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Purification and characterization of a cytochrome P-450 from insecticide susceptible and resistant strains of housefly Musca domestica L. before and after phenobarbital exposure

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Archives of Insect Biochemistry and Physiology 241-1 9 (1993)
Purification and Characterization of a
Cytochrome P-450 From Insecticide
Susceptible and Resistant Strains of Housefly,
Musca domesfica L., Before and After
Phenobarbital Exposure
Jeffrey G . Scott and Susanna S.T. Lee
Department of Entomology, Cornstock Hall, Cornell University, Ithaca, New York
A cytochrome P-450 (P-450) was purified from abdominal microsomes of
untreated and phenobarbital treated susceptible (S + ) and insecticide-resistant
(LPR) houseflies using HPLC purification procedures. The purified P-450s
exhibited the same apparent molecular masses (54,400 dalton) and could not
be distinguished from each other on the basis of HPLC chromatographic
properties, reduced CO-difference absorbance maxima (447 f 0.5 nm), or
partial NHpterminal sequences (MLLLLLLIVVTTLYIFAKL). Since these P-450s
were indistinguishable and were immunologically identical to cytochrome
P-45qP, we conclude that these P-450s are in fact cytochrome P-45QP,. The
level of P-45qp, was increased twofold after PBexposure in theS strain, while
the level of P-45QP, did not change in the LPR strain. lmmunoinhibition of
ECOD activity with anti-P-4501p, antiserum suggests that PB treatment in S
house flies induced P-4501,,, and other forms of cytochromes P-450 with high
activity toward this substrate. o 1993 Wiley-Liss, Inc.
+
+
Key words: chromatography, cytochrome b, monooxygenase, HPLC, immunoelectrophoresis
INTRODUCTION
The microsomal cytochrome P-450 monooxygenases play an essential role in
oxidative metabolism, insecticide resistance, and enzyme induction in insects
Acknowledgments: This work was supported in part by grant R 0 1 CM47835-01 from the U.S.
National Institutesof Health and Hatch Project 139414.
Received November 10, 1992; accepted February 15, 1993.
Address reprint requests to Jeffrey G. Scott, Department of Entomology, Comstock Hall, Cornell
University, Ithaca, NY 14853-0999.
0 1993 Wiley-Liss, Inc.
2
Scott and
Lee
[14]. Insects have multiple forms of P-450*with different substrate specificities,
and being differentially inducible [4-7]. Induction of P-450 monooxygenase
activity has been extensively investigated in insects; however, only limited
information is available on the profiles of individual forms of P-450s after
induction.
Previously [8], we found that treatment with PB in an insecticide susceptible
strain of housefly resulted in elevation of the total P-450s and six P-450 monooxygenase activities (methoxyresorufin 0-demethylation, ethoxyresorufin 0deethylation, aryl hydrocarbon hydroxylation, aldrin epoxidation, and
p-chloro-methylaniline N-demethylation) in microsomes from whole abdomens. In contrast, PB had no effect on the total P-450 content, increased ECOD,
and decreased MROD activities in an insecticide resistant (LPR) strain of
housefly. It is unclear if the lack of changes in the total cytochrome P-450 content
in LPR was due to the lack of PB responsiveness, or if alternatively PB induced
some P-450s and suppressed others leading to a lack of change in total P-450s.
Study of individual P-450s is necessary to clarify this point.
Herein, we report the purification and characterization of P-450s before and
after PB induction in insecticide susceptible and LPR houseflies. Since P-450s
are often differentiated from each other on the basis of chromatographic behavior, spectral properties, electrophoretic properties, immunological reactivity,
and NHrterminal sequences [9], these criteria were used to characterize the
P-450s purified in the present study. Further, we investigated the immunoinhibition of ECOD activity using anti-P-4501~~
antiserum in control and PBtreated S + housefly microsomes.
MATERIALS AND METHODS
Chemicals
Sodium phenobarbital was provided by Dr. C.F. Wilkinson. Normal rabbit
sera, 3-MC, and diaminobenzidine tetrahydrochloride were obtained from
Sigma (St. Louis, MO). Low range molecular mass markers were obtained from
Bio-Rad (Richmond, CA). Goat antiserum to rabbit gamma-globulin was purchased from Calbiochem (San Diego, CA), and horseradish peroxidase rabbit
anti-horseradish peroxidase complex was obtained from Organon Teknika
(Durham, NC). All organic solvents used for purification were HPLC grade
obtained from Fisher Scientific (Rochester, NY). HPLC buffers were prepared
as described by Wheelock and Scott [lo] and HPLC mobile phases were first
filtered through 0.22 pm acetate membrane and then degassed with helium for
at least 30 min before use.,
*Abbreviations used: BCA = macro-bicinchoninic acid; CHAPS = 3-(3-~holarnidopropyl)-dirnethylamrnonio-1-propanosulfonate; ECOD = 7-ethoxycoumarin 0-deethylation; HIC = hydrophobic
interaction chromatography; LPR, Learn-pyrethroid-resistant strain of housefly; 3-MC = 3-methylcholanthrene; MROD = methoxyresorufin0-demethylation; PB = phenobarbital; P-450 = rnicrosornal cytochrome P-450; P-4501,~= cytochrome P-4501,~P-450,+ = cytochrorne P-45Q4 PEG =
polyethyleneglycol 8000; RIE = rocket immunoelectrophoresis; 5 = phenobarbitaltreated susceptible; SDS = sodiumdodecyl sulfate.
