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Evidence for a sex pheromone metabolizing cytochrome P-450 mono-oxygenase in the housefly.

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Archives of Insect Biochemistry and Physiology 6:121-140 (1987)
Evidence for a Sex Pheromone Metabolizing
Cytochrome P-450 Mono-Oxygenase in the
Housefly
Sami Ahmad, Kenneth E. Kirkland, and Gary J. Blomquist
Department of Biochemistry, University of Nevada, Reno
,
Direct evidence is presented for the role of a cytochrome P-450 monooxygenase (called mixed-function oxidase, or polysu bstrate mono-oxygenase,
PSMO) in the metabolism of the sex pheromone (Z)-9-tricosene t o its
corresponding epoxide and ketone in the housefly. A secondary alcohol,
most likely an intermediate i n the conversion of the alkene t o the ketone,
was also tentatively identified. The results of in vivo and in vitro experiments
showed that the PSMO inhibitors, piperonyl butoxide (PB) and carbon
monoxide, markedly inhibited the formation of epoxide and ketone from
(9,10-3H)(Z)-9-tricosene.An examination of the relative rates of (a-9-tricosene
metabolism showed that males exhibited a higher rate of metabolism than
females with the antennae of males showing the highest activity of any tissue/
organ examined. The major product from all tissueslorgans was the epoxide.
Data from experiments with subcellular fractions showed that the microsomal
fraction had the majority of enzyme activity, which was strongly inhibited by
PB and CO and required NADPH and O2 for activity. A carbon monoxide
difference spectrum with reduced cytochrome showed maximal absorbance
at 450 n m and allowed quantification of the cytochrome P-450 in the
microsomal fraction of 0.410-nmol cytochrome P-450 m g - I protein. Interaction
of (Z)-9-tricosene with the cytochrome P-450 resulted in a type I spectrum,
indicating that the pheromone binds t o a hydrophobic site adjacent to the
heme moiety of the oxidized cytochrome P-450.
Key words: epoxidation, hydroxylation, Musca domestica, polysubstrate mono-oxygenase,
(2)-9-tricosene
INTRODUCTION
The sex pheromone of the housefly, Muscu domesticu, consists of (3-9tricosene (CZ3alkene) [l]and related oxygenated compounds (Z)-9,lO-epoxAcknowledgments: This work was supported in part by National Science Foundation grant
DCB-8416558 and i s a contribution of the Nevada Agricultural Experimental Station. We thank
the Biology Section, S.C. Johnson and Sons, Racine, WI, for supplying housefly pupae.
Received January29,1987; accepted May 26,1987.
Address reprint requests to S. Ahmad, Department of Biochemistry, University of Nevada,
Reno, NV 89557-0014.
0 1987 Alan R. Liss,
Inc.
122
Ahmad, Kirkland, and Blomquist
ytricosane (C23 epoxide) and (Z)-14-tricosen-lO-one (C23 ketone) [2], along
with a series of C28-30 methylalkanes [3]. Recently, the precise role for each
component of the sex pheromone was established [4]. Accordingly, (4-9tricosene is responsible for initiating the courtship ritual and especially inducing ”striking activity” in males. The C23 epoxide and ketone are ”sex
recognition” factors, whereas the methylalkanes act as ”arrestants,” and
promote and extend sexual contact and mating.
The precursors and enzymatic pathways in the biosynthesis of the hydrocarbon components [(.Z)-9-tricoseneand methylalkanes] of the pheromone
have been elucidated [5]. It was demonstrated that the oxygenated components, C23 epoxide and C23ketone, are derived from (Z)-9-tricosene [6], and
also hypothesized that these two components were products of oxidative
attack by a “mixed function oxidase type enzyme,” also called a polysubstrate mono-oxygenase.
This suggestion prompted a series of experiments on the in vivo and in
vitro metabolism of (4-9-tricosene involving cytochrome P-450 mono-oxygenase inhibitors, piperonyl butoxide and carbon monoxide. The results,
reported here, show that both C23 epoxide and C23 ketone are products of a
microsomal cytochrome P-450 attack on (2J-9-tricosene.
Aside from the well-established role of cytochrome P-450 in metabolism/
deactivation of foreign substances in organisms, these enzymes are further
adapted for participation in the biosynthesis and regulation of substances
physiologically important to insects [7,8]. This is the first report providing
direct evidence for a role of the mono-oxygenase in pheromone synthesis
and deactivation.
MATERIALS AND METHODS
Insects
The housefly, Fales 1958 strain T-11, was supplied by the Biology Section,
S.C. Johnson and Sons (Racine, WI). Within 12 h of emergence from the
pupae, males and females were separated and held at 25 5 1”C, ca. 60-80%
relative humidity, and provided ad libitum sucrose and low-fat powdered
milk (l:l,wlw) and water.
Chemicals
[9,10-3H](Z)-9-Tricosene (60 Cilmmol) was prepared and purified as described earlier [6] and dissolved in either hexane or ethanol. The radiochemical purity of (4-9-tricosene was found to be 99+% by radio-GLC* [6]. The
use of high specific activity [3H](Z)-9-tricosenehas the advantage of reflecting
the low amounts presumably transferred to males from females in courtship
and mating. In addition, since females produce (Z)-9-tricosene, the addition
*Abbreviations: BSA = bovine serum albumin (fraction V); HC = hydrocarbon; PB = piperonyl butoxide; PMSO = polysubstrate monooxygenase; NHC = nonhydrocarbon; radio-CLC
= radio-gas-liquid chromatography; TLC = thin-layer chromatography.
Pheromone Metabolismby Cytochrome P-450
123
of tracer amounts of labeled material does not significantly affect endogenous
amounts on the insect.
Unlabeled Q-9-tricosene was purchased from Sigma Chemical Co., St.
Louis, MO . The (Z)-9,10-epoxytricosane,tricosan-12-one, and tricosan-12-01
were prepared according to the procedures given by Blomquist and Jackson
[9] and Blomquist et al [6]. The ketone and secondary alcohol were used for
TLC standards, where they comigrated with (Z)-14-tricosen-lO-one and (414-tricosen-10-01.
Glucose-6-phosphate, glucose-6-phosphate dehydrogenase (450 units
mg-' protein), Tris buffer, sodium dithionite, and BSA were obtained from
Sigma Chemical Co. Piperonyl butoxide was purchased from Chem Service,
Inc., West Chester, PA. Coomassie brilliant blue G-250 was obtained from
Bio-Rad Laboratories, Richmond, CA. All other chemicals used were of
highest purity available (98+%), and all reagent grade solvents were redistilled prior to use.
