMNA N-demethylase activities of
mitochondrial and microsomal enzymes to >90%. These results suggest
that the same structural domain of P450 2E1, similar to that of P450
MT2, may be involved in interaction with both Adx and CPR. An analysis
of P450 2E1 sequence indeed suggests the occurrence of a domain similar
to the P450 MT2 peptide at sequence 231-244 (see Fig. 7C)
(34), which exhibits positional identities with Lys267,
Lys268, and Lys271 of the MT2 sequence. The 4th
positively charged residue (Lys243) of the putative G helix
of P450 2E1 is positionally removed by two residues as compared with
Arg275 of MT2 sequence.
Difference in the Secondary Structure of Microsomal and
Mitochondrial P450 2E1--
To understand reasons for the vast
differences in the affinity of P450 2E1 from the two cellular
compartments for Adx and CPR, we compared the purified proteins for
secondary structure contents by CD spectroscopy. Despite identical
primary sequence, the mitochondrial and microsomal enzymes exhibited a
marked difference in the CD spectra (Fig.
8). It is seen that the microsomal P450 2E1 preparation exhibited negative bands at 208 and 223 nm at equal
intensity, characteristic for proteins in the In this study, we have used a combination of protein purification,
enzyme reconstitution, and protein biochemistry for characterizing the
molecular and enzymatic properties of rat liver mitochondrial P450 2E1.
Our results demonstrate that N-terminally intact P450 2E1 is targeted
to mitochondria. The immunoblot analysis of P450 from crude membrane
fractions, as well as purified proteins, suggest that the mitochondrial
P450 2E1 is phosphorylated at a significantly higher level as compared
with the microsomal counterpart. These results are further supported by
MALDI-TOF analysis of gel-purified proteins (Fig. 5). The latter
results show the occurrence of a protease fingerprint product of 914.55 mass, representing the Ser129 phosphorylated fragment only
with the mitochondrial purified enzyme but not the microsomal enzyme.
These results suggest that similar to that shown for P4502B1 (P450
MT4), intact Ser129 phosphorylated P450 2E1 (P450 MT5) is
targeted to mitochondria. Despite an extensive analysis, we failed to
detect the putative 40-kDa N-terminal cleaved product (16) either in
intact mitoplasts (Fig. 1) or as part of purified P450 2E1 (Fig.
3A). Recent
results2 on the in
vivo protein targeting by cell transfection and in vitro mitochondrial import showed that the full length P450 2E1 with an intact N terminus is targeted to mitochondria. Furthermore, as
suggested previously, the mitochondrial imported P450 2E1 is a membrane
extrinsic protein based on its solubility in alkaline Na2CO3 (2, 37).
Recent studies from our laboratory demonstrated that mitochondrial
targeted P450 MT2 (+33/1A1) and P450 MT4 (phosphorylated P450 2B1)
interact in a functionally productive manner with mitochondrial specific electron donor proteins Adx+Adr (9, 28, 33), while retaining
their ability to interact with the microsomal electron donor protein,
CPR. Since the microsome-associated P450 1A1 and 2B1 showed preference
for CPR as an electron donor, we hypothesized that N-terminal
truncation, in the case of mitochondrial imported P450 MT2, and
Ser128 phosphorylation in the case of mitochondrial P450
2B1, may induce conformational changes that allow interaction with
soluble electron donor proteins. In many respects, the mitochondrial
imported P450 2E1 (P450 MT5) described in this study resembles the
similarly located P450 2B1 with a subtle difference. Reconstitution
experiments show the exclusive requirement of mitochondrial imported
P450 2E1 for Adx+Adr. These results suggest a notable structural or conformational difference between P450 molecules associated with the
microsomal and mitochondrial membrane compartments. As reported before
for P450 2B1 (9), immunoblot analysis with anti-Ser-phosphate antibody shows higher level of phosphorylation with mitochondrial P450
2E1 as compared with the microsomal form. Preliminary results also show
that, similar to that reported for P450 2B1 (9), Ser129
phosphorylation is critical for mitochondrial targeting of protein, suggesting that it may serve to activate the cryptic mitochondrial targeting signal of P450 2E1 apoprotein. MALDI-TOF analysis, however, suggests that only approximately 20-25% of the mitochondrial P450 2E1
pool may be phosphorylated at Ser129. These latter results
suggest that mitochondrial P450 forms are subject to regulation by
phosphorylation and dephosphorylation. This possibility is consistent
with increasing evidence that other mitochondrial proteins, such as
those associated with electron transport complexes, are subject to
modulation by cAMP- dependent PKA (9, 38) and protein
phosphatases (39).
