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Mechanism of microsomal mixed-function oxidase inhibitor from the housefly Musca domestica L
PESTICIDE
BIOCHEMISTRY
Mechanism
AND
PHYSIOLOGY,
(1972)
of Microsomal
Mixed-Function
the
Mosca
Housefly
T. G. WILSON Department
64-71
2,
of Entomology,
North
AND
Carolina Accepted
Oxidase domestica
ERNEST
State
from
L.’
HODGSON
University,
February
Inhibitor
Raleigh,
North
Carolina
27607
10, 1972
An inhibitor of microsomal mixed-function oxidaae activity from the head of the housefly, Musca domestica L., has been partially purified by chromatography on Sephadex G-25. Its chemical characteristics, in agreement with investigations from other laboratories, indicate that it is the eye pigment, xanthommatin. The inhibitory mechanism has been investigated using microsomal preparations from mouse liver and housefly abdomens as well as a purified microsomal NADPHcytochrome c reductase from the housefly. Kinetic expriments on the rate of p-nitroanisole demethylation, NADPH oxidation, and cytochrome c reduction indicate that the inhibitor acts by accepting electrons at the reductase level thus impeding electron flow to cytochrome P-450. The inhibitor will accept electrons from the purified NADPH-cytochrome e reductase and also stimulate the reduction of cytochrome c by this enzyme. Spectral studies of cytochrome b5 and cytochrome P-450 show that the inhibitor can prevent the reduct,ion of cytochrome bs , presumably by accepting electrons prior to the cytochrome level, but does not interact with cytochrome P-450 nor does it affect the binding of ot,her substrates to this cytochrome.
uncouples NADPH oxidation from cytochrome P-450 reduction (4, 8). The present communication describes the effects of the housefly head inhibitor on mouse liver and the housefly abdomen microsomal enzymes and also includes some observations on the interaction of this inhibitor and a purified microsomal NADPHcytochrome c reductase (9, 10) from houseflies.
INTRODUCTION
Naturally occurring inhibitors of microsomal mixed-function oxidase activity ha@ been shown to occur in insects (l-4). These inhibitors affect the microsomal enzymes of both insects and mammals. The endogenous inhibitor from housefly heads (1) has been reported to be xanthommatin, an eye pigment, which functions when added to housefly microsomes, as an electron acceptor at the NADPH-cytochrome c reductase level (5-7). The inhibitor from the larval gut of the southern armyworm, Prodenia eridania, is a proteolytic enzyme which
iMATERIALS
64 0 1972 by Academic Preaa, Inc. of reproduotion in sny form wserved.
METHODS
NADPH and lipase were obtained from Nutritional Biochemical Company, type III cytochrome c from Sigma Chemical Company and bovine serum albumin fro? Armour Pharmaceuticals. p-Nitroanisole and p-nitrophenol were obtained from Eastman Organic Chemicals and were recrystallized prior to use. Sephadex G-25 was obtained
1 Paper number 3664 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, N. C. These investigations were supported in part by Grants ES-0069 and ES-00044 from the U.S. Public Health Service. Copyright AU rights
AND
MIXED
FUNCTION
OXIDASE
INHIBITOR
from Pharmacia. All other chemicals nsed were of the highest purity available commercially and were used without further purification. Houseflies of the CSMA strain were reared at 27°C and 55 % relative humidity under a 16: S (light :dark) hour photoperiod. The larvact were fed standard CSMA medium and the adults sucrose and milk. Five- to nim-week-old mice of the North Carolina Department of Health strain were utilized throughout. This strain is an inbred one maintained in Raleigh since 1912. Mouse liver microsomes were prepared by homogenizing t’he livers from mice killed by decapitation in 11 .O ml 0.15 M potassium phosphate buffer, pH 7.8, in a cone-driven Pott#cr-Elvehjem type homogenizer fitted with a Teflon pestle. Microsomes were sedimented from the postmitochondrial supernatant fraction (10,OOOg for 10 min) by cent’rifugation at 100,OOOg for 1 hr. The microsomal pellet was resuspended in 0.1 38 potassium phosphate buffer, pH 7.8, for the estimation of 0-demethylation of p-nitroanisolr, or as previously described (9, lo), for spectral studies and NADPH oxidation measurements. In routine assays of the inhibitor during purification procedures, the inhibition of 0-demethylation of p-nitroanisole in mitochondrial supernatants was used as an assay method. The preparation of housefly microsomes and the purification of microsomal NADPHcytochrome c reductase from the housefly were carried out through the DEAEcellulose step by the methods of Wilson and Hodgson (9, 10). The 0-demethylation of p-nitroanisole was measured in a reaction mixture containing microsomes equivalent to 100 mg mouse liver or 40 flies (2-3 mg protein), 2.0 pmoles p-nitroanisole, and 2.1 fimoles NADPH in a total volume of 4.0 ml potassium phosphate buffer, pH 7.9. p-Nitrophenol release was measured at 400 nm. Difference spectra of cytochrome bb and
FROM
THE
HOUSEFLY
65
cytochrome P-450 were recorded with a Beckman DK-2 spectrophotometer. hficrosomal suspensions contained approximately 1.0 mg protein/ml phosphate buffer. Cytochrome bj was observed from an NADPHor NADH-reduced minus oxidized spectrum and cytochrome PAso from an NADPHreduced minus oxidized after first, saturating the sample cuvctte with CO. I:or anerobic studies, antxrobic cuvct t,cis were used and thoroughly flushed with N? beforcl initiating th(a reaction by the additions from the sidearm. The abnencc of NADPH oxidation by mouse liver microsomes was used as an indication of ancrobic conditions. SADPH or NADH oxidation was m(‘atHurcd by determining the decrease in absorbance at 340 nm in a Beckman DU spectrophotometer. Each assay mixt’urc contained 3.0 X 1O-7 moles of NADPH or NADH and mouse liver microsomal suspension cont,aining approximat,ely 1 mg protein prr milliliter in a tot’al volume of 2.4 ml phosphate buffer, pH 7.X. The molar extinction coefficient used for NADPH was 6.26 X lop3 JI-’ cm-’ (11). Protein waq determined by the method of Lowry ct nl. (12). All steps prior t,o incubation n-cre carried out at I-5°C. RESULTS
