cytochrome p-450 in insects: 1. differences in the forms present in insecticide resistant and...

PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY 12, 239-248 (1979)

Cytochrome P-450 in Insects

1. Differences in the Forms Present in Insecticide Resistant and Susceptible House Flies’

S. J. Yu AND L. C. TERRIERE

Received May 4. 1979, accepted August 9. 1979

Soluble cytochrome P-450 prepared from the microsomal fraction of abdomen homogenates of an insecticide resistant strain (Rutgers) and a susceptible strain (NAIDM) of the house fly, Mttsca dontesrictr L., was characterized by spectral and electrophoretic methods. Six chromatographically distinct fractions were obtained after chromatography on DEAE-cellulose and hydroxylapatite. Examination of the six fractions by difference spectrophotometry indicated that the wave lengths for maximum absorption of the cytochrome P-450-carbon monoxide complexes were at 450.451. and 452 nm for the NAIDM fractions and at 449, 450, and 451 nm for the Rutgers fractions. The type II binding spectra of the cytochrome P-450 in each fraction were measured with n-octylamine. Several of ‘these resembled spectra which, in studies of hepatic cytochrome P-450, have been shown to be due to the presence of the high spin form of this hemoprotein. Four of the fractions from the resistant strain were of this type compared to one from the susceptible strain. Elec- trophoresis experiments indicated that there were at least three hemoproteins in the 40,OOG60,000 molecular weight range in the fractions from the resistant strain while four could be detected in those from the susceptible strain. The specific aldrin epoxidase activity of the most active Rutgers fractions was considerably higher than that of similar fractions from the NAIDM microsomes in reconstitution experiments.

INTRODUCTION

The importance of the microsomal monooxygenase system as a mechanism of resistance to insecticides in the house fly and other species has been recognized for several years (1). Evidence that the increased detoxication capacity of resistant strains is due to changes in properties of cytochrome P-450 was summarized by Hodgson rt ml. (2). The major differences cited were a generally lower wave length of maximum absorption (A,,,) of the reduced cytochrome P-450-carbon monoxide binding complex by the hemoprotein of resistant strains, an increased cytochrome P-450 content in microsomes of the resistant strains, and a failure to form type I substrate binding spectra with the hemoprotein of susceptible strains. On the basis of this and other evidence. it was suggested that two different groups of cytochrome P-450 were involved, one predominating in

’ Oregon Agricultural Experiment Station Technical Paper No. 5088.

resistant strains and the other in susceptible strains.

More recently it has been reported that microsomes of both susceptible and resistant house fly strains contain at least two forms of the hemoprotein (3), while another laboratory (4) reported the detection of at least three forms of this hemoprotein in a susceptible strain. In our earlier comparison of P-450 from R and S strains (3) we found differences in the A,,,, values of the cytochrome P-450-CO complex and in the shape of the type II binding spectrum produced with n-octylamine as ligand.

A review of recent papers comparing the microsomal oxidase activity of resistant and susceptible house flies provides strong support for the theory that qualitative differences in the cytochrome P-450 of such strains are more important than the quantitative difference. In no case among the studies known to us has the cytochrome P-450 content of microsomes isolated from resistant strains been more than 2.5 times

239 004%3575/79/070239-10$02.00/0 Copyright i 1979 by Ac:idemic Prers. Ini All nghtr of reproductwn m any form rewved.

240 YU AND TERRIER!?

that of microsomes prepared from susceptible strains (2, 3, 5-8). On the other hand, differences in microsomal oxidase activity reported in such studies ranged from about 4- to 25-fold (1, 5, 6, 9, 10).

In this report we provide further evidence that the cytochrome P-450 of the house fly exists in multiple forms and that the hemoprotein from a strain which owes much of its resistance to increased microsomal oxidase activity differs in several re- spects from that of a susceptible reference strain.

MATERIALS AND METHODS

Insects Two strains of the house fly, M~~.sca

domestica L., were used in the experiments, the NAIDM strain which is susceptible to insecticides and has a normal level of microsomal oxidase activity and the Rut- gers strain which is resistant to diazinon and has a high level (about 10x) of microsomal oxidase activity. All experiments were with 7- to IO-day-old adults of mixed sex.