It is noteworthy that P450 2E1 proteins purified from the two membrane
compartments show markedly different A recent study using physical and genetic mapping of electron donor
protein binding sites on P450 MT2 and structural modeling of this P450
showed that both Adx and CPR bind to the putative G helix, but subtly
through different contact points. Notably, both Adx and CPR bound to
the same helix at 90° orientation to each other in a non-overlapping
fashion (33). In the present study, the MT2 peptide representing the
P450 site for binding to two different electron donor proteins
inhibited both the Adx supported and CPR supported enzyme activities.
These results suggest that a homologous P450 2E1 region is involved in
binding to these two evolutionarily divergent electron donor proteins.
Although not shown, a polar amino acid-rich, aqueous accessible helix
G, spanning sequence 231-244 and containing 4 positively charged amino
acids, is also conserved in P450 2E1 (see Fig. 7C). We
postulate that the conserved G-helix of P450 2E1 is involved in binding to the electron donor proteins, possibly involving Arg233,
Lys234, and Lys237 as the direct contact
points. In subtle variation from the P450 MT2 model, in
vitro reconstitution of purified enzymes suggests that the binding
to the two electron donor proteins, Adx and CPR, is exclusive of each
other. Currently, reasons for the observed preference of the microsomal
P450 2E1 for binding to CPR and the mitochondrial form for binding to
Adx remain unclear, although different conformations of the putative G
helix in enzymes from two different membrane sources is a likely possibility.
In summary, here we demonstrate that the P450 2E1 associated with the
mitochondrial membrane compartment is identical to the microsomal P450
2E1, except that the former is phosphorylated at a higher level. The
results of this study also support our previous observations with P450
MT2 and P450 MT4, and provide additional evidence for the occurrence of
multiple mechanisms for the activation of chimeric N-terminal signal
for the mitochondrial targeting of xenobiotic inducible P450 forms.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(P450 11B1), and
P450c27 (P450 27A1) contain cleavable N-terminal amphipathic
presequences that are important for the post-translational targeting of
the precursor protein to mitochondria (7).
-naphthoflavone-inducible P450 1A1 and phenobarbital-inducible P450 2B1, both known to be bona fide microsomal forms, are also
targeted to mitochondria (8-10). The mitochondrial targeted P450 forms were referred to as P450 MT2 and P450 MT4, respectively. These studies
described a new concept of chimeric N-terminal signal that was
responsible for targeting both of these proteins to two distinct
cytoplasmic organelles. In the case of P450 1A1, processing past the
4th or the 32nd amino acid residue by a cytosolic endoprotease resulted
in the activation of a cryptic mitochondrial targeting signal at amino
acid sequence 33-44, which directed the truncated proteins (P450 MT2)
to mitochondria (8, 10, 11). In the case of P450 2B1 (P450 MT4),
protein with an intact N-terminal end was targeted to mitochondria,
although PKA-dependent phosphorylation at
Ser128 was essential for mitochondrial targeting (9). We
postulated that a conformational shift induced by Ser128
phosphorylation helped expose a cryptic mitochondrial targeting signal
at amino acid sequence 21-36 of the protein. Thus, the two mechanisms
have a common theme in that both require the activation of chimeric
N-terminal signals for mitochondrial targeting (8, 9, 11), although
mechanisms of signal activation were different.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-octylamine-agarose column equilibrated with buffer A containing 0.6% sodium cholate, as described previously (20-22). The column was
then washed with buffer B (70 mM potassium phosphate, pH
7.3, containing 20% glycerol, 0.1 mM dithiothreitol, 0.1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride)
containing 0.6% sodium cholate, and the P450 proteins bound to the
column were eluted successively with buffer B containing 0.06 and 0.2%
Emulgen 911. Fractions containing P450, as determined by CO-binding
difference spectrum and immunoblot analysis, were pooled and dialyzed
overnight against two changes of 10 mM potassium phosphate
buffer, pH 7.8, containing 20% glycerol, 0.1 mM
dithiothreitol, 0.1 mM EDTA, 0.1 mM
phenylmethylsulfonyl fluoride, 0.2% sodium cholate, and 0.1% Emulgen
911 (buffer C). The dialyzed fraction was applied to a DEAE-Sephacel
column (Amersham Pharmacia Biotech) pre-equilibrated with buffer C (20,
22). The column was washed with five volumes of buffer C, and the bound P450 was eluted with a linear gradient from 0 to 0.5 M NaCl
in buffer C. Aliquots of fractions were analyzed by CO-binding
difference spectrum and subjected to SDS-polyacrylamide gel
electrophoresis. Fractions containing relatively pure P450 were pooled,
concentrated by ultrafiltration through Amicon filters, and dialyzed
against buffer A.