+
Purification of Housefly P-450s 3
Insects
+
Two strains of housefly (Muscu domesticu L.) were used in this study. S ,an
insecticide susceptible strain, was obtained from Dr. F.W. Plapp, Jr., of Texas A
& M University. LPR, a multi-resistant strain, was originally collected from a
Learn dairy in Horseheads, New York, and subsequently selected with the
pyrethroid insecticide permethrin for 40 generations [11]. The major pyrethroid
resistance mechanism in the LPR strain appears to be cytochrome P-450 monooxygenase-mediated detoxification [12].
Induction
Adult houseflies ( 3 4 days old) were fed 0.15% (w/v) sodium phenobarbital
in 17%sugar water (w/v) ad libitum for 48 h and maintained in an environmental chamber at 29°C with a relative humidity of 50% and a 12:12 (light:dark)
photoperiod. Controls were fed 17%sugar water. After 48 h of induction, flies were
stored frozen for 2 4 days at - 80°C and then used to prepare microsomes.
In one experiment, S houseflies were fed 0.5% (w/w) 3-MC in (1:l)non-fat
dry milk powder and sugar diet. Treatment solution was prepared from a 1%
(w/v) stock 3-MC solution in benzene. Five milliliters of 1%3-MC solution were
added to 5 g of non-fat dry milk powder (Carnation) and 5 g of granular sugar
in a porcelain mortar. This mixture was immediately mixed thoroughly using
a pestle until all solvent was evaporated and then transferred to a glass petri
dish and used to feed the houseflies. Control diet was prepared by using 5 ml
of benzene instead of the 3-MC solution. Distilled water was also provided to
each group. Induction conditions and subsequent procedures were the same as
described above. All diets were consumed by the flies at the termination of the
experiment. Mortality was low and not different between the 3-MC and control
groups.
+
Microsome Preparation
Female housefly (-2,400 per treatment group) abdominal microsomes were
prepared as detailed earlier [13]. Microsomes from each treatment group were
diluted to 2 mg proteidml for a total of 5 ml for total cytochrome P-450s,
SDS-PAGE, RIE, immunoblotting, and immunoinhibition assays and 3 mg
proteiniml for a total of 5 ml for P-450 purification. Microsomes were stored in
1 dram glass vials or 5 ml polypropylene tubes at -80°C and used directly as
enzyme sources.
Protein and Enzyme Assays
The Bradford [14] and the BCA protein assays (Pierce Chemical Company,
Rockford, IL) were used to determine the protein concentrations in the crude
microsomes, while only the BCA assay was used to determine the protein
content in the PEG supernatant, phenyl-5PW, and DEAE5PW column outputs
becuase the latter samples contained either detergents or other substances that
interfered with the Bradford assay. A tenfold dilution of the original crude
microsomal suspensions (500 abdomendml) with resuspension buffer was
done prior to protein determination, whereas outputs collected from different
4
Scott and lee
purification steps were assayed for protein without dilution. Bovine serum
albumin was used as the standard.
P-450s and cytochrome b5 were assayed according to Ornura and Sat0 [15].
Crude microsomal samples were diluted 1:l to a final protein concentration of
1 mg/ml with resuspension buffer while outputs collected from the purification
steps were assayed as undiluted.
Cytochrome P-450 Purification
A two-step HPLC purification scheme [lo] was used to monitor the profiles of abdominal P-450s before and after PB exposure in susceptible and
LPR houseflies. Briefly, 5 ml of microsomal suspensions at 3 mg proteidml
(-800-1,200 female abdomens) from each treatment group were first solubilized with CHAPS (0.5% final concentration) and then the P-450s were
separated from other proteins by PEG precipitation at an 8% final concentration. P-450s were then purified using hydrophobic interaction chromatography (phenyl-5PW column) followed by anion exchange chromatography
(DEAE6PW column). At each purification step, at least 500 pl of eluate were
saved and stored frozen (-SOT) for assays of protein and total cytochrome
P-450 content. For each treatment group, the cycle of P-450 purification by
HPLC was repeated three times with abdominal microsomes from three
separate rearings of houseflies. The elution profiles obtained were similar,
and the results reported in Figure 1 show one representative cycle of this
purification from each treatment group.
SDS-PAGE and Immunoblotting
SDS-PAGE was done according to Laemmli [16] as described by Lee and Scott
[8].Crude microsornes and purified P-450s were treated with 5 x sample buffer
containing P-mercaptoethanol, boiled for 5 min, and applied to each well in a
10-15% gradient or 10% nongradient polyacrylamide gels. After electrophoresis, gels were either stained for protein with Coomassie brillant blue or subjected
to immunoblotting procedures as described previously [17].
Rocket and Fused Rocket Immunoelectrophoresis
RIE of crude microsomes and purified P-450s were performed as described
by Wheelock and Scott [18] using a monospecific polyclonal anti-P-4S01p, antiserum (8-241) prepared against purified P-4501,, [lo]. Standard curves were
made using 0.25, 0.5, 1, and 2 pmol of purified P-4501,~at 5 pl per well. Four
serial dilutions from the original samples were made, and all sample dilutions
were done based on equal dilutions of the total P-450 contents (0.9-2.4 pmol).
A total of three replications (15-21 determinations) from three different batches
of microsomes were used for each treatment group.
