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Vol. 283, Issue 3, 1425-1432, 1997
Department of Pharmacology and Toxicology,
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Abstract |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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A cDNA encoding a new member of the cytochrome P450 3A subfamily, P450 3A26, has been isolated from phenobarbital-induced canine liver. The sequence encodes a protein of 503 amino acids with 33 nucleotide differences conferring 22 amino acid substitutions when compared with the previously identified canine CYP3A12 enzyme. Nine of the amino acid differences are within the substrate recognition sites (SRSs) identified for P450 family 2, with five residue substitutions clustered within SRS-6. To facilitate heterologous expression in Escherichia coli, the N-terminus of 3A26 was modified. The expressed protein comigrated with a 3A-immunoreactive protein in dog liver microsomes with a slightly greater electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than 3A12, which suggests that 3A26 corresponds to a previously noted but never characterized 3A enzyme in dogs. Functional characterization of 3A26 was undertaken with use of progesterone, testosterone and androstenedione as substrates. Assays of expressed 3A26 and 3A12 demonstrated that 3A26 displays low steroid hydroxylase activity. Identification of an additional canine 3A enzyme should increase our understanding of xenobiotic metabolism in this important animal model. These findings also suggest that 3A26 and 3A12 may be an interesting model system for the investigation of structure-function relationships involved in steroid metabolism catalyzed by members of the cytochrome P450 3A subfamily.
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Introduction |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Cytochromes 450 constitute a superfamily of hemoproteins that play a central role in
the metabolism of a wide variety of xenobiotics and endogenous
compounds. During the past decade, the study of these enzymes has been
advanced greatly by the cloning of P450 cDNAs and expression of these
proteins in heterologous systems. P450 family 3, which consists of the
single subfamily 3A, is particularly important because of its
metabolism of a wide range of pharmacologically, physiologically and
toxicologically important agents. Compounds metabolized by P450 3A
enzymes include macrolide antibiotics such as erythromycin and
triacetyloleandomycin (Wrighton et al., 1985
), the calcium
channel blockers nifedipine and diltiazem (Guengerich et
al., 1986), the immunosuppressive agent cyclosporine (Kronbach et al., 1988), steroidal compounds (Waxman et
al., 1988) and several carcinogens including benzo(a)pyrene and
aflatoxin B1 (Shimada et al., 1989;
Gallagher et al., 1994). In addition, adverse
pharmacokinetic drug interactions have been observed clinically with
the concomitant use of multiple drugs that are metabolized by 3A
enzymes (Periti et al., 1992). Despite the numerous studies,
relatively little is known about the structure-function relationships
of previously identified 3A enzymes. Unlike cytochromes P450 of family
2, 3A enzymes within or across species exhibit few dramatic substrate specificity differences that could provide obvious leads for
site-directed mutagenesis of particular residues. Identification of
substrates that can distinguish the various members of the 3A subfamily
or of novel enzymes with altered substrate specificity would be very useful in the determination of the roles that specific residues play in
conferring the distinctive catalytic activities of the 3A subfamily.
The rat, human and mouse P450 3A subfamilies consist of multiple
members, which differ in their regulation (Nelson et al., 1996). Canine models have been used extensively in drug metabolism studies, but to date, only a single canine cytochrome P450 3A enzyme,
3A12, has been isolated (Ciaccio et al., 1991). Considerable evidence suggests that multiple canine 3A forms exist and that these
may have different catalytic properties. Immunoblots of liver
microsomes from PB-treated dogs were probed with a polyclonal antibody
generated against canine hepatic 3A12. The results indicated the
presence of two distinct proteins, one with an apparent molecular weight of 51 kDa, corresponding to 3A12, and the second with an apparent molecular weight of 49.5 kDa (Ciaccio and Halpert, 1989). In
addition, previous studies demonstrated differences between the
6
-hydroxylation of steroids and TAO complex formation in canine
liver microsomes. In particular, although both reactions were induced
by PB and inhibited by antibodies to 3A12, TAO-P450 complex formation
had little effect on steroid 6-hydroxylation. These data suggested
that some PB-inducible cytochrome P450 3A other than P450 3A12 might be
responsible for TAO complex formation in dog liver microsomes. Finally,
complex Southern blot hybridization patterns were observed when canine
genomic DNA was probed with the 3A12 cDNA (Ciaccio and Halpert, 1989).