-cyano-4-hydroxycinnamic acid matrix onto the MALDI plate. The
calibration standard mixture 1 from Sequazyme peptide mass standard kit
(PE Biosystems, Foster city, CA) was used as the close external
calibration. Peptides were analyzed using a Voyager DE STR mass
spectrometer (PerSeptive Biosystems, Framingham, MA). Data base
searching using Protein Prospector UCSF was conducted to identify the
intact protein.
]MR)
is expressed in degrees cm2/dmol using a mean residue
weight of 110.
Synthetic peptides used for competition
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Purity of mitochondria and P450 induction by
pyrazole. Proteins (20 µg) from liver mitoplasts and microsomes
from control and pyrazole-treated rats were resolved by electrophoresis
on a 12% SDS-polyacrylamide gel. Proteins were transblotted to Nytran
membrane, and the blot was co-immunostained with rabbit polyclonal
antibodies against mtTFA and CPR (1:2000 each) and goat polyclonal
anti-P450 2E1 antibodies (1:3000). The blot was developed with
horseradish peroxidase conjugated secondary antibody (1:50,000) by
chemiluminescence as described under "Materials and Methods." The
bottom panel shows the relative band intensities
of the P450 protein (52 kDa) in comparison to the control mitochondrial
band intensity considered as 1.
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Fig. 2.
Effects of pyrazole treatment on the enzyme
activities of mitochondrial and microsomal fractions. Mitoplast
and microsome preparations from control and pyrazole-treated rats were
assayed for PNPH activity (A) and N-demethylation
of DMNA (B) as described under "Materials and Methods."
SKF 525-A (1 mM) was added 3 min before the initiation of
reaction by adding NADPH. Anti-Adx (5 µg of IgG) or anti-CPR (5 µg
of IgG) antibodies were pre-incubated with the enzyme as described
under "Materials and Methods" before initiating the reaction. The
mean values and standard deviations were calculated from three
different experiments.
-octylamine-agarose column showed a very
low P450 content of 0.3 nmol/mg of protein. On the other hand, the
fraction eluted with 0.2% Emulgen 911 contained approximately 45% of
input P450 with a specific activity of 4.8 nmol of P450/mg of protein
and was therefore used for further purification by DEAE-Sephacel column
chromatography. The eluates from DEAE-Sephacel column were collected as
three fractions. The first fraction contained only 5% of the input
P450, although it exhibited the highest specific activity of 10.2 nmol/mg protein. The second and third fractions showed a lower specific
activity of 4 and 7 nmol/mg protein, respectively (Table II). Although
not shown, the microsomal P450 2E1 exhibited a similar pattern of
purification and recovery.
Purification of rat liver mitochondrial P450 2E1
-octylamine-agarose step (Fig. 3A, lane
4). As indicated by different specific activities (Table
II), the three DEAE-Sephacel column fractions showed different
levels of purity. Proteins from fraction 1 resolved as a major
component of 52 kDa, and exhibited approximately 70-80% purity (Fig.
3A, lanes 5-7). Fractions 2 and 3 contained additional bands suggesting a lower level of purity (Fig.