In Vivo Assays
Three-day-old unmated male and female flies were seperated into two
batches; one batch of each sex was pretreated topically with 5 p g of PB
dissolved in 1pl acetone to the fly dorsum, the other with 1p1 acetone. Equal
batches of PB-treated and solvent control flies were held in glass beakers
covered with glass petri dishes for 30 min. Flies from both batches were then
treated with 1p1 of [9,10-3H]((Z)-9-tricosenesolution in hexane. Another batch
of flies treated with labeled(Z)-9-tricosenewere held under a gentle stream
of CO:N2:02 (60:20:20) 20 cclmin. Each fly received ca. 100,000 dpm of
[3H](Z)-9-tricosene.The flies were transferred into 10-ml glass vials chilled
over dry ice 30 and 60 min after the treatment with the labeled material. Two
ml of hexane was immediately added (2 ml) and the glass containers were
capped and stored at -20°C for later extraction. Each replicate consisted of
ten flies, and there were three to four replicates to each treatment group.
Organ Tissue Assays
Male and female flies were dissected in isotonic 1.15%KC1 solution chilled
to ca. 4°C. The following organsltissues from eight flies were collected:
abdominal and thoracic epithelia including the cuticle, antennae, fat bodies,
and legs. Tissues from eight flies comprised a replicate; the experiment was
duplicated. Each organltissue replicate was placed in 0.5 ml of the isotonic
KCl solution. The abdominal and thoracic epithelia were placed with the cell
layer facing down and in contact with the KC1 solution; the cuticle was
dorsally exposed. Approximately 1,000,000 dpm of [3H](Z)-9-tricosenein 4 pl
ethanolic solution was carefully spread over the upper surface of the pooled
tissues. The tissues were then equilibrated at 30°C in a shaker bath with
minimal agitation. After 5 min, 1.5 ml of isotonic KC1 and Tris buffer, 50 mM,
pH 7.5, was added. After 15 min, the reaction was terminated by adding 6
ml of a methanol-chloroform (2:l) mixture and the samples were capped and
stored at -20°C.
In some tissuelorgan incubations, the incubates were treated with 5 pgl
replicate PB (in 1 p1 of ethanol) 10 min prior to starting the assay by the
124
Ahmad, Kirkland, and Blomquist
addition of the labeled substrate. Controls were pretreated with 1 pl of
ethanol.
Subcellular Assays
Subcellular fractions containing mitochondria and microsomes were prepared by the following procedure. From 3-day-old unmated male and female
houseflies, 200 intact abdomens, and, in a separate experiment, 200 abdomens with guts removed, were ground in a glass homogenizer with a Teflon
pestle. The homogenization medium was 20 ml of cold (2-4°C) 50 mM Tris
buffer (pH 7.5) containing 1.15" (wh) KC1. The homogenate was immediately
fractionated into subcellular components by differential centrifugation. The
750 g (for 15 min) precipitate was discarded. The 750 g supernatant was
centrifuged at 10,800 g for 20 min at 2°C to pellet the mitochondrial fraction.
The supernatant was centrifuged at 100,900 g for 1 h at 1°C to obtain the
microsomal pellet. The mitochondrial and microsomal fractions were washed
by resuspension in the homogenization buffer and recentrifuged to minimize
cross contamination.
Protein concentration was determined by the Bio-Rad Coomassie blue
method using BSA as standard. Both mitochondrial and microsomal fractions
were adjusted to 2 mg protein ml-I with the Tris buffer-KC1 solution.
An incubation volume of 2 ml contained 100 pmol Tris Buffer, pH 7.5, 24.4
pmol KCI, 4.8 pmol NADP, 4.8 pmol glucose-6-phosphate, 0.48 units of
glucose-6-phosphate dehydrogenase, and 2 mg of mitochondrial or microsoma1 protein. The reaction was started by adding ca. 1,000,000 dpm of [3H(Z)9-tricosene in 4 pl of ethanol. Incubations were carried out aerobically for 20
min with agitation in a shaker water bath at 30°C. The reaction was terminated by adding 6 ml of methanol-chloroform (2:1), and immediately stored
at -20°C. In some samples, 5 pg of piperonyl butoxide in 1pl of ethanol was
added 10 min prior to the addition of the labeled substrate, whereas 1 pl
methanol was added to controls. Each experiment was duplicated.
In another experiment using microsomal preparations from whole abdomens, instead of the NADPH-generating system, varying amounts of
NADPH were directly added and incubations were carried out aerobically
under continuous gassing with water-saturated N2:02 (80:20). To demonstrate oxygen dependence, additional incubations were performed under
water-saturated 100% N2. The inhibition by CO was demonstrated by continuously gassing the incubation mixtures with water-saturated C0:02 (80:20),
whereas controls were gassed with N2:02(80:20).
Extraction, Partitioning, and Clean-Up of Metabolites
Insects from in vivo experiments were extracted by immersion in 2 ml of
hexane for 10 min, and then rinsed twice with 2 ml of hexane for 1min. The
rinses were combined, the volume reduced under N2, and one-tenth aliquots
were used for assaying radioactivity.
Rinsed insects from the in vivo experiment, incubated organsltissues, and
subcellular assays were extracted by the method of Bligh and Dyer [lo].
Phase separation was facilitated by the addition of ca. 50 mg anhydrous
Pheromone Metabolism by Cytochrome P-450
125
Na2S04 and by refrigeration overnight at 2°C. The aqueous and the organic
phases were each reduced in volume under a stream of N2, the organic phase
to 1ml and the aqueous phase to 5 ml, and one-tenth aliquots of each were
used for determination of radioactivity.
The organic phase was separated into HC and NHC fractions on 7 cm by
0.5-cm columns packed with activated Biosil A. The HCs were eluted with 7
ml of hexane. The NHCs were eluted with 8 ml diethyl ether-hexane: 3:2.
Each fraction was reduced in volume under N2 and reconstituted to 1ml;
one-tenth aliquots of each sample were used to assay radioactivity.