Fused rocket irnmunoelectrophoresis was determined according to
Wheelock and Scott “1, except that samples were allowed to diffuse for 5 h at
8°C in a humidified chamber and electrophoresed for 25 h. Four picomoles of
purified P-450 from S+ flies (18.2 pl) after PB exposure were diluted with buffer
to a total volume of 30 pl, and 2.64 pmol (30 pl) of purified P-450 from flies
without PB exposure were used as undiluted. Four picomoles of purified P-450
from LPR flies before (6.4 p1) and after PB exposure (7.7 pl) were diluted with
Purification of Housefly P-450s 5
Elution Volume (ml)
Fig. 1 . DEAE-5PW HPLC chromatograms of partially purified microsomes from susceptible (S? and
resistant (LPR) houseflies with ( fPB) and without PB (CK) treatment. Flow rate was 0.5 ml/min with
a 30-min linear gradient of sodium acetate from 0-0.5 M in 20 mM Tris-acetate buffer (pH 7.5)
containing 20% glycerol and 0.4% Emulgen 91 1 Peaks A, B, and C contained the void volume,
P4501,,, and cytochrome b.
buffer to a total volume of 15 pl. Fifteen and twenty microliters of purified P450
from LPR and S + microsomes, respectively, were loaded onto each well at the
agarose plates.
6
Scott and Lee
NHTTerminal Sequence Analysis
Approximately 500-1,000 pmol of purified P-450s from each treatment group
were prepared for NH2-terminal sequencing. Due to the low yield of purified
P-450s in the untreated (145 pmol) and PB-induced (604 pmol) S t houseflies,
a total of 10 or 5 HPLC cycles, respectively, were performed to obtain enough
materials for NH2-terminal sequence analysis. Purified cytochrome P-450 was
pooled and diluted 1:l with buffer [lo] and reinjected onto the DEAE anion
exchange column for further purification. Peaks from reinjected samples were
then dialyzed in 1 1 of 10% isopropanol in 50 mM ammonium acetate buffer at
4°C for 4.5 h. This sample was then frozen in a polypropylene tube for 2 h at
-80°C and lyophilized in a speed-vac which was attached to a lyophilizer to
regulate the drying temperature and pressure for at least 15 h. The drying
temperature and pressure were - 60°C and 60 torr, respectively. The dried and
oily pellet which still contained Emulgen 911, glycerol, and Tris was washed in
2 ml of (1:l) isopropanol and acetone for 10 min and was centrifuged at
maximum speed in a Fisher Scientific micro-centrifuge (model 235C) for 10 min.
The supernatant (1,900 pl) was then withdrawn and discarded and the washing
process was repeated three more times. The last traces of solvent were then
dried under a gentle stream of nitrogen. The dried pellet was resuspended in
80 p,l of (2:l) absolute alcohol and 80% formic acid and sequenced by Cornell
Biotechnology Laboratories.
TrnmunoinhibitionStudies
Seventy picomoles of total P-450 from the control and PB-treated S + crude
microsomes were preincubated with different volumes (0, 0.5, 1, 2, 4, 8, 16, and
32 p1) of anti-P-4501,~antiserum (3.2 pg protein/pl) at 32°C for 30 min. Normal
rabbit antiserum which had been partially purified twice wth ammonium
sulfate precipitation [19] was used to adjust the volume of total antisera to 32 pl
in the incubates and 68 p1 of 0.1 M sodium phosphate buffer (pH 7.5) was added.
Seventy-one microliters ( = 50 pmol of total cytochrome P-450 content) of this
incubate were then aliquoted and used for assay of ECOD activity as described
earlier.
Statistical Analysis
Student’s t test [ZO] was used to compare the appropriate treatment means.
The criterion of significance was P 6 0.05.
RESULTS AND DISCUSSION
ChromatographicBehavior
Figure 1 shows the typical DEAE-HPLC chromatograms from control and PB
induced S t and LPR houseflies. Three major peaks (A, B, and C) were seen in
all chromatograms which agrees with previous work [lo]. Peak A was the void
fraction and contained low levels of P-450s (data not shown). Peak B contained
P-450. Peak C contained cytochrome b5. The retention times for all peaks were
identical for both strains with or without PB treatment.
Purification of Housefly P-450s 7
Differences in the DEAE-HPLC profiles of P-450s were found between S +
and LPR, and between S + control or PB treatments (Fig. 1) Peak B was less
abundant in microsomes from S + flies compared to LPR. However, Peak B was
increased after PB treatment only in the S + strain. There was no clear evidence
that PB treatment resulted in the production of new forms of P-450s in the S+
and LPR houseflies based on the HPLC chromatograms. In contrast, Imaoka
and Funae [21] reported that four forms of P-450s were resolved on a DEAE5PW column (similar conditions to the present study) in rat liver microsomes,
and that PB treatment resulted in production of two new forms of P-450s
(CYP2B1 and CYP2B2).
It is possible PB induced forms of P-450s that eluted in the void fraction of the
DEAE5PW column in the S+ and LPR strains. According to Imaoka and Funae
[21], the eluate collected from the void volume of the DEAE-5PW column could be
resolved into two peaks (two forms of P-450s) using cation exchange column
chromatography such as a SP-5PW column in PB-induced rat liver microsomes.
Fisher and Mayer [22] partially purified a P-450 from PB-induced Rutgers diazinon
resistant strain of housefly that was not retained on a DEAE-cellulosecolumn. Also,
Ronis et al. [23] isolated three different forms of P450s from PB-induced Rutgers
diazinon resistant houseflies and again these P-450s did not bind to a DEAE-cehlose column. Thus, it would be interesting to see if the void fraction collected from
the DEAE5PW column could be further resolved using additional purification
steps, to determine whether any new forms of P450s were induced after PB
treatment in these strains.
