Mutagenesis Advance Access originally published online on December 8, 2006
Mutagenesis 2007 22(1):75-81; doi:10.1093/mutage/gel054
© The Author 2006. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org
The stimulatory role of human cytochrome b5 in the bioactivation activities of human CYP1A2, 2A6 and 2E1: a new cell expression system to study cytochrome P450-mediated biotransformation (a corrigendum report on Duarte et al. (2005) Mutagenesis 20, 93–100)
Maria Paula Duarte1,2,
Bernardo Brito Palma1,
Andrei A. Gilep3,
António Laires1,2,
José Santos Oliveira2,
Sergey A. Usanov3,
José Rueff1 and
Michel Kranendonk1,*
1 Department of Genetics, Faculty of Medical Sciences, Universidade Nova de Lisboa Rua da Junqueira 96, 1349-008 Lisboa, Portugal
2 Faculty of Science and Technology, Universidade Nova de Lisboa Quinta da Torre, 2829-516 Caparica, Portugal
3 Institute of Bioorganic Chemistry, National Academy of Sciences Minsk, Belarus
This corrigendum report describes the study of the comparison of human cytochrome b5 (b5) with rat b5 when coupled with human cytochrome P450 CYP1A2, 2A6 or 2E1. Results indicate a role of the N-terminal part of b5 in the coupling with CYP. Indeed, the plasmid pLCM-b5-RED used in our former study on b5 [Duarte et al. (2005) Mutagenesis, 20(2), 193–100] erroneously contained rat b5. Plasmid pLCM-b5-RED was corrected with human b5 and subsequently all experimental work was repeated as was described for the rat b5 plasmid. Although absolute values of contents and activities were lower, all key-findings as found for rat b5 could be confirmed using human b5. The physiological relevant co-expression of the members of the cytochrome P450 complex, CYP, NADPH-cytochrome P450 oxidoreductase (RED) and human b5 could be demonstrated in the different BTC strains, as was found before. The stimulatory effect of human b5 on the activity of CYP1A2, CYP2A6 and CYP2E1 was in general similar, when compared with rat b5, though less quantitatively pronounced. This was both the case when using membrane preparations as well as by the bioactivation of procarcinogens using the bacterial mutagenicity assay. Human b5 stimulated the bioactivation of all compounds as described for rat b5, except for CYP1A2 mediated bioactivation of 2-aminoanthracene (2AA), which was not stimulated by human b5. All other main findings of the effect of rat b5 were confirmed with human b5, i.e. for CYP2A6: N-nitrosodiethylamine (NNdEA): 
14-fold increase (23-fold with rat b5) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK): 3-fold (9-fold with rat b5); for CYP2E1: NNdEA: 1.5-fold increase (3-fold with rat b5); NNK: no mutagenicity with or without human b5. Both CYP2A6 and CYP2E1 demonstrated total dependence on the presence of human b5 for N-nitrosodi-n-propylamine (NNdPA) mutagenicity, as was shown before with rat b5.
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Introduction
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Subsequent experimental work, as follow-up of our publication
(Duarte et al., 2005) (1
) demonstrated that plasmid pLCM-b
5-RED
contained unexpected discrepancies. The first 15 bp as well
as the last 19 bp of the cytochrome
b5 (
b5) cDNA in pLCM-b
5-RED
aligned with the sequence of human cytochrome
b5, as annotated
by NCBI BC015182
[GenBank]
. The intermediate sequence of 370 bp aligned
completely with the rat
(Rattus norvegicus)
b5 cDNA (NCBI NM022245)
(
Figure 1). The resulting translation of the
b5 open-reading
frame in pLCM-b
5-RED resulted in a protein, which aligned totally
with the
b5 protein sequence of rat (
Figure 2). Secondly, the
DNA-linker was found to be different, instead of the expected
linker 5'-
GGGGATCCTCTAGAGTCGACCAAGGAGAGAATTCTA-3', the sequence
5'-
AAGCCGAATTCTA-3' was present (
Figure 1). Although rat and
human
b5 proteins are highly homologues, specific differences
are present (
Figure 2) which may be the cause of different effects
when coupled with human CYPs. The following report describes
the correction of these discrepancies in plasmid pLCM-b5-RED
as well as the exact repetition of all experimental work as
was described before (1
). This allowed the comparison of the
effect of human and rat
b5 on the activity of CYP1A2, 2A6 and
2E1.