3A, lanes 8 and 9,
respectively). The P450 from DEAE-Sephacel column fraction 1 was
therefore used for further characterization.
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Fig. 3.
Electrophoretic patterns of proteins at
different stages of purification. Figure shows mitochondrial P450
2E1 (A) and microsomal P450 2E1 (B). In
upper panel of A, protein fractions at
various steps of purification of mitochondrial P450 2E1 (as indicated
in Table II) were subjected to electrophoresis on a 12%
SDS-polyacrylamide gel and visualized by staining with Coomassie Blue.
Lane 1, molecular weight markers; lane 2, 25 µg
of cholate extract; lane 3, 50 µg of PEG fraction;
lane 4, 15 µg of -octylamine-agarose column fraction
(OAA); lanes 5-7, 0.5, 1, and 2 µg of
DEAE-Sephacel column fraction 1; lane 8, 2 µg of
DEAE-Sephacel column fraction 2; lane 8, 2 µg of
DEAE-Sephacel column fraction 3. In the upper
panel of B, microsomal protein fractions at
different steps of purification were processed as in A. Lane 1, molecular weight markers; lane 2, 20 µg
of microsomal protein; lane 3, 40 µg of cholate extract;
lane 4, 10 µg of
-octylamine-agarose column fraction
(OAA); lane 5, 1 µg of DEAE-Sephacel column
fraction 1; lane 6, 1 µg of DEAE-Sephacel column fraction
2; lane 7, 2 µg of DEAE-Sephacel column fraction 3. The
lower panels in A and B
represent duplicate gels subjected to immunoblot analysis using
anti-P450 2E1 antibody as described in Fig. 1. C shows the
N-terminal amino acid sequence of the mitochondrial P450 2E1, compared
with the reported sequence of microsomal P450 2E1. About 8 pmol of
purified mitochondrial P4502E1 (DEAE-Sephacel fraction 1 from Fig.
3A) were analyzed by N-terminal sequencing.
Numbers in parentheses show percentage of
recovery of residues at each cycle.
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Fig. 4.
Levels of phosphorylation of mitochondrial
and microsomal P450 2E1. Proteins solubilized from 200 µg
each of mitochondria (MT) and microsomes
(MIC) or proteins purified from these membranes (5 µg each) were subjected to immunoprecipitation with anti-P450 2E1
antibody. The immunoprecipitates were divided into two equal fractions,
and each fraction was subjected to electrophoresis on a 12%
SDS-polyacrylamide gel. One gel was subjected to immunoblot analysis
with anti P450 2E1 antibody (top panel), and the
companion gel was subjected to immunoblot analysis with mouse
monoclonal anti-phosphoserine antibody (1:1000) as
described under "Materials and Methods" and in Fig. 1.
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Fig. 5.
MALDI-TOF analysis of tryptic digests of
purified P450 2E1. Purified mitochondrial and microsomal proteins
(10 µg each) were subjected to electrophoresis on a 12%
SDS-polyacrylamide gel, stained with Coomassie Blue. Single stained
bands were excised from the gel and subjected to trypsin digestion, and
the peptides were extracted and subjected to MALDI-TOF analysis as
described under "Materials and Methods." A, mass
fingerprints of peptides from mitochondrial P4052E1 (upper
panel) and microsomal P450 2E1 (bottom
panel). Ion 835.5 in both panels represents the
non-phosphorylated peptide fragment from the Ser129 region.
Ion 914.5 in the panel for mitochondrial P450 2E1 represents
phosphorylated peptide fragment. B, selective enlargement of
chromatograms of mitochondrial (upper panel) and
microsomal (lower panel) proteins corresponding
to masses 914-918 to visualize ion 914.5, representing the
phosphorylated fragment. C, alignment of 2E1-specific
fragments identified in the mitochondrial and microsomal P4502E1
digests along the protein sequence. The N-terminal 10-amino acid
sequence region was identified by direct sequencing as shown in Fig.
3C.
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Fig. 6.
Reconstitution of DMNA
N-demethylation activity with purified P450 2E1.