Preparation and properties of inhibitor. Initial experiments indicated that the greatest’ amount of inhibit,or per unit weight was present in the housefly head when compared to the thorax, abdomen, or whole fly. All subsequent inhibitor preparations were derived from fly heads obtained from 5- to lo-day-old flies by the method of Moor&eld (13). Two grams of heads were homogenized at room temperature for 2 min in a Servall Omni-mixer with the variable resistor set at 65. Fifty milliliters of chloroform were added, homogenization continued for 1.5 min, and the yellou-brown aqueous layer freeze-dried. The rrasidue wm dissolved in 5
66
WILSON
AND
ml distilled water, applied to a 2 X 40-cm Sephadex C-25 column, and eluted with water. Three visible bands were eluted, first a gray protein-containing band, second a dark-yellow band containing the inhibitor, and third a light-yellow band. The first and third fractions were discarded and the second diluted to 20 ml with distilled water. Aliquots were used as indicated in the experiments described below. This inhibitor solution loses most of its inhibitory properties in 1 week at 5°C. Housefly heads stored for 2 weeks at - 15°C prior to preparation of the inhibitor also show a reduced level. The isolated inhibitor preparation is dark-yellow in color, with a broad peak in the absorption spectrum between 340 and 500 nm, having a maximum at 350 nm. On reduction by dithionite, the color changes from yellow to pink and the absorption in the visible region is greatly reduced. The inhibitor is not reduced nonenzymatically by NADPH or NADH. The reduced inhibitor is slowly reoxidized below pH 7.0 and rapidly at 7.8. It can also rapidly reduce cytochrome c. Upon reduction by sulfur dioxide the inhibitor turns pink and is pre-
0
I 5 TIME-minutes
I 10
I 15
FIG. 1. Effect of housejly-head inhibitor on pnitroanisole demethylation by mouse liver microsomes. Inhibitor concentration varied. Incubation jlasks contained 0.0 (control), 0.3 or 0.6 ml of inhibitor soktion prepared as described in Materials and Methods.
HODGSON
cipitated. Although it is apparently reoxidized by titration’ to pHlO.0, it can no longer be reduced by either dithionite or sulfur dioxide. E$ect
on p-nitroanisole
demethylatim.
Addition of the inhibitor preparation to mouse liver microsomes decreases both the initial velocity and the extent of this reaction (Fig. 1). If the amount of inhibitor and the amount of substrate are varied, a Lineweaver-Burk plot of the resulting reaction velocities (Fig. 2) shows noncompetitive kinetics. If the amount of inhibitor and the amount of NADPH are varied, the resulting Lineweaver-Burk plot indicates competitive kinetics (Fig. 3). The same results are obtained if housefly microsomes are substituted for mouse liver microsomes. E$ect on NADPH
and NADH
oxidation.
The inhibitor preparation increases the rate of NADPH oxidation by mouse liver microsomes. This increase also occurs in the presence of CO, which is known to complex with cytochrome P-450 and inhibit electron transport by this cytochrome (Table 1). Reaction rates for NADPH and NADH are shown in Figs. 4a and b. The inhibitor preparation increases the rate of oxidation of both pyridine nucleotides. After the reaction has gone to completion addition of NADH or NADPH again initiated the reaction indicating that the mode of action does not involve irreversible binding to an active site or denaturation of the enzyme. It is noteworthy that the stimulation of NADH oxidation is as great as that for NADPH oxidation. Addition of the inhibitor solution colors the reaction mixture slightly yellow and this does not change during the course of the react.ion if the conditions are aerobic. If NADPH is incubated with microsomes under anerobic conditions, no oxidation occurs. If inhibitor solution is added from the sidearm of the anerobic cuvette, oxidation proceeds for several minutes, then stops (Fig. 5) while at the same time the solution changes from yellow to pink. The
MIXEI:
FUNCTION
OXIDASE
INHIBITOR
FROM
THE
HOUSEFLl
67
1 p-NITROANISOLE
FIG. 2. Effect of house$y-head inhibitor on p-nitroanisole demethylation hibitor and p-nitroanisole concentration varied. Incubalionjiasks contained of inhibitor solution prepared as described in Materials and Methods.
TABLE EQ’ecf
liver microsomes. In-
by naouse 0.0 (control),
of HouseJEy Inhibitor by Mouse Liver
0.1,0.2?,
an,d 0.9 ml
1 on NADPH Microsomescl
r\dditions
htmosphere
Inhibitor 0 0.2 ml 0.5 ml 0 0.2 ml 0.5 ml
Air Air Air CO co co
Oxidation
pmoles
NADPtI
oxid.imin
a Enayme used was 1.0 ml mouse liver microsomes prepared as described in Materials and Methods
01
30 1 NADPH
FIG. 3. Effect
of housefly-head inhibitor on pnitroanisole demethylation by mouse liver microsomes. Inhibitor and NADPH concentration varied. Incubation jlasks contained 0.0 (control) 0.06, 0.1, and 0.8 ml of inhibitor solution prepared as described in Materials and Methods.
admission of air allows the oxidation to proceed to completion and, at the sametime, the inhibitor returns to the yellow, oxidized form.