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Fig. 1. DNA sequence alignments (Clustal W-1.82) of the expected human b5 cDNA in plasmid pLCM-b5-RED and the sequence result of the b5 region of the plasmid. (Asterisk: homologous bases; boxed sequences: I: 3'-sequence of pTAC promotor; II: human b5 sequence; III: rat b5 sequence; IV linker DNA; V: 5'-sequence of RED: over-lined sequence: EcoRI site.)
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Fig. 2. Protein sequence alignments (MACAW-2.0.5) of cytochrome b5. (A) human b5 (NCBI BC015182) with rat b5 (NCBI NM022245); (B) human b5 with sequence present in pLCM-rb5-RED; (C): rat b5 with sequence present in pLCM-rb5-RED. Amino acid differences between human and rat b5 shaded gray (structural similar residues) and black (structural different residues). The different b5 protein-domains are indicated as arrows based on data from Clarke et al. (8).
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Materials and methods
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All experimental work was carried out exactly as described before
(1
), except for the reconstruction of plasmid pLCM-b
5-RED, which
is described in detail in the Results section. The plasmid containing
the rat b
5 sequence from our former study (1
) will from hereon
be designated pLCM-rb
5-RED and the new human b
5 plasmid pLCM-hb
5-RED.
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Results
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Construction of plasmid pLCM-hb5-RED and the BTC-hb5-CYP strains
The following procedure was followed to construct the plasmid
pLCM-hb
5-RED, containing full-length cDNA of
Homo sapiens cytochrome
b5 (NCBI gene ID no. 1528). A cDNA (clone no. MSH1010-58144)
was obtained from Open Biosystems (Huntsville, AL, USA), containing
the clone sequence as annotated by NCBI BC015182
[GenBank]
. The cDNA was
PCR amplified with the forward primer 5'-
GATGAATTCATATGGCAGAGCAGTCGG-3'
and the reverse primer 5'-
CTAGAATTCTCTCTTCGCTGACTTCTGAGG-3'
introducing two flanking EcoRI sites. The PCR product was digested
with the restriction endonuclease EcoRI and subsequently cloned
in plasmid pUC18, which was EcoRI linearized. This pUC18_b
5 plasmid was extensively sequenced using universal M13 forward
and reverse primers, 5'-
TGTAAAACGACGGCCAGT–3' and 5'-
CAGGAAACAGCTATGACC–3',
respectively. The full-length cDNA of human
b5 could be confirmed
as well as the linker DNA sequence (5'-
ACACCTCCTCAGAAGTCAGCGAAGAGAGAATTC-3'),
following the TGA stop-codon of the
b5 sequence. The cDNA plus
linker was subsequently cloned in the unique EcoRI site of plasmid
pLCMhOR. The correct orientation of the EcoRI fragment was determined
by the diagnostic restriction analysis using the endonucleases
BamHI and NdeI. The new plasmid, pLCM-hb
5-RED was subsequently
extensively sequenced confirming both the human cytochrome
b5 and NADPH-cytochrome P450 oxidoreductase. The plasmid was subsequently
transfected to the mutagencity tester strains PD301, containing
the human cytochrome P450 expression vectors pCWh1A2, pCWh2A6
or pCWh2E1, thus creating the correct strains BTC-hb
5-1A2, BTC-hb
5-2A6
and BTC-hb
5-2E1, respectively. The former BTC-CYP strains containing
rat
b5 (1
) will be designated from hereon as BTC-rb
5-1A2, BTC-rb
5-2A6
and BTC-rb
5-2E1, respectively.
Characterization of RED-, CYP- and hb5-expression and CYP activities
Membranes from cells derived from cultures as used for the bioactivation assays were analyzed for their human b5, CYP and RED contents (Table I), as well as their CYP activities (Table II). Expression levels of both b5 and CYP could be quantified through spectrophotometric techniques (Figures 3 and 4). Human cytochrome b5 expression was further confirmed by a Western blot analysis (Figure 5). Immuno-detectable b5 was present in membranes derived from BTC-hb5-CYP strains but not in membranes derived from BTC-CYP strains. Pure human b5 used as control was histidine-tagged (four histidine residues) and, consequently, migrated slightly higher in comparison to the b5 detected in the membranes of BTC-hb5-CYP strains. The Western blot demonstrated several b5-non-specific signals, which can be easily distinguished through a comparison with the signals of the corresponding b5-void strains.