Twelve batches, 2 g each, of house fly abdomens were homogenized in 25 ml of 0.1 M sodium phosphate buffer, pH 7.8, containing 10% glycerol (v/v) in a motor- driven tissue grinder for 30 sec. The crude homogenates were filtered through four layers of cheesecloth, combined, and centrifuged at lOOOg,,, for 15 min in a Spinco Model L preparative centrifuge. The pellet (cell debris and nuclei) was discarded and the supernatant which had been filtered through glass wool was recentrifuged at lO,OOOg,,, for 15 min to remove mitochon- dria. The resulting supematant was again filtered through glass wool and centrifuged at lOS,OOO~,,, for 1 hr. The microsomal pellet was suspended by homogenization in 0.05 M Tris-HCI buffer, pH 7.5, containing 0.25 M sucrose, 1 mM dithiothreitol (DTT).” 0.1 mM EDTA, and 30% (v/v)

’ Abbreviations used: SDS. sodium dodecyl sulfate; DEAE. diethylaminoethyl; DTT, dithiothreitol; EDTA, ethylene diaminetetraacetate.

glycerol. The combined microsomal suspension was diluted to a final protein concentration of 10 mg/ml and stored overnight at -20°C under nitrogen.

Solubilixtion arid Chromcctogruphy oj Cytochrome P-450 The thawed microsomal suspension was

treated with sodium deoxycholate (0.2 mgimg protein) and stirred for 15 min. The mixture was then centrifuged at lOS,OOOg,,, for 1 hr and the pellet, which contained cytochrome P-420, was discarded. The supematant was diluted to a final concentration of 0.05% sodium deoxycholate and then applied to a DEAE-cellulose column (2.4 x 22 cm) previously equilibrated with 0.05 M Tris-HCl buffer, pH 7.5, containing 1 mM DTT, 0.1 mM EDTA, 20% (v/v) glycerol, and 0.05% sodium deoxycholate. This solution is referred to as Buffer A. The column was first washed with 150 ml of Buffer A and then eluted stepwise with increasing concentrations of KC1 (0.1,0.3, and 0.5 M) in Buffer A while collecting IO-ml fractions. Protein and heme were detected by continuous monitoring at 280 and 405 nm, respectively, with an Isco Mode1 UA-5 absorbance monitor.

The fractions absorbing at 405 nm were dialyzed for 3 hr against 20 vol of 0.01 M sodium phosphate buffer, pH 7.5, containing 0.1 mM DTT, 0.1 mM EDTA, and 20% (v/v) glycerol (designated as Buffer B). The dialyzed samples were then applied to hydroxylapatite columns (2.0 x 6.0 cm) previously equilibrated with Buffer B containing 0.05% (v/v) Emulgen 911 (Kao-Atlas Co., Ltd.). The column was first washed with 40 ml of Buffer B containing 0.05% (v/v) Emulgen 911 and then eluted stepwise with 50 ml of 0.1 and 0.3 M sodium phosphate buffer, pH 7.5, containing 0.1 mM DTT, 0.1 mM EDTA, 20% (v/v) glycerol, and 0.05% (v/v) Emulgen 911. Protein and heme were monitored as described above and lo-ml fractions were collected. The 405 nm absorbing fractions were dialyzed against 20 vol of Buffer B overnight. After dialysis, the samples were passed through Sephadex

CYTOCHROME P-450 IN HOUSE FLIES 241

LH-20 columns (2.4 x 9 cm) previously equilibrated with Buffer B to remove the excess Emulgen 911. The samples were then concentrated by ultra-filtration and stored at -20°C under nitrogen.

Anaiytical Procedures Cytochrome P-450 and P-420 concentra-

tions were determined by the method of Omura and Sato (11, 12). The n-octylamine difference spectra of cytochrome P-450 were determined as described previously (3). All spectra were measured in an Aminco DW-2 spectrophotometer in the split beam mode.

NADPH-cytochrome c’ reductase activity was measured as described previously (13). One unit of the reductase is defined as the amount of enzyme which catalyzes the re- duction of 47.6 nmol of cytochrome c per minute.