Soluble diaphorases in rat liver are known to reduce quinones and dyestuffs in the presence of reduced pyridine nucleotides (14). The possibility that contamination of the microsomesby these enzymes resulted in inhibition due to reduction in NADPH was investigated by measuring inhibition of p-nitroanisole demethylation in the presence of excws (1.2 x 1O-3 M) NADPH. The rates in the presence of inhibitor were clearly lower yet both cont,rol and inhibited reactions were linear for at least 6 min, indicating that NADPH is not being exhausted by diaphorase activity.
WILSON
68
AND
HODGSON
NADH ADDED
0.4 b--*NADPH DSm, iNHlB,TDR
-NADPH
0.3 z 0
2 2
0.2
a a
0
FIG. 4. E$ect microsomes.
IO
of houseJy-head
TABLE of
chrome
Oxidation of NADPH c Reductase with Test
No enzyme Boiled enzyme Enzyme
30
inhibitor
FIG. 5. Efect of housejZu-head oxidation o.f NADPH by mouse under anaerobic conditions.
Rate
20
40
50 60 70 TIME-minutes
on the oxidation
inhibitor on the liver microsomes
2 by NADPH-CytoInhibitor Present” nmoles NADPH oxid./lO min 8.0 8.0 37
a Inhibitor present (1.0 ~1); enzyme was 100 ~1 purified reductase (sp act 40 units/mg) in a total volume of 2.4 ml 0.015 M phosphate buffer, pH 7.8.
IO
of NADPH
20
30
40
(A)
and NADH
(B) by
liver
.Eflect on pur$ied NADPH-cytochrome c reductase. The inhibitor can act as an electron acceptor for the NADPH-cytochrome c reductase from housefly microsomes and cause a concomitant oxidation of NADPH (Table 2). The inhibitor also stimulates the reduction of cytochrome c by the purified enzyme (Fig. 6). E$ect of bovine serum albumin. Table 3 shows the effect of 1 o/o bovine serum albumin on t,he inhibition of 0-demethylation of p-nitroanisole. Possibly bovine serum albumin binds the inhibitor and prevents its interaction with the microsomal enzymes. Effect of oxidized inhibitor on microsomal cytochromes. When mouse liver microsomes are incubated aerobically with NADPH, a partially reduced cytochrome bs spectrum rapidly develops which becomes fully developed under anerobic conditions. This spectrum is due, presumably, to crossover of electrons from the NADPH-cytochrome P-450 pathway to the NADH-cytochrome b5 pathway. If inhibitor is added from the sidearm of an anerobic cuvette to a fully
MIXED
5r; 07-
FUNCTION
OXIDARE
I 20
10
i NADPH-MOLES
from
jlasks contained inhibitor solution and Methods.
0.0 (control), prepared as
TABLE Effect
of
1% BSA methylation
inhibitor
on the
c recluctase purijed microsomes. Incubation
abdomen
0.03,
and
0.10 ml
of
described in Materials
3
on Inhibition by Housefly
of pNd Inhibitor
added (ml) 0.00 0.05 0.1 0.3 0.5
O-De-
Without BSA
With BSA
-0
-5
9 11 52 77
3 7 22 42
a Mouse liver microsomes (2.5 mg protein assay) were used as enzyme.
I 400
69
HOUSEFLY
per
developed bgspectrum (Fig. 7) the spectrum is not changed. However, if air is bubbled through the incubation medium, the reduced cytochrome bg spectrum disappears and cannot be restored by the addition of more NADPH. The addition of inhibitor to a cytochrome bg spectrum produced aerobically causes its disappearance; however, if the incubation is made anerobic by bubbling nitrogen through it, the anerobic b, spectrum reappears. These observations suggest that the inhibitor is accepting electrons from the component which reduces 65 but not from bg itself, and under aerobic conditions prevents its reduction. Under anerobic condi-
I 425 WAVELENGTH
I 450 - nm
FIG. 7. Effect of housejly inhibitor on cytovhrome bg reduction by NADPH ,under aerobic (A) and anaerobic
yo Inhibition Inhibitor
THE
x IO-’
of housejly-head of NADPH-cytochrome
housejfy
FROM
I 30
FIG. 6. E$ect activity
INHIBITOR
(B)
conditions.