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Fig. 5. Immunodetection of b5 in membranes derived from different BTC strains. All lanes contained 5 µg of membrane-protein, except for the marker lane, which contained 30 ng of pure human recombinant histidine-tagged b5.
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Table I.. CYP, RED and b5 contents in membranes derived from strains BTC and BTC-hb5, expressing different human CYP forms
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Table II.. CYP activities in membranes derived from strains BTC and BTC-hb5, expressing different human CYP forms
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The three BTC-hb
5-CYP strains demonstrated similar levels of
human
b5, namely 35 (BTC-hb
5 and BTC-hb
5-1A2), 59 (BTC-hb
5-2A6)
and 66 (BTC-hb
5-2E1) pmol/mg protein. RED expression levels
were significantly lower in all the
b5 expressing strains in
comparison to the b
5-void strains (
Table I). With respect to
the CYP levels, the expression of CYP1A2 and CYP2E1 decreased
and the expression of CYP2A6 increased in the strains co-expressing
b5 in comparison with the corresponding
b5-void strains (
Table I).
To study the effect of
b5 on the catalytic rates of CYP1A2,
2A6 and 2E1, we compared the enzymatic activities with the standard
chemical probes of membranes derived from the new BTC-hb5-CYP
strains with those derived from BTC-CYP strains. CYP enzyme
activities were determined as ethoxyresorufin de-ethylase (EROD)
and methoxyresorufin demethylase (MROD) for CYP1A2 expressing
strains, as coumarin 7-hydroxylase for CYP2A6 expressing strains
and chlorzoxazone 6-hydroxylase for CYP2E1 expressing strains
(
Table II). Strain BTC1A2 presented EROD and MROD activities
7- and 10-fold higher, respectively in comparison to strain
BTC-hb5-1A2. Strains BTC1A2 and BTC-hb5-1A2 demonstrated a higher
MROD activity when compared with the EROD activity. Strain BTC2A6
demonstrated a 2-fold higher coumarin 7-hydroxylase activity
when compared with BTC-hb5-2A6 and BTC-hb5-2E1 presented a chlorzoxazone
6-hydroxylase activity 1.5-fold higher than strain BTC2E1. No
activity was detected in strain BTC
0 for all enzymatic reactions
(
Table II).
Bioactivation assays
The role of human b5 on the bioactivation activities of CYP1A2, 2A6 and 2E1 was evaluated with the procarcinogens, as used before. The five different compounds, namely 1-aminopyrene (1AP), 6-amino-chrysene (6AC), 2-amino-3-methylimidazo(4,5-f)quinoline (IQ), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 2-aminoanthracene (2AA) were tested with strains BTC-hb5-1A2 and BTC1A2. The mutagenic activities (in revertants/nmol) ranged from 1.0 for NNK to 6283 for 2AA with strain BTC1A2 and from 0.8 for NNK to 6837 for 2AA with strain BTC-hb5-1A2 (Figure 6). The carcinogens 2AA, 1AP, 6AC, IQ and NNK demonstrated similar mutagenicity in the presence or absence of human b5. As before, in order to test the effects of b5 on the bioactivation activities of CYP2A6 and CYP2E1, three different N-nitrosamines, namely N-nitrosodiethylamine (NNdEA) (Figure 7), N-nitrosodi-n-propylamine (NNdPA) and NNK (Figure 8) were assayed with strains BTC2A6, BTC-hb5-2A6, BTC2E1 and BTC-hb5-2E1. These compounds showed an increase in mutagenic activity with the strains co-expressing human b5 (BTC-hb5-2A6 and BTC-hb5-2E1) relative to the corresponding b5-null strains, except for NNK with the BTC2E1 and BTC-hb5-2E1 strains, which in both cases, did not demonstrate any mutagenicity. The CYP2A6- (Figures 7 and 8) and CYP2E1-dependent (Figure 7) bioactivation of NNdEA increased from 314 and 1658 revertants/µmol to 4288 and 2278 revertants/µmol, respectively when co-expressed with b5. The mutagenicity of NNK increased from 350 to 830 revertants/µmol when CYP2A6 was co-expressed with b5. In the case of NNdPA, human b5 was absolutely required for bioactivation with CYP2A6 and CYP2E1 (Figures 9 and 10). This procarcinogen demonstrated the mutagenic activity of 216 and 995 revertants/µmol for BTC-hb5-2A6 and BTC-hb5-2E1, respectively.