The activities of purified mitochondrial (A) and purified
microsomal (B) P450 2E1 were reconstituted with Adx+Adr or
CPR as indicated. In some reactions, SKF 525-A (1 mM) and
4-methylpyrazole (1 mM) were added 3 min before the start
of the reaction as described under "Materials and Methods." CO was
gently bubbled through the enzyme suspension for 30 s. A duplicate
reaction mixture bubbled with N2 at the same rate was used
as control. Details of reconstitution and enzyme assays were as
described under "Materials and Methods." The mean values and
standard deviations were calculated from three different
experiments.
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Fig. 7.
Effects of competing peptides on the PNPH
activity of purified mitochondrial and microsomal P450 proteins.
Purified mitochondrial (A) and purified microsomal
(B) P450 2E1 were reconstituted with Adx+Adr or CPR electron
transfer proteins and the PNPH activity was assayed as described under
"Materials and Methods." Treatments with SKF 525-A (1 mM), 4-methylpyrazole (1 mM), and CO were as
described in Fig. 6. Competing peptides, Adx-C peptide, Mut Adx-C
peptide, and MT2 peptide (Table I) were added at 30 M
excess (1.5 nmol) and pre-incubated with purified P450 2E1 for 20 min
at 37 °C before adding the substrates and NADPH. Treatment with P450
2E1 antibody (5 µg IgG) was carried out as described in Fig. 2. The
mean values and standard deviations were calculated from three
different experiments. C, sequence region of P4502E1,
representing part of the putative G-helix, showing similarity to the
Adx and CPR binding domain of P450 MT2. Positively charged amino acids,
possibly involved in binding to Adx and CPR, are highlighted in
bold.
-helical conformation
(35). Although
-helices are characterized by an additional positive
band near 190 nm, this band was apparently masked by the noise from the
inorganic salts and other additives of the solvent (buffer C). The
solvent conditions may also explain the somewhat higher than 100%
-helix content, as determined by the manual algorithm established by
Greenfield and Fasman (36). The intensity ratio of the 208 nm/223 nm
bands was greatly reduced for the mitochondrial variant, and in fact
the CD spectrum resembled more of those representing
-pleated sheets
(36). A computer-based analysis of the CD spectra for the composition
of different secondary structural elements (35) indicated that the
microsomal P450 2E1contains approximately 36%
-helix, and
relatively low
-sheet structure, while the mitochondrial P450
contains 13%
-helix, and markedly increased
-sheet structure. As
reported for P4502B1 previously (9), the mitochondrial targeted P450
2E1 contains lower helical content, suggesting a less compact
structure.
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Fig. 8.
CD spectra of purified mitochondrial P450 2E1
(solid line) and microsomal P450 2E1
(dotted line). CD spectra were
measured on a Jasco J720 instrument at room temperature, using 0.2 mg/ml enzyme suspension as described under "Materials and
Methods."
DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helical contents (Fig. 8). In
our previous study with mitochondrial P450 2B1, we rationalized that
phosphorylation might be an important contributing factor in the
manifestation of structural differences, as revealed by CD spectral
studies (9), and binding to Adx (results not shown). Since the
phosphorylated species comprise only approximately 20-25% of the
mitochondrial P450 2E1 pool, additional possibilities are likely to
account for this difference. The mode of mitochondrial entry of the
apoprotein through the N-terminal end, and its first encounter with
mitochondrial import proteins, such as HSP70 (40) may some how direct
the protein to assemble in a membrane extrinsic orientation,
differently from the signal recognition particle-directed membrane targeting and membrane insertion processes in the ER compartment. It is also likely that the P450 protein is folded differently in the mitochondrial compartment because of possible differences in the protein folding machinery or chaperones. It is known
that a single amino acid substitution in the chaperone component of the
subtilisin E causes subtle changes in the folding of its protease
domain, suggesting that the same polypeptide can be folded in different
manner (41). Phosphorylation of Ser129 may be an important
contributing factor in this process. Additional experiments are needed
to understand the molecular and cellular processes leading to the
formation of P450 2E1 with subtly different structure or conformation.