We next investigated whether Peak B was responsive to 3-MC which represents a different class of inducer in mammals. Results from the DEAE-HPLC
chromatograms (Fig. 2) indicated that 3-MC had little or no effect on the level
of Peak B and did not cause the production of any new forms of P-450s that were
resolvable by the HPLC techniques. Due to these results and the refractory
nature of cytochrome P-4501,, to PB treatment, we did not investigate the effect
of 3-MC on LPR houseflies.
A summary of the purification of cytochrome P-450s from the untreated and
PB-induced S and LPR houseflies is given in Table 1. The specific content of
purified P-450s in S and in LPR before and after PB treatment were 3.11, 6.68,
7.60, and 7.15 nmol/mg protein, respectively. The specific content of the P-4501,,
obtained in the present study (7.60 nmoVmg protein) was lower than that (14.4
nmolhg protein) reported by Wheelock and Scott [lo]. These values are less
than the theoretical maximum of 18.5 nmol/mg for protein with a molecular
mass of 54,400 daltons (see following). This could be due to the presence of
minor proteins or cytochrome P-450 apoprotein [24].
In the S + strain, a final yield of 2.3 and 5.5% of purified P-450s were obtained
in the control and PB-treated crude microsomes, respectively, while higher
yields were obtained in the control (16%)and PB-treated (13%)LPR microsomes
(Table 1). Similar yields of P-4501,, from untreated LPR microsomes have been
reported by Wheelock and Scott [lo].
+
+
Spectral Properties
All four purified P-450s exhibited peaks at about 447 2 0.5 nm in the reduced
CO-difference absorbance spectrum (Fig. 3) suggesting they are similar P-450s.
8
Scott and
Lee
Elution Volume (rnl)
Fig. 2. DEAE-5PW HPLC chromatogramsof partially purified S + microsonieswith and without 3-MC
treatment. Flow rate was 0.5 mlimin with a 30-inin linear gradient of sodium acetate from 0-0.5 M
in 0.02 M Tris-acetate buffer (pH 7.5) containing20% glycerol and 0 ~ 4 %Emulgen 91 1 . Abbreviations
as in Figure 1 .
The absorbance maximum (447 nm) of P-4501,~.from the untreated LPR
houseflies agrees with a previous report [lo]. It is interesting to note that the
absorbance maxima of the purified P45Os are lower than those obtained
from partial purification of other housefly cytochrome P-450s. For example,
a P-450 which has an absorbance maximum of 452 nm was isolated from
PB-induced, Rutgers diazinon resistant housefly strain [22,23] while three
P-450s which have an absorbance maxima of 450, 452, and 453 nm were
partially purified from an untreated insecticide susceptible housefly strain
[25]. These results reflect the presence of multiple forms of P-450s in different
housefly strains. None of our purified P-450s showed any peaks at 420 nm
in their CO-difference absorbance spectra, suggesting there was no conver-
S+
LPR
LPR
S+
S+
LPR
LPR
S+
S+
LPR
LPR
S+
LPR
LPR
S+
S+
LPR
LPR
S+
S+
Strain
Treatment
Control
PB
Control
PB
Control
PB
Control
PB
Control
PB
Control
PB
Control
PB
Control
PB
Control
PB
Control
PB
*
*
"
Total
protein
(mg)
15
0.0
f 0.0
15
f 0.0
15
f 0.0
15
22.1
f 0.3
1.2
23.2
i 2.4
25.1
22.6
k 1.6
c 0.4
10.4
12.0
f 0.6
f 0.7
12.8
11.8
-+ 1.8
1.37 f 0.05
1.72 ? 0.02
1.96 f 0.15
2.08 f 0.07
0.0472 2 0.0025
0.0899 f 0.0070
0.251
0.034
0.258 2 0.013
*
Total
P-450
(nmol)
6.25 f 0.48
11.0 f 1.0
12.3
0.8
14.0 k 1.3
6.25 f 0.48
11.0 2 1.0
12.3 f 0.8
14.0 f 1.3
4.80 f 0.43
6.17 c 0.91
7.75 f 0.61
7.94 f 0.78
2.54 f 0.23
4.00 f 0.37
4.83 k 0.59
5.26 i 0.58
0.145 & 0.012
0.604 f 0.081
1.92 f 0.31
1.84 f 0.06
7.60 f 0.17
7.15 ? 0.40
6.68 f 0.55
*
*
Specific
content
(nmoVmg)
0.42 f 0.03
0.73 0.07
0.82 f 0.05
0.93 ? 0.08
0.28 f 0.02
0.47 2 0.02
0.49 f 0.05
0.62 i 0.06
0.47 k 0.06
0.51 0.08
0.61 f 0.04
0.69 f 0.06
1.85 f 0.19
2.33 f 0.25
2.44 f 0.11
2.51 f 0.21
3.11 f 0.41
100
100
100
100
77
56
63
57
41
36
39
38
2.3
5.5
16
13
-
-
-
("/.I
-
Yield
*Dataare mean ? S.E. of three separate purification runs from three different batches of microsomes.
Trotein d u e s from crude microsomes were determined according to Bradford protein assay.
bProtein values from crude microsomes, PEG supernatant, Phenyl-5PW coIumn, and DEAE-5PW column were determined according to 3CA
protein assay.