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Discussion
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Subsequent cloning work as part of our follow-up studies on
the role of human
b5 in bioactivation activities of human CYPs
(1
) lead to the detection of discrepancies in the
b5 cDNA of
plasmid pLCM-b
5-RED. Subsequent extensive sequencing demonstrated
that this sequence encoded for the rat
b5 protein. This report
describes the correction of this discrepancy and complete repetition
of all experimental work. The plasmid was corrected by using
the cDNA of human
b5 (NCBI BC015182
[GenBank]
) leading to the consensus
human
b5 protein (NCBI gene ID no. 1528). All subsequent experimental
procedures were carried out exactly as before to safeguard the
correct evaluation of the difference between the presence of
rat and human
b5 in BTC-
b5-CYP bacteria.
RED, CYP and human b5 could be detected (Figures 3–5) and quantified (Table I) in the corrected BTC-hb5-CYP strains. Differences were observed in expression levels when compared with those found for the corresponding rat b5 containing strains (Table I). Human b5 expressed lower but similar in all three b5 strains. However, tendencies of expression levels were maintained, i.e. lower RED expression levels when co-expressed with human b5, lower expression levels of CYP1A2 and CYP2E1 as well as higher CYP2A6 levels, when in the presence of human b5. The relative human b5, CYP and RED contents of the different BTC-CYP and BTC-hb5-CYP strains demonstrated to have maintained their physiological relevance when compared with human liver microsomes (Table III) as was the case with the corresponding rat b5 strains. The low RED expression levels in BTC-hb5-1A2 and BTC-hb5-2A6 strains led to a RED:CYP ratio slightly lower than those presented in human liver microsomes (Table III). However, these ratios continue to have biological significance as in vivo RED is simultaneously involved in supporting the activity of multiple (different) CYPs resulting in an apparent lower RED availability. We and others (2,3) are able to obtain higher expression levels of RED and/or CYP, quite easily in Escherichia coli. However, induction of high expression levels of CYP and/or RED results in the activation of stress-inducible systems (such as the SOS response), which makes the tester bacteria unsuitable for mutagenicity assays (4). Moderate expression levels are thus required to maintain the bacterial cell viable for such subsequent application. The effect of human b5 on CYP activities as determined in membrane preparations demonstrated to be similar when compared with the corresponding rat b5 data, namely a reduction in EROD and MROD activities (CYP1A2) and an increase in chlorzoxazone 6-hydroxylation (CYP2E1) (Table II). The coumarin 7-hydroxylation activity (CYP2A6) with human b5 was reduced relatively to the corresponding b5-void strain. This was not the case with rat b5, which seemed not to have an effect on this CYP activity (1). It is known that CYP turnover rates are affected by several factors that are biochemically different from the CYP enzyme itself such as the concentration of the electron transfer proteins RED and/or b5. The use of high RED:CYP (
1) ratios can lead to exaggerated turnover rates (2,5). Thus, when applying in vitro systems for physiological relevant studies of the role of CYP biotransformation in xenobiotic metabolism, the relative content of the different CYP complex factors should reflect those found in human liver. That is, (relative) RED content is rate limiting for CYP reactions. The stimulatory effect of human b5 on the bioactivation activity of human CYPs could be demonstrated, as shown by the bacterial mutagenicity assays (Figures 6–10). The same procarcinogens were tested as with rat b5. Human b5 stimulated (albeit less pronounced), the bioactivation of all compounds as described for rat b5, except 2AA. The slight increase (1.5-fold) of 2AA-mutagenicity found with rat b5 could not be confirmed when human b5 was combined with CYP1A2. All other main findings of the effect of rat b5 were confirmed with human b5, i.e. for CYP2A6 (Figure 9): NNdEA: 14-fold increase (23-fold with rat b5) and NNK: 3-fold (9-fold with rat b5); for CYP2E1 (Figure 10): NNdEA: 1.5-fold increase (3-fold with rat b5); NNK: no mutagenicity with or without human b5. Both CYP2A6 and CYP2E1 demonstrated total dependence on presence of human b5 for NNdPA mutagenicity, as was shown before with rat b5.