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ACKNOWLEDGEMENTS |
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We thank members of the Avadhani laboratory for valuable suggestions and help with the preparation of the manuscript. We are also thankful to Dr. Melanie Lin of PerSeptive Biosystems for helping with the MALDI-TOF analysis.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant GM-34883.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Animal Biology and the Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce St., Philadelphia, PA 19104-6047.
Published, JBC Papers in Press, April 26, 2001, DOI 10.1074/jbc.M100363200
2 M. A. Robin, H. Anandatheerthavarada, and N. Avadhani, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: P450, cytochrome P450; ER, endoplasmic reticulum; PKA, protein kinase A; Adx, adrenodoxin; Adr, adrenodoxin reductase; CPR, NADPH cytochrome P450 reductase; PEG, polyethylene glycol (average mass 8000 Da); mtTFA, mitochondrial transcription factor A; MALDI, matrix-assisted laser desorption ionization; TOF, time of flight mass spectrometry; PNPH, p-nitrophenol hydroxylase; DMNA, dimethylnitrosamine; CD, circular dichroism; IgG, immunoglobulin G.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Niranjan, B. G.,
Wilson, N. M.,
Jekcoate, C. R.,
and Avadhani, N.
(1984)
J. Biol. Chem.
259,
12495-12501 |
2. | Anandatheerthavarada, H. K., Addya, S., Dwivedi, R. S., Biswas, G., Mullick, J., and Avadhani, N. G. (1997) Arch. Biochem. Biophys. 339, 136-150[CrossRef][Medline] [Order article via Infotrieve] |
3. | Neve, E. P. A., Eliasson, E., Prontazo, M. A., Albano, E., Marinari, U., and Ingelman-Sundberg, M. (1996) Arch. Biochem. Biophys. 333, 459-465[CrossRef][Medline] [Order article via Infotrieve] |
4. | Loeper, J., Descatoire, V., Maurice, M., Beaune, P., Feldmann, G., Larrey, D., and Pessayre, D. (1990) Hepatology 11, 850-858[Medline] [Order article via Infotrieve] |
5. |
Robin, M. A.,
Descatoire, V.,
Le. Roy, M.,
Berson, A.,
Lebreton, F. P.,
Maratrat, M.,
Ballet, F.,
Loeper, J.,
and Pessayre, D.
(2000)
J. Pharmacol. Exp. Ther.
294,
1063-1069 |
6. | Sakaguchi, M., Mihara, K., and Sato, R. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3361-3364[Abstract] |
7. | Omura, T., and Ito, A. (1991) Methods Enzymol. 206, 75-81[Medline] [Order article via Infotrieve] |
8. |
Addya, S.,
Anandatheerthavarada, H. K.,
Biswas, G.,
Bhagwat, S. V.,
Mullick, J.,
and Avadhani, N. G.
(1997)
J. Cell Biol.
139,
589-599 |
9. |
Anandatheerthavarada, H. K.,
Biswas, G.,
Mullick, J.,
Sepuri, N. B. V.,
Otvos, L.,
Pain, D.,
and Avadhani, N. G.
(1999)
EMBO J.
18,
5494-5504 |
10. |
Bhagwat, S. V.,
Biswas, G.,
Anandatheerthavarada, H. K.,
Addya, S.,
Pandak, W.,
and Avadhani, N. G.
(1999)
J. Biol. Chem.
274,
24014-24022 |
11. |
Boopathi, E.,
Anandatheerthavarada, H. K.,
Bhagwat, S. V.,
Biswas, G.,
Fang, J. K.,
and Avadhani, N. G.
(2000)
J. Biol. Chem.
275,
34415-34423 |
12. | Tanaka, E., Terada, M., and Misawa, S. (2000) J. Clin. Pharmacol. Ther. 25, 165-175[CrossRef][Medline] [Order article via Infotrieve] |
13. |
Koop, D. R.
(1992)
FASEB J.
6,
724-730 |
14. |
Sakarai, K.,
and Cederbaum, A. I.
(1998)
Mol. Pharmacol.