HPLC
DEAE-5PW columnb
HPLC
Phenyl5PW columnb
8%PEG supernatantb
Crude microsomesb
Crude microsomesa
Purification
step
TABLE 1. Purification of P450s From Abdominal Microsomes of Insecticide Susceptible (S +) and Insecticide Resistant (LPR) Strains of
HouseflyWith and Without PB Treatment*
10
Scott and
Lee
0.1
A
-0.1
~
400
450
500
Wavelength (nrn)
Fig. 3 . The reduced CO-difference absorbance spectra of the purified P-450s from untreated LPR
(A), PB-treated LPR (B), PB-treated S + (C), and untreated S f (D)housefly rnicrosomes. Abbreviations
as in Figure 1 .
sion of P-450s to cytochrome P-420 during the purification. The relative heights
of the absorbance peaks in the CO-difference absorbance spectra are consistent
with the corresponding peak heights observed in the DEAE-HPLC chrornatograms (Fig. 1).
Electrophoretic Properties
All four purified P-450s from control and PB treated houseflies represented
a single major proteiiband with an apparent molecular mass of 54,400 daltons,
although minor bands at lower molecular masses were observed (Fig. 4). In
another experiment, the wells in a 10% nongradient polyacrylamide gel were
loaded with all possible pairs of purified P-450s to further compare their
electrophoretic properties. The results (Fig. 5) showed that all of the P-450
combinations comigrated together as a single band at 54,400 dalton, indicating
that all of these P-450~were electrophoretically identical. The apparent molecular
masses of P450s isolated from the S + (insecticidesusceptible) and LPR (insecticide
resistant) strains, respectively, are similar to the P-4501,~isolated by Wheelock and
Scott [lo], but are different from that obtained with the Rutgers diazinon resistant
strain (60,000 dalton [22]; 49,000 dalton [23]) and a CSMA insecticide susceptibIe
strain (43,000, 48,000, and 53,000 daltons [El).
Purification of Housefly P-450s 11
Fig. 4. SDS-PACE of crude S + and LPR microsomes and purified P-450~with and without PB
treatment on a 10-1 5 % gradient gel. Crude microsomes(50 kg) were electrophoresed in lanes 2 (S t
CK), 3 (S + PB), 6 (LPR CK), and 7 (LPR PB)and purified P-450s (1 pg) each in lanes 1 (5 CK), 4 (S
PB), 5 (LPR CK), and 8 (LPR PB). Standard molecular protein markers (lane 9 ) : rabbit muscle
phosphorylaseb, 97,400; bovine serum albumin, 66,200; hen eggwhiteovalbumin, 42,699; bovine
carbonic anhydrase, 31,000; and soybean trypsin inhibitor, 21,500. Abbreviations as in Figure 1.
+
+
Immunological Reactivity
A monospecific polyclonal antiserum (8-241) raised against P-450lpr [lo]
was used to examine the homology between the purified P-450s. Immunoblotting analysis (Fig. 6) showed that anti-P-4501,, antiserum recognized a
single major protein band of identical molecular mass in crude microsomes
of the S + and LPR houseflies before and after PB treatment. The immunostaining intensity of this protein band was stronger in the LPR houseflies
compared to the S + strain (compare lanes 3 and 7) and was significantly
increased in S + houseflies treated with PB (compared to untreated S +
flies-for example, lanes 6 and 7), but not in the LPR houseflies (compare
lanes 2 and 3). These results suggest that these proteins share at least one
epitope recognized by the P-45Olpr antiserum, and are consistent with the
data obtained from the DEAE-HPLC chromatograms.
The purified P-450s were compared to P-450ip, for immunological relatedness using fused rocket immunoelectrophoresis. As shown in Figure 7, all
~
immunological identity with
purified P-450s fused with P - 4 5 0 1 ~indicating
P-4501p.
12
Scott and Lee
Fig. 5. SDS-PAGE of purified P-450s on a 10% nongradient gel. Lane 1: Purified P-4505+untreated(1
pgj. LaneZ: Purified P-450,+PB treated (1 pg). lane 3: Purified P-4501,~untreated (1 pg). lane4: Purified
P-45U1,,, PB treated (1 pg). lane 5: Purified P-45o5+untreated(0.5 Fg) and P-4505+PBtreated (0.5 pg).
Lane 6: Purified P-450,. untreated (0.5 p,g) and P-4501,, untreated (0.5 pgj. Lane 7: Purified P-45Q+
untreated (0.5 pg)and P-4501,~PBtreated (0.5 pg). Lanel: Purified P-4SOS+PBtreated (0.5 pg)and P-4SqP,
untreated (0.5 pg). Lane% Purified P-45OS+PBtreated (0.5 pg) and P-450prPBtreated(0.5 pg). Lanelo:
Purified P-4501,,untreated (0.5 Fg) and P-4501,~PB treated (0.5 pg). Lane 11: Standard molecular weight
markers (rabbit muscle phosphorylase b, 97,400; bovine serum albumin, 66,200; hen egg white
ovalbumin, 42,699; bovine carbonic anhydrase, 31,000; and soybean trypsin inhibitor, 21,500).
NHTTerminal Sequence
The NH2-terminal amino acid sequences of the purified P-450s were identical
to each other in the first 19 positions from their amino terminal ends:
MLLLLLLIVVTTLYIFAKL (Table 2). This agrees with the sequence previously
reported for P-4501p, [26]. This further supports the idea that these four proteins
are very closely related or identical. The P-450s purified here had a high
hydrophobic nature in the NH2-terminal region. Specifically, 15 (79%)of the 19
amino acid residues in the N-terminus were hydrophobic amino acids. Interestingly, 42% of the amino acids in this region were leucine. There was no significantly
homology of the purihed housefly P-450s with any other insect P45Os and the
known PB-inducible P-450~in mammalian species. Thus, it appears that P-4501,,
may belong to a different family of the P-450 gene superfamily, although cloning
of this P-450 will be necessary to confirm this idea.