Thus, several lines of evidence support the hypothesis that multiple
canine cytochromes P450 3A exist, and that these enzymes may differ in
their substrate specificity.
This study describes the isolation of a cDNA encoding a novel member of
the cytochrome P450 3A subfamily, P450 3A26, and its subsequent
heterologous expression and functional characterization. Degenerate
oligonucleotide PCR techniques were used to probe the cDNA library,
which resulted in the isolation of a 1.97-kbp fragment encoding the
3A26 enzyme. The cDNA encodes a protein of 503 amino acids and differs
from 3A12 at 22 amino acid positions. 3A26 was expressed in
Escherichia coli as described previously (Barnes et
al., 1991; Born et al., 1996). The different catalytic
activities ascribed to this enzyme will be useful in the elucidation of
structure-function relationships between 3A26 and the previously
identified 3A12.
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Materials and Methods |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Materials. Restriction endonucleases and media for bacterial growth were purchased from GIBCO-BRL (Grand Island, NY). The pKK233-2 and pSE380 expression plasmids were purchased from Pharmacia (Alameda, CA). Primers for PCR amplification were obtained from the University of Arizona Macromolecular Structures Facility (Tucson, AZ). PCR products were purified with the GeneClean II kit from Bio101 (Vista, CA). TOPP3 cells were obtained from Stratagene (La Jolla, CA). CHAPS, progesterone, testosterone, androstenedione, erythromycin, troleandomycin, NADPH and DOPC were purchased from Sigma Chemical Co. (St. Louis, MO). [4-14C]Testosterone was obtained from Amersham Life Science (Arlington Heights, IL). [4-14C]Progesterone and [4-14C]androstenedione were obtained from Dupont-New England Nuclear (Boston, MA). HEPES was purchased from the Calbiochem Corp. (La Jolla, CA). Thin-layer chromatography plates (silica gel, 250 µm, Si 250 PA (19C)) were purchased from Baker (Phillipsburg, NJ). All other reagents and supplies not listed were obtained from standard sources.
Isolation and sequencing of the cDNA encoding P450 3A26.
A
gt11 cDNA library previously generated and used to isolate the
canine 3A12 cDNA (Ciaccio et al., 1991) was used for the isolation of the cDNA encoding P450 3A26. The overall cloning scheme is
presented in figure 1. PCR was used in
conjunction with degenerate oligonucleotide primers to probe the canine
cDNA library for 3A sequences. The N-terminal
(5
-TTTGC(GT)GG(AGCT)TATGA(AG)AC(AC)AC(AGCT)AGCAG-3) and C-terminal
(5-CCTCAT(GT)CCAA(GT)GCA(AG)TT-3) degenerate primers were based on
highly conserved regions of mammalian P450 3A sequences, corresponding
to amino acid residues 304-311 and 441-446 of 3A12, respectively. Use
of these primers resulted in the amplification of a 0.4-kbp product
(fig. 1, Step 1). Reaction conditions were: one cycle of 94°C for 2 min, 54°C for 2 min and 72°C for 2 min, followed by 30 cycles of
94°C for 1 min, 54°C for 1 min and 72°C for 1 min. The total
reaction volume was 100 µl and all reactions were done in duplicate.
A 20-µl aliquot of each reaction was run on a 1.2% agarose gel, and
a single band at 0.4 kbp was identified and excised from the gel. The
PCR products were isolated from the gel with the GeneClean II kit
(Bio101, Vista, CA) and cloned into the pCRII cloning vector with the
TA Cloning Kit (Invitrogen, San Diego, CA). Two types of clones could be distinguished. One type was identical in sequence to 3A12, and the
other type possessed a Sau96I site and lacked an
EarI site when compared with 3A12.