Metabolite Separation and Identification
Radio-GLC was performed as described by Blomquist et al. [6]. The nonhydrocarbons (diethyl ether-hexane fractions) were separated by TLC developed in hexane-diethyl ether (85:15). With the use of standards, the zones
corresponding to the epoxide, ketone, and secondary alcohol metabolites
were ascertained, and, moreover, additional metabolites were detected by
sequential cm-by-cm scan of the TLC plates for radioactivity. The Rfs of the
identified metabolites and of unknowns were: (Z)-14-tricosen-lO-one
(0.89Ifr 0.01), (Z)-9,10-epoxytricosane (0.85~frO.Ol), unknown 3 (0.61f0.032),
(Z)-14-tricosen-l0-o1(0.461t0.03), unknown 2 (0.35+0.02), and unknown 1
(0.10 Ifr 0.01)
1
Assay for Radioactivity
Radioactivity was determined by assaying sample aliquots by liquid scintillation spectrometry at 45-50% efficiency. All data were corrected for background. Aliquots of organic phases were assayed by placing in 10 ml of a
toluene-based (PPO, 0.4%, wlv) scintillation cocktail and aqueous samples
were assayed in 10 ml Formula 963 Scintillation cocktail (NEN Research
Products, Boston, MA).
Cytochrome P-450 Spectral Analysis
The CO-difference (of the reduced cytochrome P-450-carbon monoxide
complex) spectrum was obtained essentially as described by Omura and Sat0
[ll]. The dithionite used in reducing the cytochrome P-450 often converts it
to substantial amounts of the inactive form, cytochrome P-420, also accompanied by a reduction in maximum absorbance of cytochrome P-450 at 450
nm from an erroneously apparent wavelength shift, thus resulting in incorrect estimation of the cytochrome content [7]. Microsomes were therefore
reconstituted in 50 mM Tris buffer, pH 7.75, containing 1mM EDTA and 20%
(vh) glycerol. This procedure minimized cytochrome P-450 to cytochrome P420 conversion. The amount of cytochrome P-450 was determined using the
extinction coefficient, 91 mM-' cm-', for the absorbance between 450 and
490 nm, and the cytochrome P-420 was also measured using the extinction
coefficient, 110 mM-l cm-' at 420 nm [ll]. An Aminco DW-2 spectrophotometer in the split beam mode was used to measure the absorbances of the
reduced CO-cytochromes P-450 and P-420 difference spectra.
126
Ahmad, Kirkland, and Blomquist
A difference spectrum of (Z)-9-tricosene binding to the oxidized cytochrome P-450 was obtained. The ligand was added gradually to the oxidized
cytochrome preparation as a 25 nm solution in acetone. An equal volume of
acetone was added to the reference cuvette. A difference spectrum appeared
when the ligand concentration reached 25 pM. Spectra were recorded for
microsomes from both male and female flies.
RESULTS
In Vivo Metabolism of (3-9-Tricosene
Most of the recovered radioactivity from topically administered [3H](Z)-9tricosene in both male and female houseflies was present in the hexane
extract, representing material on the surface of the insect (Table 1).In males,
between 83% and 94% of the recovered radioactivity was in external washes,
and in females, between 94% and 98%.
The metabolites of [3H](Z)-9-tricosenepresent on the surface of the insect
increased with time, from 27.4% to 36.6% in males and 5.5% to 10.8% in
females at 30 and 60 min, respectively (Table 1).Metabolism of the alkene in
males proceeded more rapidly during the first 30 min, amounting to 75% of
the metabolism observed at 60 min. In females, however, the total amount
of (3-9-tricosene metabolized by 60 min was doubled of that at 30 min,
suggesting that in females metabolism proceeds at a constant rate up to 1h.
In addition to this difference, metabolism of (3-9-tricosene was significantly
greater in males than in females (ANOVA, P > F = 0.01; metabolism ratio
ca. 37:11 at 60 min). However, direct comparison of rates of conversion in
males and females is not possible. Males do not produce the pheromone, but
the females past day 2 are producing (Z)-9-tricosene1 which dilutes the
specific activity of topically administered [3H](Z)-9-tricosene.
Effects of Cytochrome P-450 Inhibitors on the Metabolism of
[3H](2n-9-Tricosene
Pretreatment with PB (5 pglinsect) markedly inhibited [3H](Z)-9-tr*
icosene
metabolism. There was a decrease in the amount of metabolites in both males
(66% and 72% at 30 and 60 min, respectively) and females (55% and 61% at
30 and 60 min, respectively) (Table 1).Carbon monoxide also markedly
inhibited the alkene metabolism. there was a decrease in the amount of
metabolites in both males (86%) and females (55%) in 30 rnin (Table 1).
Formation of Metabolites
Radio-GLC analyses of the HC fractions from both the external and internal extracts (and those derived from control, PB, or CO-treated insects) all
indicated that (G-9-tricosene was the sole constituent. TLC was used to
separate each component of the NHC fraction. In addition to the original
position of the spot (TLC origin), six metabolites were clearly resolved. Of
the six metabolites, three cochromatographed with authentic standards of
the C23 epoxide, ketone, and secondary alcohol. The remaining were designated as unknowns (U) 1,2 and 3, and, together with the origin, are included
60
60
30
30
30
30
30
30
60
60
Incubation
time (min)
94.0
94.9
92.5
94.9
97.6
f 1.0
f 1.0
f 0.2
f 0.6
f 0.3
87.8 f 0.8
93.8 f 0.4
90.4 rt 0.5
83.4 f 0.5
91.5 f 1.6
5.3 f 0.4
2.4 f 0.2
2.4 f 0.1
10.8 f 0.8
4.2 f 0.1
27.4 f 1.6
9.3 f 2.1
6.6 rt 0.5
36.6 f 1.6
10.4 f1.4
metabolized
% Alkene
rt 2.0
f 7.6
f 2.8
f 6.7
f 6.1
53.8 f 4.4
63.2 f 5.1
63.6 f 5.4
61.4 f 5.0
54.0 f 2.8
39.8
41.7
37.3
42.6
45.1
Epoxide
21.8 f 3.9
19.1 f 5.0
16.9 ir: 0.9
24.5 f 1.4
29.9 f 2.2
14.8 f 2.6
16.6 f 1.3
17.3 f 0.8
13.3 ir: 3.4
14.8 f 4.8
4.2
4.3
4.0
2.0
5.8
4.5
4.4
3.7
2.9
3.2
ir: 2.3
f 0.5
f 0.7
f 0.4
f 0.4
f 1.6
f 0.9
f 0.3
f 1.2
f 1.8
20.2 f 4.1
13.2 ir: 0.2
15.5 f 5.7
12.1 f 3.2
10.3 f 2.0
40.9 f 6.1
37.3 f 8.3
41.7 f 2.0
41.2 f 9.4
36.9 f 5.7
% Composition of external metabolites
Secondary
Ketone
alcohol
Unknowns
*Data are means f SD derived from four replicates, except for the CO experiments, where means are derived from three replicates. A batch
of ten insects comprised a replicate, and each insect was topically treated with ca. 100,000 dpm [3H] (Z)-9-tricosene.Total average recovery
of the label including Bligh and Dyer extracts, was 98%, the range being 96-102%. PB, piperonyl butoxide.