Immunoquantitation of P - 4 5 0 1 ~ ~
Levels of the purified P-450s in crude microsomes were quantified using RIE.
The constitutive levels of the immunoreactive P-450s were 21 and 80% of the
Purification of Housefly P-450s 13
Fig. 6. lmmunoblots of purified P-450s and crude microsomes with anti-P-4501p,antiserum. lane 1 :
Purified P-450r,, from PB-treated LPR houseflies (1 pg). lane 2: Microsomes from PB-treated LPR
houseflies(50pg). Lane3: Microsomesfrom untreatedLPR houseflies(50pg). lane4: PurifiedP-4501,~
from untreated LPR houseflies (1 pg). lane 5: Purified P-450 from PB-treated S + houseflies (1 pg).
Lane 6: Microsomesfrom PB-treated S houseflies(50 pg). lane 7: Microsomesfrom untreated S+
houseflies (SO pg). Lane 8: Purified P-450 from untreated S houseflies (1 pg). Abbreviations as in
Figure 1.
+
+
total content of P-450s in the S + and LPR houseflies, respectively (Table 3).
There were 7.5-fold higher immunoreactive P-450s in LPR compared to S +
houseflies. The values for LPR are in general agreement with a previous
study; however, the level of immunoreactive P-450 reported here for S + is
higher, and thus the difference between strains was less than previously
reported [181. PB significantly increased the level of immunoreactive P-450s
in the S + houseflies by twofold, but had no effect on the level of P - 4 5 0 1 in
~~
the LPR houseflies (Table 3). This is consistent with the results of the DEAEHPLC chromatograms (Fig. l), SDS-PAGE (Fig. 4), and immunoblotting (Fig.
6). After PB induction, the level of the immunoreactive P-450s represented
40% of the total microsomal P-450s in the S + houseflies. It is interesting to
note that although the specific content of the total P-450s in the S + strain
after PB exposure (0.73 i 0.07 nmolimg protein) was increased to 89% of the
level found in the control LPR strain (0.82 -+ 0.05 nmol/mg protein), the
specific content of the immunoreactive P-450 in the S -t strain was only 40%,
which is still less than found in the LPR strain. This suggests that PB induced
an immunoreactive P-450 (i.e., P-4501,~)as well as other P-450s in the S +
strain. This is consistent with other workers’ [22,25] findings of PB-induced
14
Scott and Lee
Fig. 7. Fused rocket immunoelectrophoresisof partially purified cytochrome P-450s from 5 f and
LPR microsomeswith and without PB treatment. Each pair shows P-4501,~(4 pmol, 6.4 PI)on the left
and test samples on the right. Samples were obtained from peak B on the DEAE-SPW HPLC
chromatograms(Fig. 1). Sample volumes used were (A) P-450~,,untreated 6.4 pI(4 pmol), (B)P-4501,~
PB-treated 7.7 pI (4 pmol), (C)P-450,, untreated 20 FI (1.8 pmol), and (D) P-4505+PB-treated18.2
PI (4 pmol). Samples of P-4501,, untreated and PB treated were diluted to 15 pI and loaded onto the
wells. Twenty microliters of P-450,+ untreated was loaded as undiluted while the sample of P-450,+
PB treated was first diluted to 30 pI and 20 pI was loaded onto the wells at the agarose plates.
Abbreviations as in Figure 1.
P-450s in the void volume of DEAE column in other PB-treated housefly strains,
and for results of CYP6A1 mRNA expression [7].
After the levels of immunoreactive P-450s in the crude microsomes were
quantified by ME, we calculated the percentage recovery of the purified proteins (DEAE5PW Peak B) with respect to the starting crude microsomes. The
percentage recovery of purified P-450s from S + and LPR microsomes before
and after PB treatment were 11 2 2,13 1,ZO -+ 5, and 17 2 2%, respectively.
*
Immunoinhibition of ECOD Activity in S + Microsomes
It was previously shown [8] that metabolism of ECOD was markedly increased (tenfold) in S + housefly microsomes after PB treatment. We wanted to
determine if the induced ECOD activity was correlated with the twofold increase in immunoreactive P-450 in S + houseflies after PB exposure. As shown
in Figure 8, anti-P-4501, antiserum half-maximally (Ic50) inhibited the metabolism of this substrate at 4.4 pl of antiserum (14 pg IgG) and almost completely
inhibited (> 90%) the reaction at 16 pl of antiserum (51 pg IgG) in the untreated
S + microsomes. In contrast, a fourfold higher concentration of anti-P-4501,~
antiserum was needed to obtain half-maximally inhibited ECOD activity in the
PB-treated S + microsomes. Also, only 35 and 74% of ECOD was inhibited at 16
and 32 pI, respectively. This suggests that P-450 immunoreactive to anti-P-4501,,
Leu
Leu
Leu
Leu
Leu
Leu
3
Leu
Leu Leu
Leu
Leu
Leu
Leu
Ile
Phe
Ile
Ile
Ile
Ile
Leu
Leu
Leu
Leu
Val
Val
Val
Val
Ser
Tyr
Leu
Leu
Val
Val
Val
Val
Thr
Ala
Ala
Ala
10
Thr
Thr
Thr
Thr
Ala
Leu
Leu
Leu
Thr
Thr
Thr
Thr
Leu
Gly
Leu
Leu
Leu
Leu
Leu
Leu
Phe
Val
Val
Val
Tyr
Tyr
Tyr
Tyr
Ile
Leu
Gly
Gly
Phe
Phe
-
Ile
Ile
Ile
Ile
Val
15
Phe
Phe
Phe
Phe
Thr
Ala
Leu
Leu
Leu
Leu
-
AIa
AIa
AIa
Ala
Phe
Lys
Lys
Lys
Lys
Leu
Ser
Leu
Leu
Leu.