The epoxidation of aldrin was used as a measure of the activity of the cytochrome P-450 fractions. Unless otherwise stated, the j-ml incubation mixture contained 0.075 nmol of cytochrome P-450, 0.1 M sodium phosphate buffer (pH 7.5), 1.5 units of rat liver cytochrome L’ reductase (209 unitsimg protein, a gift of Dr. R. A. Neal, Vanderbilt University), 0.05 mg of dioleoyl phos-

0.5 -

T ’ 0.4-

L

z

s OJ 0.3-

‘j

.---I I I

t I ’ t ’ I f I

phatidyl choline, 0.2% glycerol, 250 nmol of aldrin in 0.1 ml methyl cellosolve, and an NADPH-generating system consisting of 1.8 nmol of NADP, 18 nmol of glucose-6- phosphate, and 1 unit of glucose-6- phosphate dehydrogenase. The incubations were carried out in an atmosphere of air with shaking at 34°C for 10 min. The epoxidation product, dieldrin, was extracted and analyzed by gas chromatography (14).

Protein was determined by the method of Bradford (15) using bovine serum albumin as standard.

Disc Gel Electrophoresis Sodium dodecyl sulfate (SDS)-polyacryl-

amide disc gel electrophoresis was per- formed according to the method of Laem- mli (16) except for the following modifica- tions: The IO-cm gels contained 5.6% acryl- amide and the stacking gel was omitted. The samples, approximately 20 nmol of cytochrome P-450/gel, were treated at room temperature with 1% SDS containing 10 m&I Tris-HCl (pH 8.0), 1 mM EDTA, 20% glycerol, and 0.001% bromophenol. /3- Mercaptoethanol was not used for the treatment because it inhibits the peroxidase-staining reaction (17). The samples were applied to preelectrophoresed

i i ---

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L I I I ’ : . I I

0.0 L k-o.1 M KCl+0.3M KCI+0.5M KU-j

242 YU AND TERRIERE

gels and electrophoresis was carried out with a current of 2 mA/gel under subdued light at 4°C. After electrophoresis, the gels were first stained for cytochrome P-420 peroxidase activity using tetramethyl ben- zidine and H,O, as described by Thomas et rrl. (17). They were then destained with sodium sulfite and stained for proteins using Coomasie brilliant blue according to Fairbanks et ul. (18).

Molecular weights were determined by comparison with bovine serum albumin (68,000), catalase (60,000), fumarase (50,000). alcohol dehydrogenase (41 .OOO), carbonic anhydrase (30,000). and trypsin (23,000) as standards.

RESULTS

Chronlatogmphy c!f’ Solubilized Microsomes The resolution of components released

when microsomes from the Rutgers house fly strain were solubilized is illustrated in Figs. 1. 2, 3, and 4. Elution of the mixture from DEAE-cellulose with Buffer A containing stepwise increases in KC1 concen-

0.5

-i- I

L 0.4 E

6 00

; 0.3

I I; 0.2

2 0 0

0.5

T I

L E 0.4 E

i?

“0 0.3

,’

I

j 0.2

z

z 0.1

6

0.0

,

1 I I

\ \ \ \

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f+O.l hi+0.3td+ Sodium Phosphate

I I I I

0 5 IO 15

0.5

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L 0.4

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T ; 0.2 c

:


tration results in the separation of three fractions absorbing in the 405nm region, fractions A, B, and C. Fig. 1. No additional 405nm absorbing fractions could be obtained when the KC1 concentration of the eluting buffer was increased above 0.5 M. Similar elution profiles were obtained with solubilized microsomes from the NAIDM strain.

As shown in Figs. 2, 3, and 4, each of the DEAE-column fractions is further resolved into two fractions on a hydroxylapatite column. These are referred to as fractions A,, A,: B,, B,: and C,, C,, respectively. Frac- tions A,, B,, and C, were eluted with 0.1 M phosphate buffer and A,, BZ, and C, with 0.3 M phosphate buffer. No additional cytochrome P-450 was eluted with buffer containing phosphate concentrations higher than 0.3 M. Similar elution profiles were obtained with the three DEAE-cellulose fractions from NAIDM house fly microsomes.

Spectrnl Properties of Colrrnm Fractions Tables 1 and 2 show the yield and spec-

tral properties of the cytochrome P-450 in the six fractions isolated from microsomes of the NAIDM and Rutgers house flies. The recovery of the hemoprotein in all fractions based on that present in the microsomal fraction before solubilization, was 9.67% in the case of the NAIDM strain and 13.9%, for the Rutgers strain. The greatest recovery was in the B, fraction in both cases.

The A,,, of the reduced cytochrome P- 450-CO difference spectra of the six fractions from microsomes of NAIDM flies were 450 (C,, C,), 451 (A,. B,), and 452 nm (A,, B,) whereas those from Rutgers flies were 449 (B,, C,), 450 (A,, G), and 451 nm (A.), B,). The C, fraction had the highest specific content of cytochrome P-450, amounting to three- and sixfold purification with respect to the P-450 content of the intact microsomes of NAIDM and Rutgers strains, respectively.