+
Females
+PB
+CO
PB
+PB
+co
+
Males
PB
Inhibitor
(PB or CO)
%
Recovered
in hexane
extracts
TABLE 1. The Effect of the Cytochrome P-450 Inhibitors, PB and CO, on the In Vivo Metabolism of [3H](Z)-9-Tricosene in 3-Day-Old
Male and Female Houseflies*
128
Ahrnad, Kirkland, and Blornquist
as "unknowns" in Table 1. Radio-GLC [6] was used to confirm that the
radioactivity in the epoxide and ketone fractions was entirely in the C23
components. A C23 secondary alcohol was predicted to be the precursor of
the C23 ketone [6] and its resolution and quantification here supports that
hypothesis. The relative proportions of C23 epoxide, ketone, secondary alcohol, and unknown metabolites are given in Table 1.The external metabolite
composition in both males and females was C23epoxide > ketone > secondary alcohol, but the internal composition was C23 epoxide > secondary
alcohol > ketone. These data indicate that the less polar epoxide and ketone
are more efficiently extruded to the cuticular surface than the more polar
secondary alcohol. The internal pool of secondary alcohol is apparently
designated for oxidation to the ketone, much of which is then transported to
the cuticle.
Cytochrome P-450 epoxidation and hydroxylation (in the formation of
alcohols) reactions are strongly inhibited by PB and CO 17,121, a finding
consistent with a profound decrease of (9-9-tricosene metabolism (Table 1).
The decrease in ketone formation is undoubtedly due to the inhibition of the
enzyme that forms its precursor, the secondary alcohol.
The radioactivity in the aqueous phase showed that the total water-soluble
radioactivity was much higher in males compared with females. For example,
42.0 k 2.3% of the internal radioactivity from 30-min incubations in males
was in the aqueous phase, compared with 15.2 If: 4.2% for females. This is
consistent with the greater amounts of [3H](Z)-9-tricosenemetabolized by
males to NHC, which are then presumably converted to water-soluble products by phase I1 enzymes [12].
Comparative Profile of (23-9-TricoseneMetabolism by Body Parts (Organs/
Tissues) of Male and Female Houseflies
(3-9-Tricosene metabolism by isolated organs/tissues was greater in males
than in females (Table 2). Male: female ratios, depicting metabolic rate differences were: antennae, 4.0:l.O; legs, 1.6:l.O; fat bodies, 1.5:l.O; and abdominal
epithelial tissue, 1.8:l.O. The ratio for thoracic epithelial tissue was 0.7:1.0,
indicating that in this tissue, in females, the metabolic capacity surpasses
that of males. Also, within each sex, the rate of metabolism greatly varied:
the order in males being: antennae > > legs 2 fat bodies 2 abdominal
epithelial tissue > > thoracic epithelial tissue. In females, however, the rate
of metabolism was thoracic epithelial tissue = legs > fat bodies 2 abdominal
epithelial tissue > antennae.
Data in Table 2 further show that in males, more water-soluble than NHC
metabolites were formed by antennae and legs: 67% and 154% more, respectively. In the remaining body parts, the NHC components were in excess of
water-soluble metabolites. In females, nearly equal amounts of radioactivity
were in the NHC fraction and the aqueous phase from antennae, and were
greater in the NHC fraction than other fractions in all other tissues. These
data suggest that the male antennae and legs have the highest activity of
phase-I1 enzymes [12].
Piperonyl butoxide markedly inhibited the metabolism of (Z)-9-tricosene
by all organsltissues, presumably by blocking the initial enzymatic attack on
3.9
(3.7-4.1)
6.1
(6.1-6.1)
5.2
(4.7-5.6)
4.7
(4.5-4.9)
6.1
(6.0-6.2)
15.5
(15.1-15.8)
9.9
(8.4-11.4)
9.0
(8.4-9.5)
8.5
(8.3-8.6)
4.2
(3.9-4.4)
(%I
1.9
(1.7-2.0)
3.4
(3.43.4)
4.1
(3.7-4.4)
3.8
(3.6-4.0)
4.2
(4.1-4.3)
5.8
(5.7-5.8)
2.8
(2.5-3.1)
5.0
(4.7-5.2)
4.5
(4.44.5)
2.9
(2.6-3.1)
2.1
(2.0-2.1)
2.7
(2.7-2.7)
1.1
(1.0-1.2)
0.9
(0.9-0.9)
1.9
(1.9-1.9)
9.7
(9.4-10.0)
7.1
(5.9-8.3)
4.0
(3.7-4.3)
4.0
(3.9-4.1)
1.3
(1.3-1.3)
Distribution (%) of metabolitesa
NHC
Aqueous
49.8
(43.7-55.9)
65.3
(65.0-65.0)
57.4
(56.6-58.1)
47.8
(43.0-52.6)
56.8
(56.6-56.9)
75.2
(74.1-76.3)
72.0
(71.4-72.6)
60.5
(60.4-60.6)
56.2
(54.6-57.8)
62.3
(62.3-63.9)
54.2
(51.4-56.1)
69.8
(68.7-70.1)
37.5
(33.7-41.3)
54.6
(48.6-60.6)
75.2
(74.6-75.8)
83.3
(82.6-84.0)
79.6
(78.4-80.7)
68.3
(67.5-69.1)
65.3
(63.4-67.2)
78.7
(77.7-79.6)
Inhibition (%) by PBa
NHC
Aqueous
*Metabolism of [3H] (Z)-9-tricosene directly applied to separate pools of antennae, legs, fat bodies, abdominal epithelium (abd. ep.), and
thoracic epithelium from eight insects, and incubated in isotonic KC1-Tris, pH 7.5, solution for 20 min at 30°C. The total recovery of 3H was
in the 96-99% range. Data in the last column are transformed to show percentage inhibition of 3H metabolites partitioning either in NHC or
aqueous fractions. PB, piperonyl butoxide; NHC, nonhydrocarbon.
aData are averages of duplicate determinations, with the range given in parentheses.
Thoriac ep.
Abd. ep.
Fat Bodies
Legs
Females
Antennae
Thoriac ep.
Abd. ep.