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Table III.. Relative contents of RED, CYP and b5 in strains BTC and BTC-hb5 expressing different human CYP forms and in human liver microsomes expressed in mean values and intervals
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Advantages for application of the BTC
in vitro model in biotransformation
studies, containing different human CYP enzyme complexes, becomes
apparent when compared with the use of metabolic systems derived
from human tissues. Although the use of human liver microsomes
in combination with BTC strains, void of CYPs could shed light
on CYP bioactivation of chemical compounds, the unequivocal
determination of the involvement of a specific CYP enzyme and
the effect of
b5 is hampered. This is due to the presence of
multiple enzymes, involved both in bioactivation and bioinactivation
reactions and the permanent presence of
b5 in these extracts.
Moreover, the metabolic activation under these conditions occurs
outside the target cell and the sensitivity for detection of
highly reactive, short-lived metabolic intermediates will be
lower, due to limited trans-membrane migration (4
).
The encountered less pronounced effects of human b5 on CYP1A2, 2A6 and 2E1 relative to rat b5 (see Figures 9 and 10), seem to indicate a more efficient coupling of the rat protein with human CYPs. This is corroborated by studies of others, demonstrating the same effect with human CYP3A4 (6). The tighter coupling of the rat–human protein complex holds also true when CYP and b5 are reversed: human b5 seem to form a tighter complex with rat CYP1A2 when compared with human CYP1A2 (7). The alignment of the rat and human b5 sequence reveals 16 amino acids differences (out of 134) between the two proteins (Figure 2A). A highly conserved region is present between Lys29 and Glu74, comprising the two axial ligands of the heme prosthetic group (His44 and His68) and surrounding residues which are important for the orientation of the heme, involved in the interactions of b5 with other proteins, including CYP (8). It has been suggested that the anionic residues around heme, namely Glu48, Glu49, Asp58 and Glu61, are involved in complementary charge pairing (9–11). Seven of the amino acids differences between human and rat b5 result in the change of physico-chemical proprieties of the residues (Fig. 2A), three (Pro90Ser, Asn93Ala and Pro96Ser) located in the linker region and 4 (Glu7Lys, Ala8Asp, Asn21Lys and His22Asp) in the N-terminal domain. Amino acid composition changes of the linker domain have shown to cause few affects on the stimulatory role of b5 but it is rather the length of this domain, which is of importance (8). Taking this into account, it seems that the 4 N-terminal amino acid differences between rat and human b5 is the cause of the observed activity differences between these two proteins. This implicates a role of the N-terminal part of b5 in the coupling with CYP. This result is corroborated by studies of others, using human CYP17 and chimeric (human/Xenopus) b5 proteins, demonstrating the importance of human b5 residues 16–41 for the coupling with this human CYP (12). Two of the main differences between human and rat b5 are exactly located in this region, namely Asn21Lys and His22Asp.
In conclusion, we described here the correction of the work published in Duarte et al. (2005) (1). Although absolute values of contents and activities were lower, all key-findings as found for rat b5 could be confirmed using human b5. In general, the physiological relevant co-expression of the cytochrome P450 complex factors CYP, RED and human b5 could be demonstrated in the different BTC strains, as was found with rat b5. The effect of human b5 on the three CYPs was in general similar, both as measured in membrane preparations as well as by the bioactivation of procarcinogens using the bacterial mutagenicity assay, albeit less pronounced. The only exception is the slight stimulatory effect of rat b5 on the CYP1A2 bioactivation of 2AA, which could not be confirmed with human b5. The current study allowed us to compare the effect of human and rat b5 with 3 human CYPs, indicating a role of the N-terminal domain of b5 in its coupling with CYP.