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gt11 cDNA library that
produced the 3A12 cDNA (Ciaccio et al., 1991) (fig. 1, Step 2). The cDNA inserts were amplified from the phage isolates by use of
gt11 forward and reverse primers. Two types of phage
inserts were isolated in these experiments, the previously identified 3A12 and a variant clone lacking an EarI site and possessing
a Sau96I site. However, because none of the variant clones
were full length, it was necessary to go back to the library to isolate a cDNA encoding the complete 3A enzyme coding region (fig.1, Step 3). A
region after the translation stop site was used to design a primer
specific for the novel cDNA. That primer, 5-AACCGGATAGGTTGAGTCTAC-3, was used in conjunction with the forward gt11 primer to produce a
1.8-kbp PCR product, which was cloned into the pCRII cloning vector and
sequenced.
N-terminal modification and heterologous expression of 3A26.
The N-termini of canine 3A26 and 3A12 are identical in sequence until
the first variation is encountered at base pair 295. Modifications to
the N-terminus of 3A12 have been described previously (Born et
al., 1996). Restriction endonucleases and subcloning were used in
the modification of 3A26 for expression in E. coli. The
unmodified N-terminus of 3A26 was removed using NcoI and
ScaI restriction endonuclease sites and replaced with the
corresponding fragment from the modified N-terminus of 3A12. These
alterations removed 10 amino acids in the signal anchor sequence of
3A26 and changed the second amino acid residue from aspartic acid to
alanine, changes that have been shown to facilitate expression in
E. coli (Barnes et al., 1991; Born et
al., 1996). Both 3A26 and 3A12 constructs were subsequently
subcloned into the pSE380 expression vector with an NcoI
site at the 5-end and BamHI or HindIII sites at the 3-end, respectively.
). pSE3A26- and pSE3A12-containing TOPP3
E. coli cells were grown at 37°C with 240 rpm shaking in
250 ml liquid TB media (12 g Bacto tryptone, 24 g Bacto yeast
extract, 4 ml glycerol/l to mid log phase. IPTG (final concentration,
1.0 mM) and 80 mg/l 
-ALA were added, and cells were harvested after
an additional incubation at 30°C with 190 rpm shaking. Maximum
expression of 3A26 was observed at 42 h after IPTG/-ALA
addition, and typically 5 to 8 nmol of P450 3A26 were recovered per
liter of culture. Maximum expression of 3A12 was observed at 72 h
after IPTG/-ALA addition and yields of 40 to 55 nmol/l of culture
were routine.
Immunochemical detection of canine cytochromes P450 3A from
heterologous expression systems and PB-induced canine liver
microsomes.
Polyclonal antibodies raised against canine cytochrome
P450 3A12 were isolated and characterized previously (Ciaccio and
Halpert, 1989). Analyses of purified proteins, heterologously expressed proteins and microsomes by SDS-PAGE (8% polyacrylamide gels) were performed essentially as described by Laemmli (1970) and resolved proteins were stained with Coomassie blue or transferred to
nitrocellulose membranes.
Functional characterization of E. coli-expressed
canine P450s 3A26 and 3A12.
Steroid hydroxylase assays were
performed with CHAPS-solubilized E. coli membrane
preparations of 3A26 and 3A12 as described previously (John et
al., 1994; Born et al., 1996). Ten picomoles P450 were
reconstituted with 40 pmol E. coli expressed rat NADPH-P450 reductase, 10 pmol rat cytochrome b5 and 0.1 mg/ml DOPC in a minimal reaction volume. Assays were performed in 100 µl of 50 mM HEPES (pH 7.6) with 15 mM MgCl2 and
100 µM EDTA, 0.1 mg/ml DOPC and 0.06% CHAPS. Steroid stock solutions
were made in 100% methanol. Methanol concentrations in all reactions
were equivalent and did not exceed 1% of the total reaction volume.
Individual testosterone, progesterone and androstenedione
concentrations were 250 µM for all catalytic assays performed.
Identification of metabolites was by relative mobility on TLC and by
comparison to authentic standards (Waxman, 1991).