tions, the inhibitor becomes fully reduced and cytochrome bgcan then be reduced even in the presence of the inhibitor. The inhibitor apparently does not. interact. with cgtochrome P-450. Addition of inhibit’or t’o the CO-complex has no effect nor can a sub&rate binding spectrum be observed in the absence of CO. The inhibitor neither prevents a pyridinc type II substrate-binding spectrum from being formed nor displaces one previously formed. Efect oj reduced inhibitor on ,microsQmes, Housefly inhibitor, reduced with dithionite and introduced ancrobically into microsomal suspensions, will not reduce either cytochrome bj or P450. Under appropriate conditions either of these reduced spectra can be produced by NADPH. DISCUSSION
These investigations suggest that the inhibitor from housefly heads affects mixedfunction oxidase reactions in mouse liver by accepting electrons from an enzyme in the NADPH-dependent, electron transport thus diminishing the flow of electrons to cytochrome P-450. Schonbrod and Terriere (6) in a study of the effect, of the houseflyhead inhibitor on aldrin epoxidatBion by
70
WILSON
AND
housefly microsomes reached essentially the same conclusion. Both the rate and extent of p-nitroanisole demethylation by mouse liver microsomes is reduced by the inhibitor, effects which could be caused by the inhibitor oxidizing the reduced flavoprotein, NADPH-cytochrome c reductase. Moreover, the rate of NADPH oxidation is increased, further suggesting that this reductase is the site of action. This hypothesis is also supported by the competitive nature of the inhibition of p-nitroanisole demethylation when the NADPH concentration is varied. Moreover the inhibitor acts as an electron acceptor for NADPH-cytochrome c reductase purified from housefly abdomen microsomes. The similarity of the competitive inhibition of p-nitroanisole demethylation by the inhibitor, when NADPH concentration is varied, in both mouse liver and housefly microsomes, suggests that the mode of action is the same in both organisms. Stimulation of the reaction with cytochrome c is more difficult to explain, perhaps the reduced inhibitor “fits” the active site of cytochrome better than the reduced flavoprotein or possibly it acts as an allosteric activator. Since NADH is not a good electron donor for microsomal oxidations involving cytochrome P-450, the stimulation of NADPH oxidation suggests that the inhibit,or will also accept electrons from NADH-cytochrome c reductase, particularly when the stimulation is greater than that for NADPHoxidation. The effect of bovine serum albumin in reducing the inhibition, presumably by binding the inhibitor, is in conflict with the findings of Schonbrod and Terriere (6). If binding of xanthommatin is involved, this may be due to variations in the extent to which binding sites are already saturated in different commercial samples of bovine albumin. Goodman (16) demonstrated such variations in human serum albumin.
HODGSON
The spectral studies with cytochrome bj and cytochrome P-450 indicate that the inhibitor does not interact with either as an electron donor or acceptor or, in the case of cytochrome P-450, as a substrate. The equilibrium between oxidized and reduced forms of the inhibitor is such that it remains primarily in the oxidized state during NADPH oxidation and p-nitroanisole demethylation, since the yellow color imparted to the microsomal suspension persists. Under anerobic conditions, however, the inhibitor becomes primarily reduced and the color changes from yellow to pink. It is not known at present whether the inhibitor under aerobic conditions becomes fully reduced and then is rapidly reoxidized by molecular oxygen, or whether it is reduced to a semiquinone which undergoes rapid oxidation and reduction. Schonbrod and Terriere (5, 6) and Nagatsugawa (7) have both indicated that this inhibitor is xanthommatin. The data from this laboratory, while not extensive, tend to confirm this identification. REFERENCES
1. H.
2.
3.
4.
5.
6.
B. Matthews and E. Hodgson, Naturally occurring inhibitor(s) of microsomal inhibitors from the housefly, J. Econ. Entomol. 59, 1286 (1964). J. Chakroborty, C. H. Sissons, and J. N. Smith, Inhibition of microsomal oxidations in insect homogenates, Biochem. J. 102, 492 (1967). M. Tsukamoto and J. E. Casida, Metabolism of methylcarbamate insecticides by the NADPH-requiring enzyme system from houseflies, Nature (London) 213, 49 (1967). R. I. Krieger and C. F. Wilkinson, Microsomal mixed function oxidases in insects. 1. Localization and properties of an enzyme system effecting aldrin epoxidation in larvae of the southern armyworm, Biochem. Pharmacol. 18, 1403 (1969). R. D. Schonbrod and L. C. Terriere, Eye pigments as inhibitors of microsomal aldrin epoxidase in the housefly, J. Econ. Entomol. 64, 44 (1971). R. D. Sconbrod and L. C. Terriere, Inhibition of housefly microsomal epoxidase by the eye
MIXED
pigment, Physiol.
FUNCTIOK
xanthommat,in,
OXIDARE
Pest.
INHIBITOR
Biochem.
in press.
7. T. Nakatsugawa, personal communication. 8. S. Orrenius, M. Berggen, P. Moldeus, and R. I. Kreiger, Mechanism of inhibition of microsomal mixed fun&ion oxidases by the gutcont,ent’s inhibit)or of the southern army worm (Pfvdenia eridaflia), Biochem. J. 124, 427 (1971). 9. T. Wilson and E. Hodgson, Microsomal NADPH-cytochrome c reduct,ase from the housefly, MlLsca domestica: solubilization and purification, Insect Biochem. 1, 19 (1971). 10. T. Wilson and E. Hodgson, Microsomal NADPH-cytochrome G reduct,ase from the housefly, Musca domestica: properties of the purified enzyme, Insect Biochem. 1, 171 (1971). 11. A. Komberg and B. L. Horecker, in “Bio-
12.
13.
14.
15.
FROM
THE
HOUREFLl
71
chemical Preparations” (E. E. Snell, Ed.), * I J Vol. 3, p. 24, Wiley New York 1953. 0. H. Lowry, N. J. Rosebrough, A. 1,. Farr, and R. J. Randall, Protein measurement with the Folin phenol reagent, J. Riol. Chem. 193, 265 (1951). H. H. Moorefield, Improved method for harvesting housefly heads for use in cholinesterase studies, Contrib. Boyce l’h,rttttp,son Inst. 13, 463 (1957). L. Ernster, L. Danielson, and M. Ljunggren, DT diaphorase 1. Purification from the soluble fract,ion of rat liver cytoplasm, atId properties, Hiochi)n. Rioph~/s. .4cln 3, 171 (1962). I). S. Goodman, Preparation of human serum albumin free of long chain fatty acids, Sricnce 12.5, 1296 (1957 1.