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Notes
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*To whom correspondence should be addressed. Tel: +351213610297; Fax: +351213622018; Email:
mkranendonk.gene{at}fcm.unl.pt
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References
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1. Duarte M.P., Palma B.B., Gilep A.A., Laires A., Oliveira J.S., Usanov S.A., Rueff J, Kranendonk M. (2005) The stimulatory role of human cytochrome b5 in the bioactivation activities of human CYP1A2, 2A6, and 2E1: a new cell expression system to study cytochrome P450 mediated biotransformation. Mutagenesis 20:93–100.[Abstract/Free Full Text]2. Friedberg T., Pritchard M.P., Bandera M., Hanlon S.P., Yao D., Mclaughlin L.A., Ding S., Burchell B., Wolf C.R. (1999) Merits and limitations of recombinant models for the study of human P450-mediated drug metabolism and toxicity: an intra-laboratory comparison. Drug Metab. Rev. 31:523–544.[CrossRef][ISI][Medline]
3. Parikh A., Gillam E.M., Guengerich F.P. (1997) Drug metabolism by Escherichia coli expressing human cytochromes P450. Nat. Biotechnol. 15:784–788.[CrossRef][ISI][Medline]
4. Kranendonk M., Laires A., Rueff J., Estabrook R.W., Vermeulen N.P.E. (2000) Heterologous expression of xenobiotic mammalian-metabolizing enzymes in mutagenicity tester bacteria: an update and practical considerations. Crit. Rev. Toxicol. 30:287–306.[CrossRef][ISI][Medline]
5. Yamazaki H., Nakamura M., Komatsu T., et al. (2002) Roles of NADPH-P450 reductase and apo-and holo-cytochrome b5 on xenobiotic oxidations catalyzed by 12 recombinant human cytochrome P450s expressed in membranes of Escherichia coli. Prot. Expres. Purif. 24:329–337.
6. Holmans P.L., Shet M.S., Martin-Wixtrom C.A., Fisher C.W., Estabrook R.W. (1994) The high-level expression in Escherichia coli of the membrane-bound form of human and rat cytochrome b5 and studies on their mechanism of function. Arch. Biochem. Biophys. 2:554–565.
7. Shimada T., Mernaugh R.L., Guengerich F.P. (2005) Interactions of mammalian cytochrome P450 NADPH-cytochrome P450 reductase, and cytochrome b5 enzymes. Arch. Biochem. Biophys. 435:207–216.[CrossRef][ISI][Medline]
8. Clarke T.A., Im S.C., Bidwai A., Waskell L. (2004) The role of the length and sequence of the linker domain of cytochrome b5 in stimulating cytochrome P450 2B4 catalysis. J. Biol. Chem. 279:36809–36818.[Abstract/Free Full Text]
9. Naffin-Olivos J.L. and Auchus R.J. (2006) Human cytochrome b5 requires residues E48 and E49 to stimulate the 17,20-lyase activity of cytochrome P450c17. Biochemistry 45:755–762.[CrossRef][Medline]
10. Gao Q., Doneanu C.E., Shaffer S.A., Adman E.T., Goodlett D.R., Nelson S.D. (2006) Identification of the interactions between cytochrome P450 2E1 and cytochrome b5 by mass spectrometry and site-directed mutagenesis. J. Biol. Chem. 281:20404–20417.[Abstract/Free Full Text]
11. Schenkman J.B. and Jansson I. (1999) Interactions between cytochrome P450 and cytochrome b5. Drug Metab. Rev. 31:351–364.[CrossRef][ISI][Medline]
12. Yang W.H. and Hammes S.R. (2005) Xenopus leavis CYP17 regulates androgen biosynthesis independent of the cofactor cytochrome b5. J. Biol. Chem. 280:10196–10201.[Abstract/Free Full Text]
13. Venkatakrishnan K., Von Moltke L.L., Court J.S., Harmatz J.S., Crespi C.L., Greenblatt D.J. (2000) Comparison between cytochrome P450 (CYP) content and relative activity approaches to scaling from cDNA-expressed CYPs to human liver microsomes: ratios of accessory proteins as sources of discrepancies between approaches. Drug Metab. Dispos. 28:1493–1504.
14. Paine M.F., Khalighi M., Fisher J.M., Shen D.D., Kunze K.L., Marsh C.L., Perkins J.D., Thummel K.E. (1997) Characterization of interintestinal and intraintestinal variations in human CYP3A4-dependent metabolism. J. Pharmacol. Exp. Ther. 283:1552–1562.[Abstract/Free Full Text]
Received on October 12, 2006;
revised on October 16, 2006;
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