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Results |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Isolation of a cDNA encoding canine cytochrome P450 3A26. The isolation of a cDNA clone encoding P450 3A26 was described under "Materials and Methods" and outlined in figure 1. Notably, both 3A12 and 3A26 were isolated from a cDNA library created with RNA from a single PB-induced dog liver, eliminating any possibility that the differences identified were caused by strain or inter-individual variations. In addition, PCR reactions were performed in duplicate, and one 3A26 clone from each reaction was isolated and analyzed via dideoxy-sequencing. These sequences were found to be identical, which indicates that differences in DNA sequence between 3A26 and 3A12 are extremely unlikely to be the result of PCR error.
A comparison of P450 3A26 with the previously published canine P450 3A12 sequence is shown in figure 2. Both enzymes are 503 amino acids in length and share 95.6% amino acid identity. The two enzymes exhibit 33 nucleotide and 22 amino acid differences. Most of the amino acid differences are found in the C-terminal half of the sequence. It is also interesting to note that the 5-untranslated region of 3A26 is identical in sequence to
that found in the 3A12 clone, whereas significant variations are
present in the 3-untranslated regions of 3A26 and 3A12. These
differences allowed for the design of a primer selective for 3A26, thus
facilitating the isolation of the clone encoding the entire sequence
from the gt11 cDNA library (fig. 1, Step 3).
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) for P450 family 2 enzymes are predicted to
contain key residues involved in enzyme-substrate interactions. The
limited number of amino acid differences between 3A26 and 3A12 present
interesting experimental possibilities in the investigation of
structure-function relationships of P450 enzymes. Figure
3 graphically represents the positions of
amino acid sequence differences between 3A12 and 3A26. Putative 3A SRSs
have been indicated, and the specific residue differences between 3A12
and 3A26 within these regions are noted by showing the one-letter code
for the 3A12 residue, the position and the single letter code for the 3A26 residue. Changes in residues outside of the SRSs are noted with
asterisks. Most of the changes found in the SRSs occur in sites 5 and
6, with only one difference in each of SRSs 2 and 4. No differences in
amino acid sequence were observed in SRSs 1 or 3.
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Comparison of differences found in P450 3A26 with analogous residues in other mammalian members of the cytochrome P450 3A subfamily. Many of the residue differences identified between cytochromes P450 3A26 and 3A12 occur at positions that are generally well conserved in other mammalian P450 3A enzymes. Figure 4 represents a comparison of residue differences within the putative SRSs found in 3A26 with other mammalian 3A enzymes. 3A26 has significant alterations in amino acid sequence in terms of residue volume, charge and hydrophilicity in these SRSs. For example, serine residues are found at positions 368 and 474 in 3A26, whereas proline is found at those positions in all other mammalian 3A sequences. In addition, the highly conserved lysine residue at position 476 has been replaced by a larger arginine residue in 3A26. These differences may prove to be significant in determining the metabolic profile of this enzyme.
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Heterologous expression of the modified cytochrome P450 3A26 enzyme
in E. coli.
Heterologous expression and preparation of
CHAPS-solubilized membranes of the modified 3A26 and 3A12 in E. coli were performed as described previously (John et
al., 1994). Expression levels for 3A26 were relatively low when
compared with levels obtained for 2B or 3A constructs (John et
al., 1994; Born et al., 1996; Harlow et al.,
1997), resulting in maximal recovery of 5 to 8 nmol P450 per liter of
culture. Maximal expression of P450 3A26 was found to occur at 42 h after addition of IPTG and -ALA when cultures were grown at
30°C. Culture temperatures of 24°C and 37°C resulted in reduced
levels of recovered protein. In addition, variation of IPTG and -ALA
concentrations as well as time of induction and order of addition of
compounds were all examined and resulted in no increase in expression
of P450 3A26 from the parameters described under "Materials and
Methods."
Immunoblot analyses of heterologously expressed P450s 3A26 and 3A12
and PB-induced canine liver microsomes.