Fat Bodies
Legs
Males
Antennae
OrgandTissues
Total (Z)-9-tricosene
metabolizeda
TABLE 2. Profile of [3H] (Z)-g-Tricosene Metabolism by Isolated OrganslTissues of 3-Day-Old Male and Female Houseflies*
Ahmad, Kirkland, and Blornquist
130
60y
i
"1-7
40:
Male
0
-
~~~
2
2
c
20
3
m
01
-
40-
z
*
u)
I
Female
ANTENNAE
ANTENNAE
0
a
00
-
-
~
- a a -1
-
1
LEGS
LEGS
+
+
-
_.
__c_
&
FAT BODIES
-
+
~
-
~
1
-
~
a
-
FQl===I
--c
FAT BODIES
-
Fig. 1. Relative percent distribution of the metabolites from selected organsitissuesof 3-dayold male and female houseflies incubated with [3H](Z)-9-tricosenefor 20 min at 30° C.
Metabolites shown are: epoxide (E), ketone (K), secondary alcohol (A), unknowns (UI-U3),
and radioactivity at the original thin-layer chromatography spot (0).Epithelium is abbreviated
as Ep. In each data bar, the upper and lower segments represent the relative amount of
metabolite formed in the control and piperonyl butoxide (PBI-treated tissues, respectively.
Each determination contained a pool of organs/tissues from eight insects, and the data are
averages of duplicate determinations with the error bars representing the range.
Pheromone Metabolism by Cytochrome P-450
131
indicate relative proportions of each of the metabolites. In all organsltissues
of the males, epoxide was by far the most abundant metabolite, followed by
secondary alcohol and ketone. In females, the relative amounts of epoxide,
ketone, and secondary alcohol were more variable. The relative metabolite
composition from antennae, legs, and thoracic epithelial tissue is essentially
the same in males (Fig. 1).A different pattern was observed for fat bodies
and abdominal epithelial tissue. These two tissues form significantly greater
(P > F = 0.05) amounts of C23 ketone, compared with antennae and legs. In
females, the antennae and legs produce high levels of C23 ketone, whereas
the fat bodies and the abdominal and thoracic epithelial tissues produce high
amounts of the epoxide, followed by the ketone and secondary alcohol.
Subcellular Assays
Metabolism of (Z)-9-tricosene was much more active in microsomal than
in mitochondria1 preparations from 3-day-old male and female houseflies.
Data presented in Table 3 show that the microsomal preparation from whole
abdomens converted 22.1% and 17.2% of the alkene to oxygenated products
in 20 min by males and females, respectively. The difference in the metabolic
rate between the two sexes was significant (P > F = 0.05). The metabolic
rate by microsomes from abdomens without the gut tissues was 10.7%
(males) and 12.9% (females), the decrease in the rate indicating that the gut
component of the enzyme in the intact abdominal microsomes contributed
to its high activity. (Z)-9-Tricosene metabolism by the microsomal enzymes
was markedly inhibited by PB (Table 3), with at least a 50% inhibition of the
formation of NHC metabolites and between 27.6% and 82.6% inhibition of
formation of aqueous metabolites. Carbon monoxide inhibition of the microsoma1 metabolism of the alkene was even more pronounced (Table 4), with
87.6% to 89.6% of inhibition of the formation of NHCs and between 86.4%
and 91.1% inhibition of formation of water-soluble metabolites.
The relative profiles of metabolites formed, largely in the NHC fraction,
are shown in Figure 2. Common to both sexes is the following pattern of
metabolite formation by the microsomal enzymes:
1. The C23 epoxide comprised an average of 85% of all NHC components,
whereas the secondary alcohol averaged 7.5%. This indicates that the epoxidation, rather than hydroxylation, is a more favored reaction.
2. All other metabolites were formed in very small quantities, including
the C23 ketone. Apparently the ketone is a further oxidation product of the
secondary alcohol, by another enzyme. The inhibition of the ketone formation by PB parallels the inhibition of its precursor.
Both PB and CO inhibited the formation of all metabolites of the NHC
fraction, indicating that they all arise from mono-oxygenation reaction directly, or, like the ketone, via secondary metabolism of primary products
from a mono-oxygenase-catalyzed reaction. Furthermore, the microsomal
preparation containing only the peripheral tissues’ enzyme, compared to the
other preparation containing the gut tissues, was less sensitive to the inhibition of the epoxidation reaction by PB but more sensitive to the inhibition of
hydroxylation. This pattern suggests the possibility of a qualitative difference
17.2
(16.7-17.7)
12.9
(12.5-13.3)
22.1
(20.8-23.4)
10.7
(10.4-11.1)
Total (Z)-9-tricosene
metabolized (%)
14.3
(13.8-14.7)
10.2
(10.0-10.4)
17.6
(16.7-18.4)
8.8
(8.5-9.2)
2.9
(2.9-2.9)
2.7
(2.5-2.9)
4.5
(4.1-5.0)
1.9
(1.9-1.9)
Distribution (%)
of metabolites
NHC
Aqueous
60.6
(59.6-61.6)
57.0
(49.3-52.6)
53.1
(51.5-54.6)
51.2
(51.1-51.3)
NHC
49.5
(43.0-55.9)
82.6
(79.3-85.9)
27.6
(28.0-37.2)
68.7
(64.6-72.8)
Aqueous
Inhibition by PB (%)b
*Labeled substrate was incubated with 2 mg microsomal protein for 20 min at 30°C, in total volume of 2.0 ml and buffered with KC1-Tris, pH
7.5.
aAbd.+G, intact abdomen; abd. -G, abdomen without guts.
bData are averages of duplicate determinations, with the range given in parentheses. NHC, nonhydrocarbons; PB, piperonyl butoxide.
Abd.-G
Females
Abd.+G
Abd.-G
Males
Abd. +G
Source of
microsomesa
TABLE 3. Profile of [3H](Z)-9-TricoseneMetabolism by Microsomal Preparations From Intact Abdomens and Abdomens Without Guts of
3-Day-Old Male and Female Houseflies*
Pheromone Metabolism by Cytochrome P-450
133
TABLE 4. Effect of CO on Microsomal Metabolism of [3H](Z)-9-Tricosene by Microsomal
Preparations From Whole Abdomens of 3-Day-Old Male and Female Houseflies*
%(Z)-9Tricosene
metabolized
Incubation
atmosphere
Males
N2:02 (80:20)
C0:02 (80120)
Females
N2:02 (80:20)
C0:Oz (80:20)
Relative (%)
partitioning of metabolites
NHC
Aqueous
YO Inhibition by CO
NHC
Aqueous
20.9 f 0.2
2.2
< 0.1
*
16.4 f 0.2
1.7 f 0.1
4.5 f 0.1
0.4 f < 0.1
89.6
91.1
18.3 f 0.3
2.3 f 0.1
16.1 f 0.4
2.0 f 0.1
2.2 f 0.1
0.3 I < 0.1
87.6
86.4
*All values are means f SD derived from three replicates. A batch of ten insects comprised a replicate,
and each insect was topically treated with [3H](Z)-9-tricosene.Values below 0.05 are shown as < 0.1.
