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Fatty acyl-CoA elongation in Blatella germanica integumental microsomes.

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170
Juárez
Archives of Insect Biochemistry and Physiology 56:170–178 (2004)
Fatty Acyl-CoA Elongation in Blatella germanica
Integumental Microsomes
M. Patricia Juárez*
Insect cuticular hydrocarbons are synthesized de novo in integumental tissue through the concerted action of fatty acid synthases
(FASs), fatty acyl-CoA elongases, a reductase, and a decarboxylase to produce hydrocarbons and CO2. Elongation of fatty acylCoAs to very long chain fatty acids was studied in the integumental microsomes of the German cockroach, Blatella germanica.
Incubation of [1-14C]palmitoyl-CoA, malonyl-CoA, and NADPH resulted in the production of 18-CoA with minor amounts of
C20, C22, C24, C30, and C32 labeled acyl-CoA moieties. Similar experiments with [1-14C]stearoyl-CoA rendered C20-CoA as
the major product, and lesser amounts of C22 and C24-CoAs were also detected. After solubilization of the microsomal FAS,
kinetic parameters were determined radiochemically or by measuring NADPH consumption. The reaction velocity was linear for
up to 3 min incubation time, and with a protein concentration up to 0.025 µg/µl. The effect of the chain length on the
reaction velocity was compared for palmitoyl-CoA, stearoyl-CoA, and eicosanoyl-CoA. The optimal substrate concentration was
10 µM for C16-CoA, between 8 and 12 µM for C18-CoA, and close to 3 µM for C20-CoA. In vivo hydrocarbon biosynthesis
was inhibited from 55.5 to 72.5% in the presence of 1 mM trichloroacetic acid, a known inhibitor of elongation reactions.
Arch. Insect Biochem. Physiol. 56:170–178, 2004. © 2004 Wiley-Liss, Inc.
KEYWORDS: elongases; acyl-CoA; hydrocarbons; German cockroach; sex pheromone
INTRODUCTION
Hydrocarbons are the most abundant cuticular
lipids of the German cockroach, Blattella germanica.
In addition to their critical role in restricting water
loss, certain components serve as precursors to biologically active compounds, including the major
contact sex pheromone, 3,11-dimethyl-2-nonacosanone. This oxygenated derivative is derived
from the major hydrocarbon component, 3,11dimethylnonacosane (Chase et al., 1992). Small
amounts of saturated n-alkanes up to 29 carbons
(<10%) are also present in the epicuticular wax
(Jurenka et al., 1989). Two integumental fatty acid
synthetases (FAS) involved in the synthesis of n- and
methyl-branched fatty acids, precursors to cuticular
hydrocarbons, were first shown in B. germanica
(Juárez et al., 1992). These enzymes have been
studied also in the blood-sucking insect Triatoma
infestans (Juárez et.al, 1996) and both the microsomal and soluble FAS purified to homogeneity in
the housefly (Gu et al., 1993, 1997). When incubation was performed in the presence of acetylCoA and malonyl-CoA, both FAS’s from B. germanica
produced straight chain C16 acid, whereas in the
presence of methylmalonyl-CoA, the major product was a branched C16 acid (Juárez et al., 1992,
1996).
The membrane-associated nature of the acylCoA elongation enzymes has hindered investigation of their biochemistry. Fatty acid elongation
in plants and animals occurs by a four-step mechanism that is similar to fatty acid synthesis, except
that CoA, rather than ACP, is the acyl carrier
(Bernert and Sprecher, 1979; Cassagne et al.,
1994b; Fehling and Mukherjee, 1991; Stumpf and
Pollard, 1983). The first step in the cycle is catalyzed by the β-ketoacyl-CoA synthase (KCS) and
Instituto de Investigaciones Bioquímicas de La Plata, CONICET, Facultad Ciencias Médicas, UNLP, La Plata, Argentina.
*Correspondence to: M. Patricia Juárez, Instituto de Investigaciones Bioquímicas de La Plata, CONICET, Facultad Ciencias Médicas, UNLP, calles 60 y 120, La Plata
1900, Argentina. E-mail: mjuarez@isis.unlp.edu.ar
Received 6 October 2003; Accepted 20 February 2004
© 2004 Wiley-Liss, Inc.