Previous studies in this
laboratory (Ciaccio and Halpert, 1989; Ciaccio et al., 1991)
resulted in the isolation, identification and characterization of the
canine cytochrome P450 3A12 enzyme. It was noted at that time that
immunoblots of liver microsomes from PB-induced dogs with use of a
polyclonal antibody to 3A12 produced two distinct immunoreactive bands.
It was therefore of interest to examine the electrophoretic mobility of
the expressed 3A26 enzyme. Immunoblot data of canine liver microsomes
as well as heterologously expressed P450s 3A26 and 3A12 are shown in
figure 5. Differences in electrophoretic
mobility of these two cytochromes are evident, with 3A26 having greater
mobility than 3A12. In microsomes from PB-induced dogs, two distinct
bands were discernible, with the upper band corresponding in
electrophoretic mobility to 3A12 and the lower band corresponding to
3A26. The possibility existed that, because of N-terminal modifications
that removed 10 residues of the signal-anchor sequence, electrophoretic
mobility of the expressed cytochrome P450 3A enzymes was altered
significantly from that observed for their microsomal counterparts.
However, no difference was observed between heterologously expressed
3A12 and the purified hepatic enzyme. These findings demonstrate that the differences in electrophoretic mobility observed for 3A26 and 3A12
are not likely to be the result of modifications that were introduced
for heterologous expression. To demonstrate that other constituents in
the samples had no influence on electrophoretic mobility differences,
samples of 3A26 and 3A12 were combined and run along side of induced
microsomal samples. It is evident from these experiments that P450 3A12
has decreased electrophoretic mobility relative to P450 3A26 on
SDS-PAGE, and that each enzyme corresponds to immunoreactive bands
identified in dog liver microsomes.
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Characterization of the catalytic activity of cytochrome P450
3A26.
Steroid-hydroxylase assays were performed with solubilized
E. coli membrane preparations containing 3A26 or 3A12. The
major steroid metabolite formed by other mammalian 3A enzymes is the 6-OH product (Waxman et al., 1988; Ciaccio and Halpert,
1989; Born et al., 1996). The studies performed here (table
1) indicate a significant reduction in
steroid hydroxylase activity of P450 3A26 when compared with P450 3A12.
Three different steroid substrates, androstenedione, testosterone and
progesterone, were examined to determine the relative activities of
heterologously expressed P450s 3A26 and 3A12. P450 3A12 exhibited high
rates of steroid hydroxylase activity for all of the steroids employed,
whereas the relative rates of steroid hydroxylation for 3A26 did not
exceed 22% of 3A12 activity for any individual hydroxylated steroid
metabolite. Interestingly, although steroid hydroxylase rates for 3A26
are low and determination of exact metabolite ratios is therefore difficult, metabolite profiles do seem to differ between 3A12 and 3A26.
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Discussion |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Previous studies have suggested that multiple canine 3A forms
exist and have distinct metabolic profiles (Ciaccio et al., 1989). The results presented here describe the isolation and initial characterization of a new canine cytochrome P450 3A enzyme, P450 3A26,
from a cDNA library generated from PB-induced canine hepatic tissue.
The 1.9-kbp cDNA encoding 3A26 exhibited 33 nucleotide and 22 amino
acid differences when compared with canine P450 3A12. The sequence
identity between CYP3A12 and CYP3A26 at the N-terminal and
5-untranslated region, and the sequence differences found mostly at
the C-terminal and 3-untranslated region suggest that CYP3A26 might be
the product of a recent "gene conversion" or "unequal crossing
over" event involving CYP3A12. According to this hypothesis, the 5
portion of the CYP3A26 gene would be derived from CYP3A12 and the 3
portion would be derived from either an ancestral CYP3A26 gene, which
was subsequently lost, or from a putative third canine CYP 3A gene.
Southern blot evidence does not allow us to exclude the existence of a
third, related CYP3A gene (Ciaccio et al., 1989).
Recombination between closely related genes of the CYP2 family has been
documented previously in the rat CYP2D subfamily (Matsunaga et
al., 1990).
