Purification and characterization of a cytochrome P-450 from insecticide susceptible and resistant strains of housefly Musca domestica L. before and after phenobarbital exposureкод для вставкиСкачать
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 . 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 , 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 , 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 . The major pyrethroid resistance mechanism in the LPR strain appears to be cytochrome P-450 monooxygenase-mediated detoxification . 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 . 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  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 . 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  as described by Lee and Scott .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 . Rocket and Fused Rocket Immunoelectrophoresis RIE of crude microsomes and purified P-450s were performed as described by Wheelock and Scott  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  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  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 , 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  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.  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 . 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 . 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 ; 49,000 dalton ) 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, . 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 . 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  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 . 'Amino-terminal sequence of cDNA clone isolated from hypertrehalosemic hormone-induced cockroach (Blabwus)strain . dAmino-terminalsequence of a cDNA clone isolated from PB-induced Rutgers diazinon resistant strain of housefly ( M u m dumestica) . eAmino-terminalsequences of purified rat liver P-450~were taken from Guengerich . 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 . 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]. 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