We used the n-octylamine-cytochrome P-450 difference spectrum as described by Jefcoate et trl. (19) to detect high and low spin forms of cytochrome P-450 in the six fractions. By this method a spectrum with a

244 YU AND TERRIERE

hmin at 392 nm and a h,,, at 427 nm indicates the presence of the high spin form while a Amin at 410 nm and a A,,, at 432 nm indicates the low spin form.

The results of the spin state experiments are summarized in Tables 1 and 2. Only one of the six column fractions obtained from the NAIDM microsomes contained a cytochrome P-450 with a distinct trough in the 392 region (fraction C,) whereas four of the fractions prepared from the Rutgers strain microsomes exhibited this charac- teristic (A,, B,, C,, and C,). The difference in the spin states of the P-450s from the two strains is confirmed by the n-octylamine binding spectrum of the intact microsomes (first entry in Tables 1 and 2). The trough in the case of the NAIDM microsomes is at 410 nm and in the case of the Rutgers microsomes, at 392 nm. For the most part, the h,,, values observed are in agreement with those reported by Jefcoate et ~1. (19). The exceptions are fractions C, and C,, Table 1, and fraction B,, Table 2.

Jefcoate er al. (19) have used the absorption differences, aA (392 nm-500 nm) and aA (410 nm-500 nm), to estimate the relative amounts of high and low spin cytochrome P-450 in a mixture. Their data, based on studies of rabbit liver microsomes, show a near linear relationship between the ratio LA 4lOihA 392 and the proportion of high spin to low spin form. We made similar calculations with the spectral differences found in our study of the six fractions to obtain the ratios shown in Tables 1 and 2. The significance of these values will be dis- cussed later in this report.

The degree of contamination of the six chromatographic fractions by cytochrome P-420 and by NADPH-cytochrome c reductase is also shown in Tables 1 and 2. The cytochrome P-420 content is relatively low in all of the NAIDM fractions except C, and C, where its concentration is approximately equal to that of the cytochrome P-450. The same result was obtained with the Rutgers fractions and, in addition, the B, fraction


contained relatively high concentrations of this impurity. The reductase was detected in all NAIDM fractions except C, and in all Rutgers fractions except A,. Higher amounts were found in the A,, B,, and C, fractions from both preparations indicating that a higher concentration of phosphate is required to elute this enzyme from the hydroxylapatite column.

In a further examination of the cytochrome in the column fractions we per- formed SDS -poIyacrylamide gel electrophoresis with fractions which contain sufficient hemoprotein for this purpose, i.e.. B,, B.,, C,, and C,. As seen in Table 3, several bands in each fraction responded to both heme sensitive and protein sensitive stains. Assuming that, as reported in the literature (4, 20, 21), heme-staining bands with molecular weights less than 31,000 daltons are probably artifacts of the method, only four of the bands detected in the NAIDM experiments and three in the Rutgers experiments are due to cytochrome P-450.

We made repeated attempts to detect a hemoprotein in the 40,000-60,000 molecu-

TABLE 3

Apparent molecular weights

of hemoprotein bands”

Fraction NAIDM strain Rutgers strain -- ~~

B, 29.300 29.600 23.000 18.000”

18,000”

B, 51.300 47,700

44.100 30,600

30.000

Cl 19.800 50.500

28.600

C, 55.500 49.300 46.400 76.800

3 1.100

76.200 -

” Average of two or three experiments, each with

duplicate determinations.

A Diffuse band behind the tracking dye.

lar weight range in the B, fractions which, according to the spectral data and their activity on reconstitution (Tables 4 and 5) contained the most cytochrome P-450. This included the use of lower concentrations of SDS (0.02% and 0.1%) on the assumption that the hemoprotein in these fractions was more unstable than that in the other fractions. During these experiments the band migrating to the 30,000-dalton region was always detected but there was no trace of bands at higher molecular weights. This result can be explained in two ways, the cytochrome P-450 in fraction B, is too unstable for the conditions imposed, or this fraction does contain a low molecular weight cytochrome P-450.