The recovery of heterologously expressed 3A26 was relatively limited
and may be a limiting factor in future assays investigating its
metabolic profile. However, it may be possible to augment the recovery
of expressed 3A26. Recent mutagenesis studies examining the key
residues involved in P450 3A4 substrate recognition (He et
al., 1997) may give some indication of the cause of relatively low
levels of expression of 3A26 in E. coli. These
studies noted that the substitution of Pro-368 with Ser led to a
30-fold lower expression level of 3A4 than that of wild-type 3A4.
Because residue 368 of 3A26 is a serine and all other mammalian P450s
3A examined contain a proline at this site, it is possible that
expression of 3A26 is hampered by this single difference in amino acid
sequence. Another serine for highly conserved proline substitution is
observed at residue 474 of 3A26, which suggests a second potential site which could have a negative impact on expression. Although no direct
evidence from expression studies with 3A26 indicates that these
particular residues are contributing to lower expression levels in
E. coli, these 3A4 mutagenesis findings do suggest that Ser-368 may be responsible for the low levels of expression of 3A26 in
E. coli.
Immunoblot data presented here indicate that 3A26 has electrophoretic
mobility characteristics similar to those of a canine 3A band
identified in previous studies (Ciaccio et al., 1989). Specifically, the heterologously expressed 3A26 enzyme has slightly greater mobility than expressed 3A12 on SDS-PAGE. These differences are
analogous to those seen in PB-induced canine liver microsomes in which
two separate immunoreactive bands are discernible, which suggests that
the 3A26 enzyme is analogous to the lower immunoreactive band and 3A12
corresponds to the upper band. These two enzymes are each 503 amino
acids in length and share only 22 amino acid differences between them.
Moreover, the molecular masses of 3A26 and 3A12, 57,689 Da and 57,684 Da, respectively, differ by only 5 Da. As observed in figure 4, in
which microsomal preparations and purified hepatic 3A12 were compared
with heterologously expressed enzyme samples, it was found that the
N-terminal modifications that deleted 10 amino acids and reduced the
size of each heterologously expressed enzyme by 1160 Da had no
discernible effect on electrophoretic mobility. Taken together, these
data indicate that differences in molecular weight cannot account for
the differences in electrophoretic mobility observed for 3A12 and 3A26.
Similar findings have been reported for cytochromes P450 2B1 and 2B2,
which differ by only 14 amino acids from a total of 491 and have been
found to generate two distinct immunoreactive bands (Ryan et
al., 1982; Waxman et al., 1983). The molecular masses
of these two enzymes are also quite similar to one another, differing
by only 13 Da from a total of more than 55,900 Da each. These
observations suggest that some secondary protein structure remains
intact even after samples were boiled in SDS and may account for
differences in electrophoretic mobility between these enzyme homologs.
In addition to the differences in electrophoretic mobility and
heterologous expression levels of P450s 3A26 and 3A12, several distinctions in catalytic activity have been identified between these
two enzymes. The major differences in steroid hydroxylase activities
identified here clearly demonstrate that cytochrome P450 3A26 is
uniformly less active than 3A12. Previous work on human cytochromes
P450 3A4 and 3A5 has shown some parallels when compared with canine
P450s 3A12 and 3A26. P450s 3A4 and 3A5 exhibit 84% amino acid sequence
identity and metabolize many of the same substrates. Both P450s 3A4 and
3A5 have been found to catalyze 6-hydroxylation of testosterone,
progesterone and androstenedione, although minor hydroxylation products
such as 16
-hydroxyprogesterone comprised approximately 20% of the
total metabolites of 3A4 but not 3A5 (Aoyama et al., 1989).
Recent site-directed mutagenesis studies have demonstrated that the
replacement of Ile-369 in P450 3A4 with the corresponding Val in 3A5
caused the suppression of progesterone 16-hydroxylase activity (He
et al., 1997). Interestingly, P450 3A26 also has a Val at
residue 369 and has no appreciable 16-hydroxylase activity. Taken
together, these experiments suggest that differences in residues of
SRS5 found between P450s 3A12 and 3A26 may play major roles in
determining steroid hydroxylase activity differences observed for these
enzymes.