Leu
Leu
Leu
Leu
Phe
19
Control
PB
Control
PB
S+
k
0.14(15)
0.42 f 0.03 (9)
0.73 f 0.07(9)
0.82 k 0.05 (9)
0.93 0.08 (9)
0.087 f 0.01 (21)
0.30 f 0.03(26)
0.65 2 0.07(16)
0.76
Total P450sb
(nmoYmgprotein)
p-454p:
(nmoVmgprotein)
P-450~~~
20.7 f 1.2
40.8 f 2.9*
80.3 t 8.5
80.9 f 9.5
(% of total P-450s)
'Data represent mean t S.E. of three replications using three different batches of microsomes. Number in parentheses indicates the number
of determinations.
aP-4501, is the untreated LPR houseflies, or P450s immunoreactive to anti-P-4501p,antiserum in the untreated S i,
PEtreated S + , and PBtreated LPR houseflies.
bSpecific activity of total P-450~with protein determination based on Bradford assay.
'Signhcantly different from 1(P < 0.05) compared to its corresponding control as determined by Student's t test.
LPR
Treatment
Strain
TABLE 3. Rocket ImmunoelectroDhoresis of Crude Microsomes From S + and LPR Houseflies With and Without PB Treatment'
*The underlined amino acids represent homology between the purified housefly P-450~and the selected P-450s. Amino-terminal sequences of
P-45OS+, P-4501,,, and f-45o1,, (PB) were determined from one replication while amino-terminal sequence of P-45OS+(PB) was determined from
two replications.
"(PB)represents P-450s purified from PB-treated S + or LPR microsomes.
m e amino-terminal sequence of P-4501,, has already been reported [17].
'Amino-terminal sequence of cDNA clone isolated from hypertrehalosemic hormone-induced cockroach (Blabwus)strain [30].
dAmino-terminalsequence of a cDNA clone isolated from PB-induced Rutgers diazinon resistant strain of housefly ( M u m dumestica) [31].
eAmino-terminalsequences of purified rat liver P-450~were taken from Guengerich [9].
Met
Met
Met
Leu
Leu
Leu
Leu
Ile
Gly
Ser
Ser
Leu
Leu
Leu
Leu
Thr
Ser
Ile
Ile
Leu
Leu
Leu
Leu
Phe
Phe
Pro
Pro
Met
Met
Met
Met
P450s+
P45OS+(PB)"
PW,,~
P4501pr(PB)a
CYP4ClC
CYP6Ald
CYP2B1'
CYPZB2u2e
Leu
Leu
Leu
Leu
Glu
Asp
Glu
Glu
5
1
Cytochrome
TABLE 2. Comparison of NHTTenninal Sequences of Untreated and PB-Treated S + and LPR Housefly P450s With Other Selected P45Os*
16
Scott and Lee
100
n
80
E
0
.L
.
ICI
C
60
.cI
r
L
8
40
20
0
0
5
10
15
20
25
30
35
pl antisera
Fig. 8. lmmunoinhibition of microsomal ECOD activity with anti-P-4501,, antiserum. The control
microsomes (50 pmol) were incubated with various volumes of anti-P-45O1,,
and PB-treated S
antisera at 32°C for 30 rnin. This was followed by estimationof ECOD activity as described in Materials
and Methods. The open and closed symbols represent incubation of control and PB-treated microsomes, respectively. The basal levels of uninhibited ECOD activity in the control and PB-treated S +
microsorne~were32.5 f 5.1 and243 2 20prnol/min/nmol P-450s, respectively. Abbreviationsasin
Figure 1.
+
played a major role in the metabolism of 7-ethoxycoumarin in the uninduced
S + houseflies, but not in the PB-treated S+ flies. If PB increased only the
immunoreactive P-450s in S microsomes by twofold, one should expect to see
a curve similar to that obtained in untreated S+ microsomes, but shifted to its
right by twofold. However, this was not the case. One possible explanation may
be that treatment of S + houseflies with PB not only increased the level of
immunoreactive P-450, but also increased other non-immunoreactive P-450s
which were involved in metabolism of this substrate. This agrees with other
data we have presented and suggests that ECOD is not a P-4501,, isoforrn
specific assay in housefly microsomes.
It has been pointed out that insecticide resistance and phenobarbital induction have several characteristics in common [27]. However, P-450 isoforrn
specific assays were not avaiable until recently to check this idea. Our results
suggest that this idea may have merit, as P-45Olpr is inducible by PB in susceptible houseflies, but not in the insecticide resistant LPR strain. Recently, the gene
for a resistance related P-450, CYP6A1, has been shown to be PB inducible in
both susceptible and resistant houseflies. If CYP6A1 is actually involved in
conferring insecticide resistance, then it may be possible that overexpression of
CYP6A1 in the resistant Rutgers strain is controlled differently than the overexpression of P-4501,, in the resistant LPR strain.