The electrophoresis experiments also indicate, in the case of the NAIDM microsomes, at least, that the chromatographic procedures followed do not result in the complete resolution of the cytochrome P- 450s in the solubilized microsomes. Thus we detected two hemoproteins, molecular weight, 52,300 and 44,100 in the B, fraction and two such bands, 55,500 and 46,400 in the C, fraction. At the same time, the C, fraction did not contain a hemoprotein in the accepted molecular weight range either because none was present originally or, as we suggest in the case of the B, fraction, because it was destroyed by the electrophoresis procedures.

Table 4 shows that all of the fractions prepared from Rutgers microsomes re- tained some aldrin epoxidase activity with the possible exception of fraction A,. We used rat liver NADPH-cytochrome c reductase and fraction B, and C, which were devoid of the house fly reductase (Table 2) to establish the requirement for this com- ponent. Apparently the house fly reductase already present in fraction B, was sufficient for limited epoxidase activity (1.24 k 0.05 nmol dieldrin produced compared to 2.35 2 0.19 nmol when 10 units of the rat liver reductase was added). As indicated in experiments with fraction B,, either dioleoyl

246 YU AND TERRIERE

Components included in incubations”

Fraction

A, - A,

B,

B,

B,

B, B,

B,

c,

C,

C1

Cytochrome

P-450

tnmol)

NADPH-Cytochrome reductase” Phospholipid’

(unit) (ma

Aldrin epoxidase”

(nmol dieldrini

nmol P-450ihrI

1.0 10 0.13 0.47 c 0.16

1.0 10 0.13 0.15

1.5 0 0.13 0.15

1.5 IO 0 1.35 I 0.04

1.5 IO 0. I3 2.22 k 0.14

1.5 10 0.13’ 2.14 r 0.16

1.5 0 0.13 1.24 + 0.05

1.5 IO 0.13 2.35 t 0.19

1.5 0 0.13 0.09 2 0.00

I.5 10 0.13 1.96 t 0.05

1.5 IO 0.13 0.86 t 0.03

” Incubations were for 1 hr and contained 14% glycerol. Otherwise, except as noted above, they were as de-

scribed under Materials and Methods.

’ Purified rat liver NADPH-cytochrome (’ reductase.

” Synthetic dioleoyl I.-cu-phosphatidyl choline.

” Mean t SD of two determinations except AI and B, with 0 reductase.

’ Synthetic dilauroyl L-u-phosphatidyl cholme.

or dilauroyl phosphatidyl choline, also im- proved the system but was not an absolute requirement.

Only two of the column fractions obtained after solubilizing NAIDM house fly microsomes contain sufficient cytochrome P-450 for reconstitution studies. These fractions, B, and C,, were compared with similar fractions from Rutgers house flies under conditions in which the hemoprotein was limiting and the reaction rate was linear

with time. The spin state composition of the hemoprotein in the four fractions was also determined using the n-octylamine binding method. It is clear from the results of these experiments (Table 5) that the cytochrome P-450 obtained from Rutgers microsomes is considerably more active in the epoxidation of aldrin than the hemoprotein prepared from NAIDM microsomes. Of considerable interest is the relatively larger amount of high spin P-450 in the Rutgers fractions.

Hourse fly P-450 Aldrin epoxidase

strain fraction tpmolinmol P-450/min)“.”

0-Octylamine

difference spectrum

(AA 410iAA 392)”

Rutgers B, 297.9 t 39.8 0.79 + 0.05

C, 160.0 i- 38.4 0.64 +- 0.13

NAIDM B, 1.15’ 1.98 t 0.09

C, 41.6 2 16.7 1.25 + 0.06

” Incubation conditions as described under Materials and Methods.

” Mean ? SE of four or five assays.

‘. Based on a I-hr incubation with 1.5 nmol of cytochrome P-450. No dieldrin could be detected after the

standard incubations.


DISCUSSION

Although it is clear from the chromatographic, spectrophotometric, and electrophoretic studies just described that both strains of house flies possess multiple forms of cytochrome P-450, the actual number of such forms is still uncertain. Capdevila and Agosin (4) believe that the susceptible house fly strain they studied contains at least three forms of the hemoprotein, a number with which we can agree. Inasmuch as we were able to recover only 14% of the cytochrome P-450 originally present in the microsomal membranes, however, it would be hazardous to assume that all of the possible forms of this enzyme were included in our experiments.