BIOCHEMISTRY
Mechanism
AND
PHYSIOLOGY,
(1972)
of Microsomal
Mixed-Function
the
Mosca
Housefly
T. G. WILSON Department
64-71
2,
of Entomology,
North
AND
Carolina Accepted
Oxidase domestica
ERNEST
State
from
L.’
HODGSON
University,
February
Inhibitor
Raleigh,
North
Carolina
27607
10, 1972
An inhibitor of microsomal mixed-function oxidaae activity from the head of the housefly, Musca domestica L., has been partially purified by chromatography on Sephadex G-25. Its chemical characteristics, in agreement with investigations from other laboratories, indicate that it is the eye pigment, xanthommatin. The inhibitory mechanism has been investigated using microsomal preparations from mouse liver and housefly abdomens as well as a purified microsomal NADPHcytochrome c reductase from the housefly. Kinetic expriments on the rate of p-nitroanisole demethylation, NADPH oxidation, and cytochrome c reduction indicate that the inhibitor acts by accepting electrons at the reductase level thus impeding electron flow to cytochrome P-450. The inhibitor will accept electrons from the purified NADPH-cytochrome e reductase and also stimulate the reduction of cytochrome c by this enzyme. Spectral studies of cytochrome b5 and cytochrome P-450 show that the inhibitor can prevent the reduct,ion of cytochrome bs , presumably by accepting electrons prior to the cytochrome level, but does not interact with cytochrome P-450 nor does it affect the binding of ot,her substrates to this cytochrome.
uncouples NADPH oxidation from cytochrome P-450 reduction (4, 8). The present communication describes the effects of the housefly head inhibitor on mouse liver and the housefly abdomen microsomal enzymes and also includes some observations on the interaction of this inhibitor and a purified microsomal NADPHcytochrome c reductase (9, 10) from houseflies.
INTRODUCTION
Naturally occurring inhibitors of microsomal mixed-function oxidase activity ha@ been shown to occur in insects (l-4). These inhibitors affect the microsomal enzymes of both insects and mammals. The endogenous inhibitor from housefly heads (1) has been reported to be xanthommatin, an eye pigment, which functions when added to housefly microsomes, as an electron acceptor at the NADPH-cytochrome c reductase level (5-7). The inhibitor from the larval gut of the southern armyworm, Prodenia eridania, is a proteolytic enzyme which
iMATERIALS
64 0 1972 by Academic Preaa, Inc. of reproduotion in sny form wserved.
METHODS
NADPH and lipase were obtained from Nutritional Biochemical Company, type III cytochrome c from Sigma Chemical Company and bovine serum albumin fro? Armour Pharmaceuticals. p-Nitroanisole and p-nitrophenol were obtained from Eastman Organic Chemicals and were recrystallized prior to use. Sephadex G-25 was obtained
1 Paper number 3664 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, N. C. These investigations were supported in part by Grants ES-0069 and ES-00044 from the U.S. Public Health Service. Copyright AU rights
AND
MIXED
FUNCTION
OXIDASE
INHIBITOR
from Pharmacia. All other chemicals nsed were of the highest purity available commercially and were used without further purification. Houseflies of the CSMA strain were reared at 27°C and 55 % relative humidity under a 16: S (light :dark) hour photoperiod. The larvact were fed standard CSMA medium and the adults sucrose and milk. Five- to nim-week-old mice of the North Carolina Department of Health strain were utilized throughout. This strain is an inbred one maintained in Raleigh since 1912. Mouse liver microsomes were prepared by homogenizing t’he livers from mice killed by decapitation in 11 .O ml 0.15 M potassium phosphate buffer, pH 7.8, in a cone-driven Pott#cr-Elvehjem type homogenizer fitted with a Teflon pestle. Microsomes were sedimented from the postmitochondrial supernatant fraction (10,OOOg for 10 min) by cent’rifugation at 100,OOOg for 1 hr. The microsomal pellet was resuspended in 0.1 38 potassium phosphate buffer, pH 7.8, for the estimation of 0-demethylation of p-nitroanisolr, or as previously described (9, lo), for spectral studies and NADPH oxidation measurements. In routine assays of the inhibitor during purification procedures, the inhibition of 0-demethylation of p-nitroanisole in mitochondrial supernatants was used as an assay method. The preparation of housefly microsomes and the purification of microsomal NADPHcytochrome c reductase from the housefly were carried out through the DEAEcellulose step by the methods of Wilson and Hodgson (9, 10). The 0-demethylation of p-nitroanisole was measured in a reaction mixture containing microsomes equivalent to 100 mg mouse liver or 40 flies (2-3 mg protein), 2.0 pmoles p-nitroanisole, and 2.1 fimoles NADPH in a total volume of 4.0 ml potassium phosphate buffer, pH 7.9. p-Nitrophenol release was measured at 400 nm. Difference spectra of cytochrome bb and
FROM
THE
HOUSEFLY
65
cytochrome P-450 were recorded with a Beckman DK-2 spectrophotometer. hficrosomal suspensions contained approximately 1.0 mg protein/ml phosphate buffer. Cytochrome bj was observed from an NADPHor NADH-reduced minus oxidized spectrum and cytochrome PAso from an NADPHreduced minus oxidized after first, saturating the sample cuvctte with CO. I:or anerobic studies, antxrobic cuvct t,cis were used and thoroughly flushed with N? beforcl initiating th(a reaction by the additions from the sidearm. The abnencc of NADPH oxidation by mouse liver microsomes was used as an indication of ancrobic conditions. SADPH or NADH oxidation was m(‘atHurcd by determining the decrease in absorbance at 340 nm in a Beckman DU spectrophotometer. Each assay mixt’urc contained 3.0 X 1O-7 moles of NADPH or NADH and mouse liver microsomal suspension cont,aining approximat,ely 1 mg protein prr milliliter in a tot’al volume of 2.4 ml phosphate buffer, pH 7.X. The molar extinction coefficient used for NADPH was 6.26 X lop3 JI-’ cm-’ (11). Protein waq determined by the method of Lowry ct nl. (12). All steps prior t,o incubation n-cre carried out at I-5°C. RESULTS