DOI: 10.1002/arch.20007
Published online in Wiley InterScience (www.interscience.wiley.com)
Archives of Insect Biochemistry and Physiology
Fatty Acyl-CoA Elongation in B. germanica
condenses malonyl-CoA with a long-chain acylCoA to yield a β-ketoacyl-CoA. Subsequent reactions
are: reduction to β-hydroxyacyl-CoA, dehydration
to an enoyl-CoA, and a second reduction to yield
the elongated acyl-CoA. In both mammalian and
plant systems where the relative activities of the
four enzymes have been studied, the initial condensation reaction is the rate-limiting step (Suneja
et al., 1991; Cassagne et al., 1994a). In analogy,
insect very long chain fatty acids (VLCFA) were postulated to be synthesized by a microsomal fatty acid
elongation (FAE) system by sequential additions
of C2 moieties to C16 fatty acids derived from the
de novo fatty acid synthesis (FAS) (Blomquist et al.,
1989). Evidence of elongation reactions in insects
was first obtained after incubation of microsomal
preparations from the housefly with [14C]stearoylCoA (Vaz et al., 1989) or incubation of epidermalenriched tissues of T. infestans with [14C] stearic acid
(Juárez and Brenner, 1989). In both systems, the
major product was a C20 fatty acid moiety, together
with elongated chains up to 30 carbons. Integumental oenocytes were recently shown to be the
cells responsible for hydrocarbon synthesis in the
german cockroach (Fan et al., 2003). As a preliminary step in the study of the complex reactions
leading to the formation of hydrocarbons and the
contact sex pheromone in the german cockroach,
we studied the elongation reactions occurring in
the integumental microsomal fraction.
MATERIALS AND METHODS
Insects
German cockroaches were reared in glass jars
and fed dog chow and water ad libitum. They were
kept at 27°C with a 12:12 light:dark cycle.
Labeled Substrates
[2-14C]Malonyl-CoA (51 mCi/mmol), [1-14C]
propionate (55 mCi/mmol), [1-14C] palmitic acid
(60 mCi/mmol), and [14C]stearic acid (56.7 mCi/
mmol) were purchased from New England Nuclear,
DuPont (Boston, MA). [1-14C] Palmitoyl-CoA (50
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171
mCi/mmol) and [14C]stearoyl-CoA (50 mCi/mmol)
were prepared from [14C]palmitic acid and [14C]stearic
acid, respectively, and coenzyme A according to
Bishop and Hajra (1980).
Chemicals
Unlabeled coenzyme A derivatives, dithioerythritol (DTE), ethylendiamine tetraacetic acid
(EDTA), potassium phosphate, magnesium chloride, ascorbic acid, fatty acid standards, and silica
gel type G were purchased from Sigma Chemical
Company, St. Louis, MO.
Tissue Preparation
Cockroaches were chilled at 4°C and the abdomens were isolated. A lateral cut along one side of
the abdomen freed the integument from the internal abdominal organs and integument tissue was
placed in a saline solution BG-SSA containing 10.3
g NaCl, 1.46 g KCl, 0.36 g NaHCO 3, 0.21 g
NaH2PO4.H2O, 1.34 g Na2HPO4, and 3 g glucose
per liter, pH 7.4 (Kurtii and Brooks, 1976). All the
procedure and separation steps were as previously
described (Juárez et al., 1992).
Preparation of Subcellular Fractions
The integuments were homogenized in a glass
homogenizer in buffered phosphate (50 mM, pH
7.4) containing 1 mM DTE and 1 mM EDTA. The
homogenate was filtered through 3 layers of cheesecloth. After sequential centrifugation at 500g for 5
min and 1,200g for 10 min, the sediment was discarded. The supernatant was centrifuged at 14,000g
for 20 min, the pellet removed and the supernatant further centrifuged at 105,000g for 60 min.
The microsomal pellet was washed by resuspension
in the buffer and recentrifuged. Protein concentrations were measured as described by Bradford (1976).