Our data are more conclusive in regard to differences in the properties of the hemoproteins isolated from the susceptible and insecticide resistant strains of this species. This is seen in the cytochrome P-450-CO A,,, values and in the n-octylamine binding spectra (Tables 1 and 2). It is quite clear from the latter that the hemoprotein of the highly resistant Rutgers strain possesses considerably more high spin character than that of the susceptible strain. Four of the six chromatographically distinct fractions from the Rutgers strain (Table 2) exhibit a trough at 392 nm in the n-octylamine binding spectrum compared to only one for the six fractions isolated from the NAIDM strain (Table 1).

Differences in the hemoprotein from the two strains is also indicated by the electrophoretic results, Table 3. Four heme- positive bands were detected in the fractions from the NAIDM strain compared to three in the same fractions from the Rutgers strain. Furthermore, the molecular weights estimated for these bands appear to be sig- nificantly different. ranging from 44,100 to 55,500 for the NAIDM fractions and from 47,700 to 50,500 for the Rutgers fractions.

The electrophoresis results are difficult to interpret because not all of the fractions contain sufficient cytochrome P-450 for the use of this technique and because three of the fractions which could be tested (B, and

C, of the NAIDM strain and B, of the Rut- gers strain) appear to contain only low molecular weight hemoproteins. The latter result is especially confusing because the B, and C, fractions contain the most highly purified and refined amounts of cytochrome P-450. The most likely explanation for the failure to detect high molecular weight bands in the electrophoresis experiments with these fractions is their lack of stability. There is some evidence of this in the case of the C, fraction of the NAIDM flies where relatively large amounts of cytochrome P-420 were present (Table 1). There was much less cytochrome P-420 in the C, fraction of the Rutgers flies (Table 2) which, in the electrophoresis experiments (Table 3) did exhibit a hemoprotein band of 50,500 daltons. This relationship does not hold with the B, fractions, however, because the cytochrome P-450 content of the Rutgers fraction (Table 2) is relatively high while that of the NAIDM strain (Table 1) is low.

Capdevila and Agosin (4) also detected hemoprotein bands of low molecular weight in their study of the cytochrome P-450 of a susceptible strain. They attribute this to artifacts, i.e., the release of heme during electrophoresis which is then bound to nonspecific low molecular weight protein, resulting in detection as a hemoprotein. They reported earlier (22) that one of the cytochrome P-450s isolated from microsomes of the insecticide resistant Fc strain of house flies was quite unstable.

Assuming that the relationships derived by Jefcoate rt al. (19) for estimating relative amounts of high and low spin cytochrome P-450 in rabbit liver microsomes have some validity in making similar estimates for house fly microsomes, we can obtain some idea of the amount of high spin P-450 present in the chromatographic fractions evaluated in Tables 1 and 2. Extrapolating from the standard curve of Jefcoate et (11. (19) a spectral ratio of 0.7 indicates a spin state ratio of 0.3, approximately 1 part high spin P-450 to 3 parts low spin P-450. Simi- larly, spectral ratios above 1 .O indicate spin state ratios of less than 0.2, i.e., less than I

248 YU AND TERRIERE

part high spin to 5 parts low spin P-450. According to some calculations by Nebert et af. (23) in their study of the induction of the microsomal oxidase system in mice, a relatively small increase in high spin P-450 can result in a large increase in microsomal oxidase activity. Thus our results indicating a change in the spin state of the house fly hemoprotein can explain the increased microsomal oxidase activity of the Rutgers compared to the NAIDM strain.

ACKNOWLEDGMENTS

This work is supported by USPHS Grant No. 5- ROI-00362-20. The authors are grateful to Dr. Robert Neal, Vanderbilt University, for supplying the NADPH-cytochrome c reductase used in the experiments. The technical assistance of Mr. Dan Farnsworth is also appreciated.

REFERENCES

1. F. W. Plapp. Biochemical genetics of insecticide resistance,Annrt. Rci,. Entomol. 21, 179 (1976).

2. E. Hodgson. L. G. Tate. A. P. Kulkarni. and F. W. Plapp. Jr., Microsomal cytochrome P-450: Characterization and possible role in insecticide resistance in Mnscrr dr~mr.stir~cc, J. Agr. Food CIwm. 22, 360 ( 1974).

16.

17.

3. R. D. Schonbrod and L. C. Terriere, The solubilization and separation of two forms of microsomal cytochrome f-450 from the house fly, MIIS(.N domasrkn L, Bioc,hcm. Biophys. Res. Cr,mmrrn. 64, 829 (1975).

18.