Incubation compositions and conditions were the same as given in the footnote of Table 3. NHC,
nonhydrocarbons .
Male microsornes
U
U
6
A
iti
h
E
K
A U l U 2 U 3 0
Female microsornes
U
c>
A
0
E
K
A U l U 2 U 3 0
E
R
RR
K A
UlU2U30
0
E
K
A
UlU2U3 0
Fig. 2. Relative percent distribution of the metabolites from incubations of microsomal
preparations from whole abdomens (A) and abdomens without the gut tissues (B) of 3-dayold-houseflies, with [3H](Z)-9-tricosene.Labeled substrate was incubated with 2 mg microsoma1 protein for 20 min at 30OC. Metabolites shown are; epoxide (E), ketone (K), secondary
alcohol (A), unknowns (UI-U3), and the radioactivity at the origin thin-layer chromatography
spot (0).In each data bar, the upper and lower segments represent the relative amount of
metabolite formed in the control and piperonyl butoxide (PB)-treated microsomal preparatons, respectively. Data are averages of duplicate determinations with the error bars representing the range.
134
Ahrnad, Kirkland, and Blornquist
between the peripheral cytochrome P-450 monooxygenase vs. the gut enzyme in (2)-9-tricosene metabolism in both sexes.
The monooxygenations catalyzed by microsomes prepared from both male
and female houseflies was highly dependent on NADPH (optimal amount
was 2.0 pmol) and molecular oxygen, as shown in Figure 3. In the absence
of any exogenous NADPH, the alkene metabolism was only 0.9% (males)
and 0.6% (females); these values were subtracted from each data point in
depicting the NADPH-dependency curves (Fig. 3). It is also evident from
Figure 1that there is an absolute requirement of 0 2 in the mono-oxygenation
reactions, because under N2 the metabolism of (9-q-tricosene was drastically
reduced. Minimal metabolism observed under gassing with N2 could be from
incomplete elimination of 0 2 from the incubation mixture.
The mitochondrial fraction produced much lower amounts of oxygenated
products of (3H](z)-9-tricosene (between 2.6% and 4.3% of recovered radioactivity) than did the microsomal fraction. Similar products were produced
by both fractions, but the mitochondrial activity was not inhibited by piperonyl butoxide (data not shown). Because of the low activity in the mitochondrial fraction, it was not further characterized.
22 20 24
zf
!2
$
N2:02* 80:20
0
la-
i-
Male
Female
16-
I
w
6
3
I-
6
0
6
a.
141210-
a6 -
4-
0
I
I
I
I
1
2
3
4
prnol NADPH
Fig. 3. NADPH and O2 dependency computer-generated plots for the metabolism of 13H](D9-tricosene by microsomes prepared from whole abdomens of 3-day old male and female
houseflies. The incubations were conducted in total volume of 2.0 ml buffered by KCI-Tris,
pH 7.5, and contained 2 mg microsomal protein labeled substrate, and varying amounts of
NADPH. Incubations were carried out at 3OoC and terminated at 20 min. NADPH dependency
of alkene metabolism was established under aerobic conditions by continuous gassing withsaturated N2:02 (80:20). Oxygen dependency was demonstrated by gassing incubation mixtures with 100% N2. The data points are means derived from three replicates. The SDs of the
means were all less than 10% and the majority were under 2% of the means.
Pheromone Metabolism by Cytochrome P-450
135
Difference Spectra: Evidence of Cytochrorne P-450 and Its Binding to
(Z)-f)-Tricosene
As shown in Figure 4, microsomal preparations from 3-day-old male and
female houseflies produced a CO-difference spectrum with reduced cytochrome with maximal absorbance at 450 nm. This feature is typical of cytochrome P-450 and forms the basis of its quantification. The absorbance mg-'
of the Fales 1958 strain I1 is 0.0375, amounting to 0.41 nmol cytochrome P450 m8-l protein; it compares well with absorbances of 0.0335 and 0.0310
established for the FC and CSMA strains of the housefly, respectively [q.
There was some conversion of cytochrome P-450 to the inactive form, cytochrome P-420, which also complexes with CO. The amount was calculated
to be 0.06 nmol cytochrome P-420 mg-' protein. The conversion is affected
by sulfuric acid formed by the oxidation of the cytochrome reductant, sodium
dithionite
Figure 4 also shows the (.Z)-9-tricosene-oxidized cytochrome P-450 difference spectrum, which resembles a type I spectrum. The absorbance peaks
and troughs vary for type I spectra, but the peak for both male and female
enzymes is at ca. 385 nm, which is usual for type I spectra [7] and is
accompanied by a broad trough (400-450 nm) that varies for the two sexes.
A type I spectrum indicates that (2)-9-tricosene resembles the majority of
lipophilic substrates in binding to a hydrophobic site adjacent to the heme
moeity of the oxidized (Fe3') cytochrome P-450. In a complete cytochrome
P-450 system substrate complexed with oxidized cytochrome P-450 under-
[n.
0.1 -
0.1
A
B
w
0
z
a
E
51
m
a
0.0
i
400
i
350
I
I
I
I
450
500
400
450
I
I
i
425
500
350
WAVELENGTH
(nm)
i
_
_
_
.
.
.
A
500
_
_
425
-
i
500
WAVELENGTH (nm)
Fig. 4. Difference spectra of the microsomal cytochrome P-450 of 3-day-old houseflies. A and
B depict spectra of male and female reduced (Fe+') cytochrome P-450-carbon monoxide
complex. C (males) and D (females) show type I binding spectra of the oxidized cytochrome
P-450 (Fe+3)with the ligand, (Z)-9-tricosene. Baseline for the spectra are also given.
136
Ahmad, Kirkland, and Blomquist
goes reduction. The reduced complex then binds with molecular oxygen,
and following oxygen activation the substrate is mono-oxygenated 1121. Evidence that (3-9-tricosene complex with the cytochrome follows this sequence is apparent from the data from the in vivo as well as in vitro studies.