Preparation and properties of inhibitor. Initial experiments indicated that the greatest’ amount of inhibit,or per unit weight was present in the housefly head when compared to the thorax, abdomen, or whole fly. All subsequent inhibitor preparations were derived from fly heads obtained from 5- to lo-day-old flies by the method of Moor&eld (13). Two grams of heads were homogenized at room temperature for 2 min in a Servall Omni-mixer with the variable resistor set at 65. Fifty milliliters of chloroform were added, homogenization continued for 1.5 min, and the yellou-brown aqueous layer freeze-dried. The rrasidue wm dissolved in 5
66
WILSON
AND
ml distilled water, applied to a 2 X 40-cm Sephadex C-25 column, and eluted with water. Three visible bands were eluted, first a gray protein-containing band, second a dark-yellow band containing the inhibitor, and third a light-yellow band. The first and third fractions were discarded and the second diluted to 20 ml with distilled water. Aliquots were used as indicated in the experiments described below. This inhibitor solution loses most of its inhibitory properties in 1 week at 5°C. Housefly heads stored for 2 weeks at - 15°C prior to preparation of the inhibitor also show a reduced level. The isolated inhibitor preparation is dark-yellow in color, with a broad peak in the absorption spectrum between 340 and 500 nm, having a maximum at 350 nm. On reduction by dithionite, the color changes from yellow to pink and the absorption in the visible region is greatly reduced. The inhibitor is not reduced nonenzymatically by NADPH or NADH. The reduced inhibitor is slowly reoxidized below pH 7.0 and rapidly at 7.8. It can also rapidly reduce cytochrome c. Upon reduction by sulfur dioxide the inhibitor turns pink and is pre-
0
I 5 TIME-minutes
I 10
I 15
FIG. 1. Effect of housejly-head inhibitor on pnitroanisole demethylation by mouse liver microsomes. Inhibitor concentration varied. Incubation jlasks contained 0.0 (control), 0.3 or 0.6 ml of inhibitor soktion prepared as described in Materials and Methods.
HODGSON
cipitated. Although it is apparently reoxidized by titration’ to pHlO.0, it can no longer be reduced by either dithionite or sulfur dioxide. E$ect
on p-nitroanisole
demethylatim.
Addition of the inhibitor preparation to mouse liver microsomes decreases both the initial velocity and the extent of this reaction (Fig. 1). If the amount of inhibitor and the amount of substrate are varied, a Lineweaver-Burk plot of the resulting reaction velocities (Fig. 2) shows noncompetitive kinetics. If the amount of inhibitor and the amount of NADPH are varied, the resulting Lineweaver-Burk plot indicates competitive kinetics (Fig. 3). The same results are obtained if housefly microsomes are substituted for mouse liver microsomes. E$ect on NADPH
and NADH
oxidation.
The inhibitor preparation increases the rate of NADPH oxidation by mouse liver microsomes. This increase also occurs in the presence of CO, which is known to complex with cytochrome P-450 and inhibit electron transport by this cytochrome (Table 1). Reaction rates for NADPH and NADH are shown in Figs. 4a and b. The inhibitor preparation increases the rate of oxidation of both pyridine nucleotides. After the reaction has gone to completion addition of NADH or NADPH again initiated the reaction indicating that the mode of action does not involve irreversible binding to an active site or denaturation of the enzyme. It is noteworthy that the stimulation of NADH oxidation is as great as that for NADPH oxidation. Addition of the inhibitor solution colors the reaction mixture slightly yellow and this does not change during the course of the react.ion if the conditions are aerobic. If NADPH is incubated with microsomes under anerobic conditions, no oxidation occurs. If inhibitor solution is added from the sidearm of the anerobic cuvette, oxidation proceeds for several minutes, then stops (Fig. 5) while at the same time the solution changes from yellow to pink. The
MIXEI:
FUNCTION
OXIDASE
INHIBITOR
FROM
THE
HOUSEFLl
67
1 p-NITROANISOLE
FIG. 2. Effect of house$y-head inhibitor on p-nitroanisole demethylation hibitor and p-nitroanisole concentration varied. Incubalionjiasks contained of inhibitor solution prepared as described in Materials and Methods.
TABLE EQ’ecf
liver microsomes. In-
by naouse 0.0 (control),
of HouseJEy Inhibitor by Mouse Liver
0.1,0.2?,
an,d 0.9 ml
1 on NADPH Microsomescl
r\dditions
htmosphere
Inhibitor 0 0.2 ml 0.5 ml 0 0.2 ml 0.5 ml
Air Air Air CO co co
Oxidation
pmoles
NADPtI
oxid.imin
a Enayme used was 1.0 ml mouse liver microsomes prepared as described in Materials and Methods
01
30 1 NADPH
FIG. 3. Effect
of housefly-head inhibitor on pnitroanisole demethylation by mouse liver microsomes. Inhibitor and NADPH concentration varied. Incubation jlasks contained 0.0 (control) 0.06, 0.1, and 0.8 ml of inhibitor solution prepared as described in Materials and Methods.
admission of air allows the oxidation to proceed to completion and, at the sametime, the inhibitor returns to the yellow, oxidized form.