4. J. Capdevila and M. Agosin. Multiple forms of house fly cytochrome P-450, in “Microsomes and Drug Oxidations,” pp. 144. Pergamon. En- gland ( 1977).

19.

5. H. B. Matthews and J. E. Casida, Properties of house fly microsomal cytochromes in relation to sex, strain. substrate specificity, and apparent inhibition and induction by synergist and insecticide chemicals, L(#>> Sci. 9, 989 ( 1970).

6. A. S. Perry, W. E. Dale, and A. J. Buckner, In- duction and repression of microsomal mixed- functon oxidases and cytochrome P-450 in resistant and susceptible house flies, Prsric. Biochem. Physid. 1, 131 (1971).

7. L. G. Tate, F. W. Plapp, and E. Hodgson. Cyto- chrome P-4.50 difference spectra of microsomes from several insecticide-resistant and -susceptible strains of the house fly. Mtcst,ci domasticu L, Chem. -Bid. ftlter. 6, 237 (1973).

8. A. P. Kulkarni and E. Hodgson, Microsomal cytochrome f-450 from the house fly, M/l.sc.tr domcsrictr: Assay and spectral characterization. I~~ec,f Bicwham. 5. 679 (1975).

9. L. C. Terriere and S. J. Yu, The induction of de- toxifying enzymes in insects. J. Agr. Food Chc~m. 22, 366 (1974).

20.

21.

22.

23.

10.

II.

12.

13.

14.

15.

L. L. Chang and E. Hodgson. Biochemistry of detoxication in insects: Microsomal mixed- function oxidase activity in the house fly. Mnsctr domesfku. lnsrc~ Biwhcm. 5, 93 ( 1975).

T. Omura and R. Sato, The carbon monoxide- binding pigment of liver microsomes, J. Biol. Cbrm. 239, 2370 (1964).

f. Omura and R. Sato, Fractional solubilization of hemoproteins and partial purification of carbon monoxide-binding cytochrome from liver microsomes, Biochem. Biophys. Ar,tcr 71. 224

( 1963). R. D. Schonbrod and L. C. Terriere, Inhibition of

house fly microsomal epoxidase by the eye pigment, xanthommatin. Pcstic.. BicJchcm. Physiol. 1, 409 (1971).

S. J. Yu. U. Kiigemagi, and L. C. Terriere. Oxidative metabolism of aldrin and isodrin by bean root fraction, J. Agr. Food Chrm. 19, 5 (1971).

M. M. Bradford. A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anti/. Biochem. 72, 248 (1976).

U. K. Laemmli. Cleavage of structural proteins during the assembly of the head of bac- teriophage T4. Ntrrlrrc, (L~~nd~~ni 227, 680 (1970).

P. E. Thomas, D. Ryan, and W. Levin. An im- proved staining procedure for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate-polyacrylamide gels, Anti/. Biochem. 75. 168 (1976).

G. Fairbanks. T. L. Steck. and D. F. H. Wallach. Electrophoretic analysis of the major polypep- tides of the human erythrocyte membrane, Biwhemistry 10. 2606 ( 1971).

C. R. E. Jefcoate, R. L. Calabrese. and J. L. Gaylor. Ligand interaction with hemoprotein P-450. III. The use of n-octylamine and ethyl isocyanide difference spectroscopy in the quantitative determination of high- and low-spin P-450. Md. Phc/rmcrco/. 6. 391 (1970).

A. F. Welton and S. D. Aust. Multiplicity of cytochrome P-450 hemoproteins in rat liver microsomes. Bioc,hcm. Bioph?s. Rev. Cr~mmutl. 56, 898 (1974).

D. A. Haugen, T. A. Van der Hoeven, and M. J.

Coon, Purified liver microsomal cytochrome 1’.450. Separation and characterization of multiple forms, J. Bid. Chrm. 250. 3567 (1975).

J. Capdevila. N. Ahmad, and M. Agosin, Soluble cytochrome P-450 from house fly microsomes. Partial purification and characterization of two hemoprotein forms. .I. Biol. Clrrm. 250. 1048 (1975).

D. W. Nebert. J. E. Gielen. and F. M. Goujon. Genetic expression of aryl hydrocarbon hy- droxylase induction. III. Changes in the binding of rr-octylamine to cytochrome P-450. MO/. Pbcirm~~crd. 8, 651 ( 1972).

cytochrome p-450 in insects: 1. differences in the forms present in insecticide resistant and...

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