This study has resulted in the isolation, expression and functional characterization of a cDNA encoding the canine cytochrome P450 3A26 enzyme. This enzyme exhibits marked differences in the respective rates of hydroxylation of steroid substrates. Immunoblot data also confirm the presence of multiple 3A proteins in canine microsomes, with 3A26 corresponding to a previously unknown enzyme of lower apparent molecular weight identified in previous studies. Future experiments will use site-directed mutagenesis techniques to facilitate increased heterologous expression of 3A26 and to identify the residues responsible for conferring specific metabolism profiles to 3A26 and 3A12. These studies should be invaluable in the determination of P450 3A substrate specificity.
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Footnotes |
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Accepted for publication August 11, 1997.
Received for publication April 7, 1997.
1 Supported by Procter & Gamble Pharmaceuticals, Inc., a fellowship from the Flinn Foundation, NIH Grant GM 54995, and Core Center Grant ES 06694. Presented in part at the XIth International Symposium on Microsomes and Drug Oxidations, Los Angeles, CA, July 1996 and the American Society for Pharmacology and Experimental Therapeutics Annual Meeting, San Diego, CA, March 1997.
Send reprint requests to: David J. Fraser, Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, AZ 85721.
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Abbreviations |
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P450, cytochrome P450;
PB, phenobarbital;
TAO, troleandomycin;
SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel
electrophoresis;
PCR, polymerase chain reaction;
androstenedione, androst-4-ene-3,17-dione;
DOPC, dioleoylphosphatidylcholine;
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
IPTG, isopropyl--D-thiogalactopyranoside;
ALA, -aminolevulinic acid;
CHAPS, 3-((3-cholamidopropyl)-dimethylammonio)-1-propanesulfonate;
EDTA, (ethylenedinitrilo)-tetraacetic acid;
TLC, thin layer chromatography;
SRS, substrate recognition site;
-OH, hydroxy;
kbp, kilobase
pairs.
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References |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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-hydroxylase in Escherichia coli.
Proc. Natl. Acad. Sci. U.S.A.
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M. Shou, R. Norcross, G. Sandig, P. Lu, Y. Li, Y. Lin, Q. Mei, A. D. Rodrigues, and T. H. Rushmore SUBSTRATE SPECIFICITY AND KINETIC PROPERTIES OF SEVEN HETEROLOGOUSLY EXPRESSED DOG CYTOCHROMES P450 Drug Metab. Dispos., September 1, 2003; 31(9): 1161 - 1169. [Abstract] [Full Text] [PDF]
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R. A. Graham, A. Downey, D. Mudra, L. Krueger, K. Carroll, C. Chengelis, A. Madan, and A. Parkinson In Vivo and in Vitro Induction of Cytochrome P450 Enzymes in Beagle Dogs Drug Metab. Dispos., November 1, 2002; 30(11): 1206 - 1213. [Abstract] [Full Text] [PDF]
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S. M. Lankford, S. A. Bai, and J. A. Goldstein Cloning of Canine Cytochrome P450 2E1 cDNA: Identification and Characterization of Two Variant Alleles Drug Metab. Dispos., August 1, 2000; 28(8): 981 - 986. [Abstract] [Full Text]
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S. K. Paulson, L. Engel, B. Reitz, S. Bolten, E. G. Burton, T. J. Maziasz, B. Yan, and G. L. Schoenhard Evidence for Polymorphism in the Canine Metabolism of the Cyclooxygenase 2 Inhibitor, Celecoxib Drug Metab. Dispos., October 1, 1999; 27(10): 1133 - 1142. [Abstract] [Full Text]
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 D. J. Fraser, Y. Q. He, G. R. Harlow, and J. R. Halpert Use of Chimeric Enzymes and Site-Directed Mutagenesis for Identification of Three Key Residues Responsible for Differences in Steroid Hydroxylation between Canine Cytochromes P-450 3A12 and 3A26 Mol. Pharmacol., February 1, 1999; 55(2): 241 - 247. [Abstract] [Full Text]
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