DISCUSSION
The pathways for the biosynthesis of the housefly pheromone components, the alkene (Z)-9-tricosene, and C28-3 methyl-branched alkanes, have
been previously established [13,14]. Accordingly, the alkene is synthesized
by the elongation of oleic acid followed by conversion to the alkene, and the
methyl-branched alkanes originate by the elongation of methyl-branched
precursors. Except for the suggestion that a mixed-function oxidase may be
involved [6], until now there has been no direct evidence for the enzymatic
pathway responsible for the formation of the remaining two oxygenated
hydrocarbon pheromone components, the C23 epoxide and the C23 ketone.
The data reported here strongly implicate a cytochrome P-450-dependent
microsomal PSMO (E.C. 1.14.14.1) in the synthesis of these two pheromone
components. Thus we now have a more complete profile on the precursors
and pathways for the biosynthesis of all pheromone components of the
housefly.
A number of criteria necessary to ascertain the involvement of cytochrome
P-450 biotransformation [12,15] in (3-9-tricosene metabolism in the housefly
have been met. The in vitro and in vivo products (the epoxide and secondary
alcohol) are typical of a PSMO-catalyzed reaction. The inhibition by the
PSMO inhibitors, i.e., PB and CO, both in vivo and in vitro, are consistent
with a cytochrome P-450 involvement. Further support for the involvement
of cytochrome P-450 is provided by NADPH and 0 2 dependence for the
enzymatic activity. Likewise, the occurrence of a type I binding spectrum in
both male and female microsomes with the addition of (3-9-tricosene is
consistent with a cytochrome P-450 dependent PSMO, since most lipophilic
substrates and some competitive inhibitors (that are slowly metabolized) bind
at a hydrophobic site in the cytochrome P-450 protein in close enough
proximity to the heme iron to allow both perturbations of the absorbance
spectrum (type I) and interaction with the activated oxygen [7].
Most of the enzyme activity was microsomal rather than mitochondrial.
Cytochrome P-450 PSMO apparently metabolizes the C23 olefin to the corresponding epoxide and ketone, the latter through an intermediate PSMOcatalyzed hydroxylated product, the secondary alcohol, (Z)-9-tricosene-lO-o1.
Reuttinger and Fulco 1161 concluded that in a bacterium, Bucillus meguterium,
a single PSMO was capable of both epoxidizing double bonds and hydroxylating saturated bonds. This also appears to be the case with the housefly in
which both PSMO reactions occur; the epoxidation of the olefin (at carbons
9 and 10) is predominant over hydroxylation (at carbon 10 from the other end
of the molecule). Well-known and commonly used inhibitors of the microsoma1 PSMO, a methylene dioxyphenyl compound, PB, and CO inhibit the in
vivo and in vitro formation of C23 epoxide, secondary alcohol, and the
ketone; formation of the latter is suppressed, presumbaly due to the inhibi-
Pheromone Metabolismby Cytochrome P-450
137
tion of its alcohol precursor. In addition, both PB and CO inhibited the
formation of the organic soluble unknowns and water-soluble metabolites,
thus indicating that these products were also either direct products or due to
further metabolism of a PSMO reaction on (2)-9-tricosene.
Recent studies have established that an epoxide hydrolase (E.C.3.3.2.3.)
and a glutathione-S-transferase (E.C.2.5.1.18) readily convert epoxides to
corresponding diols and glutathione conjugates (while opening the epoxide
ring)/ respectively 112,201. Thus the epoxide formation is a convenient way
to deactivate the main sex-attractant and to mobilize it in a highly polar form
for excretion. In this context, we have tentatively identified a polar metabolite, unknown 1, as a diol. The glutathione conjugate could well be a component of the uncharacterized metabolites in the aqueous phase. Both
enzymes occur ubiquitously in insects. Because the secondary alcohol is quite
polar, it may be excreted as such, or after conjugation (12). As to how the
ketone is deactivated, one possibility is a cytosolic ketone reductase-mediated
conversion to an alcohol [8]. The alcohol could then be disposed of in a
manner described above.
The distinct differences between the PSMO activity ratios of microsomes
prepared from whole abdomens and abdomens without gut tissues suggests
that the housefly gut PSMO is very active in oxidizing (Z)-Ptricosene. This
is not surprising, for insect gut is known to contain high levels of cytochrome
P-450 activity, and other tissues have lower enzyme activities, including the
PSMO in corpora allata, which catalyzes the synthesis of the epoxide juvenile
hormone 17,121. Furthermore, the gut PSMO of the southern armyworm,
Spodoptera evidunia, is capable of epoxidating an olefin to an epoxide, disparlure, which is the sex pheromone of the gypsy moth, Lyrnantvia dispar [ l q .
Nevertheless: 1)there was PSMO activity in housefly tissues peripheral to
the gut; 2) the organltissue assays indicated substantial PSMO activity in the
abdominal tissue; and 3) the cuticle-related tissues are recognized sites for
pheromone synthesis in the housefly [18]. We therefore suggest that a cuticular enzyme rather than the gut enzyme is responsible for the synthesis of
the oxygenated pheromone components from (Z)-9-tricosene.
Each of the housefly tissues investigated, i.e., abdominal epithelium,
thoracic epithelium, fat bodyr legs, and antennae, metabolized (3-9-tricosene to essentially the same metabolites, although the capacity varied from
one organltissue to another. The substantial microsomal PSMO activity in
abdominal tissues peripheral to the gut tissues in all probability represents
the sum of enzyme activities of the cuticle-related abdominal epithelium and
fat body. The dectection of PSMO activity in an insect’s antennae and legs
has important implications in catabolism of the pheromone. Consideration
of the data showing differences among PSMO activities of organsltissues and
between sexes, reveals possible interpretations in addition to the possibility
that separate cytochrome P-450s are responsible. One alternative would
include the same cytochrome P-450, but differing in specific activity and
working in concert with other enzyme to yield different product distributions. This is probably the case and explains: 1)high malelfemale ratios in all
organsltissues and especially the antennae; and 2) higher ketonelepoxide
ratio in the female’s antennae and legs, reflecting a shunt through either a
138
Ahrnad, Kirkland, and Blomquist
more abundant or higher specific activity enzyme that oxidizes the alcohol to
the ketone. In addition, there is evidence for the existence of separate cytochrome P-450s in the abdomen’s peripheral vs. gut microsomal PSMO.
Although much of the (3-9-tricosene synthesis takes place in abdominal
cuticle tissue, it is likely that synthesis also occurs elsewhere. Also, grooming
redistributes (Z)-Ptricosene to other body parts. The presence of large
amounts of pheromone on female housefly legs has been indicated to occur
in this manner [6]. Thus, the metabolism of (Z)-9-tricosene could be useful
for the females to maintain an effective ratio of the pheromone components
for optimizing activity.