Soluble diaphorases in rat liver are known to reduce quinones and dyestuffs in the presence of reduced pyridine nucleotides (14). The possibility that contamination of the microsomesby these enzymes resulted in inhibition due to reduction in NADPH was investigated by measuring inhibition of p-nitroanisole demethylation in the presence of excws (1.2 x 1O-3 M) NADPH. The rates in the presence of inhibitor were clearly lower yet both cont,rol and inhibited reactions were linear for at least 6 min, indicating that NADPH is not being exhausted by diaphorase activity.
WILSON
68
AND
HODGSON
NADH ADDED
0.4 b--*NADPH DSm, iNHlB,TDR
-NADPH
0.3 z 0
2 2
0.2
a a
0
FIG. 4. E$ect microsomes.
IO
of houseJy-head
TABLE of
chrome
Oxidation of NADPH c Reductase with Test
No enzyme Boiled enzyme Enzyme
30
inhibitor
FIG. 5. Efect of housejZu-head oxidation o.f NADPH by mouse under anaerobic conditions.
Rate
20
40
50 60 70 TIME-minutes
on the oxidation
inhibitor on the liver microsomes
2 by NADPH-CytoInhibitor Present” nmoles NADPH oxid./lO min 8.0 8.0 37
a Inhibitor present (1.0 ~1); enzyme was 100 ~1 purified reductase (sp act 40 units/mg) in a total volume of 2.4 ml 0.015 M phosphate buffer, pH 7.8.
IO
of NADPH
20
30
40
(A)
and NADH
(B) by
liver
.Eflect on pur$ied NADPH-cytochrome c reductase. The inhibitor can act as an electron acceptor for the NADPH-cytochrome c reductase from housefly microsomes and cause a concomitant oxidation of NADPH (Table 2). The inhibitor also stimulates the reduction of cytochrome c by the purified enzyme (Fig. 6). E$ect of bovine serum albumin. Table 3 shows the effect of 1 o/o bovine serum albumin on t,he inhibition of 0-demethylation of p-nitroanisole. Possibly bovine serum albumin binds the inhibitor and prevents its interaction with the microsomal enzymes. Effect of oxidized inhibitor on microsomal cytochromes. When mouse liver microsomes are incubated aerobically with NADPH, a partially reduced cytochrome bs spectrum rapidly develops which becomes fully developed under anerobic conditions. This spectrum is due, presumably, to crossover of electrons from the NADPH-cytochrome P-450 pathway to the NADH-cytochrome b5 pathway. If inhibitor is added from the sidearm of an anerobic cuvette to a fully
MIXED
5r; 07-
FUNCTION
OXIDARE
I 20
10
i NADPH-MOLES
from
jlasks contained inhibitor solution and Methods.
0.0 (control), prepared as
TABLE Effect
of
1% BSA methylation
inhibitor
on the
c recluctase purijed microsomes. Incubation
abdomen
0.03,
and
0.10 ml
of
described in Materials
3
on Inhibition by Housefly
of pNd Inhibitor
added (ml) 0.00 0.05 0.1 0.3 0.5
O-De-
Without BSA
With BSA
-0
-5
9 11 52 77
3 7 22 42
a Mouse liver microsomes (2.5 mg protein assay) were used as enzyme.
I 400
69
HOUSEFLY
per
developed bgspectrum (Fig. 7) the spectrum is not changed. However, if air is bubbled through the incubation medium, the reduced cytochrome bg spectrum disappears and cannot be restored by the addition of more NADPH. The addition of inhibitor to a cytochrome bg spectrum produced aerobically causes its disappearance; however, if the incubation is made anerobic by bubbling nitrogen through it, the anerobic b, spectrum reappears. These observations suggest that the inhibitor is accepting electrons from the component which reduces 65 but not from bg itself, and under aerobic conditions prevents its reduction. Under anerobic condi-
I 425 WAVELENGTH
I 450 - nm
FIG. 7. Effect of housejly inhibitor on cytovhrome bg reduction by NADPH ,under aerobic (A) and anaerobic
yo Inhibition Inhibitor
THE
x IO-’
of housejly-head of NADPH-cytochrome
housejfy
FROM
I 30
FIG. 6. E$ect activity
INHIBITOR
(B)
conditions.