54,
1024-1035 |
15. | Fataccioli, V., Andraud, E., Gentil, M., French, S. W., and Rouach, H. (1999) Hepatology 29, 14-20[Medline] [Order article via Infotrieve] |
16. | Neve, E. P. A., and Ingelman-Sundberg, M. (1999) FEBS Lett. 460, 309-314[CrossRef][Medline] [Order article via Infotrieve] |
17. | Bhat, N. K., Niranjan, B. G., and Avadhani, N. G. (1982) Biochemistry 21, 2452-2460[Medline] [Order article via Infotrieve] |
18. | Matsubara, T., Koike, M., Touchi, A., Tochino, Y., and Sugeno, K. (1976) Anal. Biochem. 76, 596-603 |
19. |
Lowry, O. H.,
Rosebrough, N. J.,
Farr, A. L.,
and Randall, R. J.
(1951)
J. Biol. Chem.
193,
265-275 |
20. |
Raza, H.,
and Avadhani, N. G.
(1988)
J. Biol. Chem.
263,
9533-9541 |
21. | Guenguerich, F. P., Dannan, G. A., Wright, S. T., Martin, M. V., and Kaminsky, L. S. (1982) Biochemistry 21, 6019-6030[Medline] [Order article via Infotrieve] |
22. | Shayiq, R. M., Addya, S., and Avadhani, N. G. (1991) Methods Enzymol. 206, 587-594[Medline] [Order article via Infotrieve] |
23. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
24. | Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354[Abstract] |
25. | Reinke, L. A., and Moyer, M. J. (1985) Drug. Metab. Dispos. 13, 548-552[Abstract] |
26. | Addya, S., Mullick, J., Fang, J. K., and Avadhani, N. G. (1994) Arch. Biochem. Biophys. 310, 82-88[CrossRef][Medline] [Order article via Infotrieve] |
27. | Nash, T. (1953) Biochem. J. 55, 416-421 |
28. | Anandatheerthavarada, H. K., Addya, S., Mullick, J., and Avadhani, N. G. (1998) Biochemistry 37, 1150-1160[CrossRef][Medline] [Order article via Infotrieve] |
29. | Tu, Y. Y., and Yang, C. S. (1983) Cancer Res. 43, 623-629[Abstract] |
30. | Koch, J. A., and Waxman, D. J. (1991) Methods Enzymol. 206, 305-315[Medline] [Order article via Infotrieve] |
31. |
Coghlan, V. M.,
and Vickery, L. E.
(1991)
J. Biol. Chem.
266,
18606-18612 |
32. | Mittal, S., Zhu, Y., and Vickery, L. E. (1988) Arch. Biochem. Biophys. 264, 383-391[Medline] [Order article via Infotrieve] |
33. |
Anandatheerthavarada, H. K.,
Amuthan, G.,
Biswas, G.,
Robin, M. A.,
Murali, R.,
Waterman, M. R.,
and Avadhani, N. G.
(2001)
EMBO J.
20,
2394-2403 |
34. |
Nelson, D. R.,
and Strobel, H. W.
(1988)
J. Biol. Chem.
263,
6038-6050 |
35. | Johnson, W. C. (1988) Annu. Rev. Biophys. Biophys. Chem. 17, 145-166[CrossRef][Medline] [Order article via Infotrieve] |
36. | Greenfield, N., and Fasman, G. D. (1969) Biochemistry 8, 4108-4116[Medline] [Order article via Infotrieve] |
37. | Von Wachenfeldt, C., and Johnson, E. (1995) in Cytochrome P450: Structure, Mechanisms and Biochemistry (Ortiz de Montellano, P. R., ed) , pp. 183-224, Plenum Publishing Corp., New York |
38. | Francis, S. H., and Corbi, J. D. (1994) Annu. Rev. Physiol. 56, 237-272[CrossRef][Medline] [Order article via Infotrieve] |
39. |
Ruvulo, P. P.,
Deng, X.,
Ito, T.,
Carr, B. K.,
and May, W. S.
(1999)
J. Biol. Chem.
274,
20296-20300 |
40. | Glick, B. S. (1995) Cell 80, 11-14[Medline] [Order article via Infotrieve] |
41. | Shinde, U. P., Liu, J. J., and Inouye, M. (1997) Nature 389, 520-522[CrossRef][Medline] [Order article via Infotrieve] |