We have shown that the oxidative pathway in the synthesis of the oxygenated hydrocarbon pheromone components is dominated by a PSMO, and
that the enzyme is active in both males and females, despite the fact that
females, not males, synthesize the pheromone. The production of the pheromone components in females occurs in synchrony with vitellogenesis and
concommital increase in ecdysteriod titer [5]. The ecdysteriod initiates (23-9tricosene synthesis probably by inducing the synthesis of an enzyme that
transforms a 24-carbon fatty acid moiety to an alkene one carbon shorter.
Thus the oxidative pathway in the synthesis of the epoxide and ketone
pheromone components is not under direct humoral control.
The very active PSMO in males probably serves to deactivate the olefinic
pheromone to prevent attenuation of the pheromone receptors. We suggest
that the same pathway the females use for the synthesis of the oxygenated
pheromone components is fortuitously used by the males for deactivation.
(Z)-9-Tricosene, being highly lipophilic and lacking functional groups, is
ideally suited as a PSMO substrate. All of the NHC products may represent
early steps in the deactivation processes involving other enzymes that act in
concert with the PSMO and convert (23-9-tricoseneto water-soluble products.
Males of several insect species readily metabolize the female ester sexpheromones by esterase(s)present in sensillae of antennae, legs, and located
elsewhere, and several body parts including wings [21]. Tibia of housefly
legs are reported to possess receptors for the sex pheromone [22], and, based
on the metabolic profile of the antennae, it also seems likely that such
receptors are present on this sensilla-rich organ. Thus the deactivation most
likely occurs in both body parts; this suggestion is consistent with the high
metabolic capacity of these organs.
The PSMO system of insects has been shown to play a central role in the
primary degradation of xenobiotics, both man-made and naturally occurring
phytochemicals [12,23]. Recently, the PSMO’s participation in the biogenesis
of physiologically important substances to insects was reviewed [7,8]. Brattsten [17] previously speculated that in danaid butterflies, pyrrolizidine alkaloids are converted to male courtship pheromones by a PSMO-catalyzed
reaction. A PSMO has been postulated to convert an olefin to an epoxide,
disparlure, a sex pheromone of the female gypsy month. To date, however,
neither one has been experimentally validated, except that the formation of
disparlure was shown to occur with the gut PSMO of the southern armyworm larva, which does not normally produce this pheromone. Thus our
finding that a cytochrome P-450 PSMO is indeed involved in the biogenesis
Pheromone Metabolism by Cytochrome P-450
139
of the pheromone components in insects is pioneering. Another novel aspect
is the finding that the PSMO has a function in the deactivation of pheromone
molecules. These findings should prompt similar research in other insects
and would help explain the occurrence of high PSMO activity in nonphytophagous species that are not exposed as much to toxic lipophilic allelochemicals as are the phytophagous ones.
LITERATURE CITED
1. Carlson DA, Mayer MS, Silhacek DL, James JD, Beroza M, Bier1 BA: Sex attractant
pheromone of the housefly: Isolation, identification and synthesis. Science 174, 76 (1971).
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activity by combination of (Z)-9-tricosene with branch saturated hydrocarbons. J Econ
Entomol5, 905 (1976).
4. Adams TS, Holt GG: Effect of pheromone components when applied to different models
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GJ, eds. Academic Press, New York, pp 217-250 (1987).
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279 (1984).
7. Hodgson E: Microsomal mono-oxygenases. In: Comprehensive Insect Physiology, Biochemistry and Pharmacology. Kerkut GA, Gilbert LI, eds. Pergamon Press, Oxford, Vol.
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8. Ahmad S: Enzymatic adaptations of herbivorous insects and mites to phytochemicals. J
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9. Blomquist GJ, Jackson LL: Incorporation of labelled dietary n-alkanes into cuticular lipids
of the grasshopper, Melanoplus sunguinipes. J Insect Physiol 19, 1639 (1973).
10. Bligh EG, Dyer WJ: A rapid method of total lipid extraction and purification. Can J
Biochem Physiol37, 911 (1959).
11. Omura T,Sat0 R: The carbon monoxide-binding pigment of liver microsomes. I. Evidence
for its hemo-protein nature. J Biol Chem 239,2370 (1964).
12. Ahmad S, Brattsten LB, Mullin CA, Yu SJ: Enzymes involved in the metabolism of plant
allelochemicals. In: Molecular Aspects in Insect-Plant Associations. Brattsten LB, Ahmad
S, eds. Plenum Press, New York, pp 73-151 (1986).
13. Dillwith JW, Blomquist GJ, Nelson DR: Biosynthesis of the hydrocarbon components of
the sex pheromone of the housefly, Muscu domesticu L. Insect Biochem 11,247 (1981).
14. Dillwith JW, Nelson JH, Pomonis JG, Nelson DR, Blomquist GJ: A "C NMR study of
methyl-branched hydrocarbon biosynthesis in the housefly. J Biol Chem 25,11305 (1982).
15. Wislocki PG, Miwa GT, Lu AYH: Reactions catalyzed by the cytochrome P-450 system. In:
Enzymatic Basis of Detoxification. Jacoby WB, ed. Academic Press, New York, Vol 1, pp
136-182 (1980).
16. Reuttinger RT, Fulco AJ: Epoxidation of unsaturated fatty acids by a soluble cytochrome
P-450-dependent system from Bacillus meguferium. J Biol Chem 256, 5728 (1981).
17. Brattsten LB: Ecological significance of mixed-function oxidations. Drug Metab Rev 10, 35
(1979).
18. Dillwith JW, Blomquist GJ: Site of sex pheromone production in the housefly, Muscu
domesficu L. Experientia 38, 471, (1982).
19. Dillwith JW, Blomquist GJ: Cuticular lipids. In: Comprehensive Insect Physiology, Biochemistry and Pharmacology. Kerkut GA, Gilbert LI, eds. Pergamon Press, Oxford, Vol.
3, pp 117-154 (1985).
20. Ottea JA, Hammock BD: Optimization of assay conditions for epoxide metabolizing
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Ahmad, Kirkland, and Blomquist
21. Vogt RG: The molecular basis of pheromone reception: Its influence on behavior. In:
Pheromone Biochemistry. Prestwich GD, Blomquist GJ, eds. Academic Press, New York,
pp 385-431, (1987).
22. Schlein Y, Galun R, Ben-Elisher MN: Receptors of sex pheromones and reactions in Musm
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