tions, the inhibitor becomes fully reduced and cytochrome bgcan then be reduced even in the presence of the inhibitor. The inhibitor apparently does not. interact. with cgtochrome P-450. Addition of inhibit’or t’o the CO-complex has no effect nor can a sub&rate binding spectrum be observed in the absence of CO. The inhibitor neither prevents a pyridinc type II substrate-binding spectrum from being formed nor displaces one previously formed. Efect oj reduced inhibitor on ,microsQmes, Housefly inhibitor, reduced with dithionite and introduced ancrobically into microsomal suspensions, will not reduce either cytochrome bj or P450. Under appropriate conditions either of these reduced spectra can be produced by NADPH. DISCUSSION
These investigations suggest that the inhibitor from housefly heads affects mixedfunction oxidase reactions in mouse liver by accepting electrons from an enzyme in the NADPH-dependent, electron transport thus diminishing the flow of electrons to cytochrome P-450. Schonbrod and Terriere (6) in a study of the effect, of the houseflyhead inhibitor on aldrin epoxidatBion by
70
WILSON
AND
housefly microsomes reached essentially the same conclusion. Both the rate and extent of p-nitroanisole demethylation by mouse liver microsomes is reduced by the inhibitor, effects which could be caused by the inhibitor oxidizing the reduced flavoprotein, NADPH-cytochrome c reductase. Moreover, the rate of NADPH oxidation is increased, further suggesting that this reductase is the site of action. This hypothesis is also supported by the competitive nature of the inhibition of p-nitroanisole demethylation when the NADPH concentration is varied. Moreover the inhibitor acts as an electron acceptor for NADPH-cytochrome c reductase purified from housefly abdomen microsomes. The similarity of the competitive inhibition of p-nitroanisole demethylation by the inhibitor, when NADPH concentration is varied, in both mouse liver and housefly microsomes, suggests that the mode of action is the same in both organisms. Stimulation of the reaction with cytochrome c is more difficult to explain, perhaps the reduced inhibitor “fits” the active site of cytochrome better than the reduced flavoprotein or possibly it acts as an allosteric activator. Since NADH is not a good electron donor for microsomal oxidations involving cytochrome P-450, the stimulation of NADPH oxidation suggests that the inhibit,or will also accept electrons from NADH-cytochrome c reductase, particularly when the stimulation is greater than that for NADPHoxidation. The effect of bovine serum albumin in reducing the inhibition, presumably by binding the inhibitor, is in conflict with the findings of Schonbrod and Terriere (6). If binding of xanthommatin is involved, this may be due to variations in the extent to which binding sites are already saturated in different commercial samples of bovine albumin. Goodman (16) demonstrated such variations in human serum albumin.
HODGSON
The spectral studies with cytochrome bj and cytochrome P-450 indicate that the inhibitor does not interact with either as an electron donor or acceptor or, in the case of cytochrome P-450, as a substrate. The equilibrium between oxidized and reduced forms of the inhibitor is such that it remains primarily in the oxidized state during NADPH oxidation and p-nitroanisole demethylation, since the yellow color imparted to the microsomal suspension persists. Under anerobic conditions, however, the inhibitor becomes primarily reduced and the color changes from yellow to pink. It is not known at present whether the inhibitor under aerobic conditions becomes fully reduced and then is rapidly reoxidized by molecular oxygen, or whether it is reduced to a semiquinone which undergoes rapid oxidation and reduction. Schonbrod and Terriere (5, 6) and Nagatsugawa (7) have both indicated that this inhibitor is xanthommatin. The data from this laboratory, while not extensive, tend to confirm this identification. REFERENCES
1. H.
2.
3.
4.
5.
6.
B. Matthews and E. Hodgson, Naturally occurring inhibitor(s) of microsomal inhibitors from the housefly, J. Econ. Entomol. 59, 1286 (1964). J. Chakroborty, C. H. Sissons, and J. N. Smith, Inhibition of microsomal oxidations in insect homogenates, Biochem. J. 102, 492 (1967). M. Tsukamoto and J. E. Casida, Metabolism of methylcarbamate insecticides by the NADPH-requiring enzyme system from houseflies, Nature (London) 213, 49 (1967). R. I. Krieger and C. F. Wilkinson, Microsomal mixed function oxidases in insects. 1. Localization and properties of an enzyme system effecting aldrin epoxidation in larvae of the southern armyworm, Biochem. Pharmacol. 18, 1403 (1969). R. D. Schonbrod and L. C. Terriere, Eye pigments as inhibitors of microsomal aldrin epoxidase in the housefly, J. Econ. Entomol. 64, 44 (1971). R. D. Sconbrod and L. C. Terriere, Inhibition of housefly microsomal epoxidase by the eye
MIXED
pigment, Physiol.
FUNCTIOK
xanthommat,in,
OXIDARE
Pest.
INHIBITOR
Biochem.
in press.
7. T. Nakatsugawa, personal communication. 8. S. Orrenius, M. Berggen, P. Moldeus, and R. I. Kreiger, Mechanism of inhibition of microsomal mixed fun&ion oxidases by the gutcont,ent’s inhibit)or of the southern army worm (Pfvdenia eridaflia), Biochem. J. 124, 427 (1971). 9. T. Wilson and E. Hodgson, Microsomal NADPH-cytochrome c reduct,ase from the housefly, MlLsca domestica: solubilization and purification, Insect Biochem. 1, 19 (1971). 10. T. Wilson and E. Hodgson, Microsomal NADPH-cytochrome G reduct,ase from the housefly, Musca domestica: properties of the purified enzyme, Insect Biochem. 1, 171 (1971). 11. A. Komberg and B. L. Horecker, in “Bio-
12.
13.
14.
15.
FROM
THE
HOUREFLl
71
chemical Preparations” (E. E. Snell, Ed.), * I J Vol. 3, p. 24, Wiley New York 1953. 0. H. Lowry, N. J. Rosebrough, A. 1,. Farr, and R. J. Randall, Protein measurement with the Folin phenol reagent, J. Riol. Chem. 193, 265 (1951). H. H. Moorefield, Improved method for harvesting housefly heads for use in cholinesterase studies, Contrib. Boyce l’h,rttttp,son Inst. 13, 463 (1957). L. Ernster, L. Danielson, and M. Ljunggren, DT diaphorase 1. Purification from the soluble fract,ion of rat liver cytoplasm, atId properties, Hiochi)n. Rioph~/s. .4cln 3, 171 (1962). I). S. Goodman, Preparation of human serum albumin free of long chain fatty acids, Sricnce 12.5, 1296 (1957 1.