In conclusion, a P-450, indistinguishable from P-4501,~was purified from S +
and LPR strains of housefly. The purified P-450s could not be differentiated
based on the chromatographic behavior, spectral properties, electrophoretic
properties, immunological reactivity, or partial NH2-terminal sequences, indicating that these P-450s are likely the same. P-45Olpr was inducible by PB in S +
but not in LPR flies. Although the four purified P-450s could not be distin-
+
Purification of Housefly P-450s 17
guished, there is a small possibility that they could be different forms. For
example, in rat livers, CYP2B1 and CYP2B2 (PB-inducible cytochrome P-450s)
had similar chromatographic behavior, molecular masses, and NH2-terminal
sequences [9,28,29]. Yet CYP2B1 and CYP2B2 have different catalytic properties
due to differences in 13 of 491 amino acids. Therefore, full protein sequences
will be needed to further confirm the identity of the P-450s purified in the
present study.
LITERATURE CITED
1. Wilkinson CF: Role of mixed-function oxidases in insecticide resistance. In: Pest Resistance to
Pesticides. Georghiou GP, Saito T, eds. Plenum Press, New York, pp 175205 (1983).
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3. Agosin M: Role of microsomal oxidations in insecticide degradation. In: Comprehensive Insect
Physiology Biochemistry and Pharmacology. Kerkut GA, Gilbert LI, eds. Pergamon Press,
New York, Vol 12, pp 647-712 (1985).
4. Hodgson E: Microsomal mono-oxygenases. In: Comprehensive Insect Physiology Biochemistry and Pharmacology. Kerkut GA, Gilbert LI, eds. Pergamon Press, New York, Vol 11, p p
293-321 (1985).
5. Schonbrod RD, Terriere LC: The solubilization and separation of two forms of cytochrome
P-450 from the house fly, Musca Domestica L. Biochem Biophys Res Commun 64, 829 (1975).
6. Naquira C, White RA, Agosin M: Multiple forms of Dvosophila cytochrome P-450. In: Biochemistry, Biophysics and Regulation of Cytochrome P-450. Gustafsson JA, Carlstedt-Duke J, Mode
A, Rafter J, eds. Elsevier, Amsterdam, pp 105-108 (1980).
7. Carino F, Koener JF, Plapp FW Jr, Feyereisen R: Expression of the cytochrome P450 gene
CYP6A1 in the housefly, Musca domestica. In: Molecular Mechanisms of Insecticide Resistance
Diversity Among Insects. Mullin CA, Scott JG, eds. American Chemistry Society Washington,
DC, pp 3140 (1992).
8. Lee SST, Scott JG: Microsomal cytochrome P450 monooxygenases in the house fly (Musca
domestica L.): Biochemical changes associated with pyrethroid resistance and phenobarbital
induction. Pestic Biochem Physiol35, 1(1989).
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78, 316 (1985).
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(Diptera: Muscidae) microsomes. J Econ Entomol 82, 1559 (1989).
18
Scott and Lee
14. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248
(1976).
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its hemoprotein nature. J Biol Chem 239, 2370 (1964).
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their inducibility by phenobarbital in the house fly, Muscu domesticu. Insect Biochem Mol Biol
22, 699 (1992).
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633 pp (1980).
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P-450 from an insecticide resistant strain of house fly (Musca domesticu). Pestic Biochem
Physiol32, 74 (1988).
24. Lange R, Perin F, Larroque C, Zajdela F: Isolation and partial characterization of a cytochrome
P-450 isoenzyme (cytochrome P-45OtU)from mouse liver tumors. Biochem Biophys Acta 1038,
130 (1990).
25. Capdevila J, Agosin M: Multiple forms of housefly cytochrome P-450. In: Microsomes and
Drug Oxidations. Ullrich V, ed. Pergamon Press, New York, pp 144-151 (1976).
26. Scott JG, Wheelock GD: Characterization of a cytochrome P-450 responsible for pyrethroid
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Among Insects. Mullin CA, Scott JG, eds. American Chemical Society, Washington, DC, pp
16-30, 1992).
27. Terriere LC: Enzyme induction, gene amplification and insect resistance to insecticides. In:
Pest Resistance to Pesticides. Georghiou GP, Saito T, eds. Plenum Press, New York, p p 265-297
(1983).
28. Fujii-Kuriyama Y, Mizukami Y, Kamajiri K, Sogawa K, Muramatsu M: Primary structure of a
cytochrome P-450: Coding nucleotide sequence of phenobarbital-inducible cytochrome P450
cDNA from rat liver. Proc Natl Acad Sci USA 79, 2793 (1982).
29. Yuan PM, Ryan DE, Levin W, Shively JE: Identification and localization of amino acid
substitutions between two phenobarbital-inducible rat hepatic microsomal cytochromes P450 by micro sequence analyses. Proc Natl Acad Sci USA 80, 1169 (1983).
Purification of Housefly P-450s 19
30. Bradfield JY, Lee Y-H, Keeley LL: Cytochrome P450 family 4 in a cockroach: Molecular cloning
and regulation by hypertrehalosemic hormone. Proc Natl Acad Sci USA 88, 4558 (1991).
31. Feyereisen R, Koener JF, Farnsworth DE, Nebert DW: Isolation and sequence of cDNA
encoding a cytochrome P-450 from an insecticide-resistant strain of the house fly, Muscu
dornesticu. Proc Natl Acad Sci USA 86, 1465 (1989).
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