For some experiments, partially purified microsomal
preparations were obtained after solubilization of the
microsomal FAS with 1M KCl, in buffer phosphate
0.01M, as described in Gu et al. (1997).
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Juárez
Elongation Assays
For elongation of endogenous primers, the reaction mixture contained 1 mM DTE, 1 mM EDTA,
1 mM MgCl2, 2 mM ascorbic acid, ATP (6.6 mM),
CoA (0.2 mM), malonyl-CoA (to 30 mM), and [214
C]malonyl-CoA (3.3 mM) plus 50 mg of microsomal protein in a total volume of 500 µl. For assays
employing exogenous primers, the elongation activity was determined in a similar reaction mixture
with DTE, EDTA, MgCl2, and ascorbic acid, in the
same concentrations as described above, plus NADPH
(200 µM), malonyl-CoA and acyl-CoA’s (C16, C18,
or C20-CoA) in the presence of albumin (acyl-CoA:
albumin, 2:1), incubated with varying amounts of
microsomal protein. NADPH consumption was
measured at 340 nm in 1-cm light pass quartz
cuvets. A blank was run for other microsomal enzymes consuming NADPH (including microsomal
FAS) by measuring NADPH consumption prior to
the addition of acyl-CoA, and then it was subtracted
from the value obtained after adding the appropriate acyl-CoA. In some experiments, partially purified microsomal preparations that were mostly free
of FAS activity were used. Alternatively, when malonyl-CoA incorporation was monitored following
the incorporation of [14C]malonyl-CoA, the reaction was stopped after 10 min by the addition of
methanolic KOH (0.5 N), saponified at 80°C for
30 min, followed by acidification with HCl (0.5 N).
Lipids were extracted three times with hexane. Radioactivity was counted and the results expressed
as picomoles of [14C]malonyl-CoA incorporated.
mance-liquid chromatography (radio-HPLC). A C8
reverse-phase column (particle size 3 mm, 15 cm
× 4.6 mm) coupled to a Spectra Physis SP8700 solvent delivery system was used, set at a flow rate of
1 ml/min, with the mobile phase (acetonitrile:water,
80:20). Radioactivity was detected with a Radiomatic Instruments Flo-one/Beta radioactive flow
detector. Absorbance at 215 nm was used to detect
the FAME. A Beckman LS 1701 was employed to
determine radioactivity by liquid scintillation
counting in 0.4% diphenyloxazol in toluene, at
90% efficiency. Radioactivity was assayed and the
results expressed as picomoles of [14C]malonyl-CoA
or [14C]stearoyl-CoA incorporated into fatty acids.
Inhibition of Elongation Reactions:
The in vivo inhibition of the formation of methyl branched chains was analyzed following the
incorporation of labeled propionate into female
cuticular lipids after injection of 2 µl of 1 mM sodium trichloroacetate (NaTCA) containing 1 µCi
of [1-14C]propionate and compared to control insects where the label was administered in the same
volume of saline. Alternatively, 2 µl of NaTCA were
topically applied 2 h prior to injection of label in
saline. Cuticular lipids were extracted with hexane
after 12 h, radioactivity was determined by liquid
scintillation in an aliquot, and distribution of the
radioactivity into lipids was determined by radioTLC after separation in hexane:ethyl ether (90:10).
RESULTS
Elongation Studies
ELONGATION PRODUCTS ANALYSIS
[1-14C]Stearoyl-CoA (56 mCi/mmol) or [114
C]palmitoyl-CoA (50 mCi/mmol) were used as
the radioactive primers. After appropriate incubation times, the reactions were stopped by adding
10% NaOH in methanol. Samples were saponified
(80°C, 30 min), acidified (1N HCl), and fatty acids esterified with diazomethane. Fatty acid methyl esters (FAMEs) were purified by radio-TLC on
silicagel plates developed in hexane:ether:acetic
acid (80:20:2) and analyzed by radio-high perfor-
Elongation of endogenous primers was measured
by the incorporation of 14C from [2-14C]malonylCoA into elongated fatty acid products. Elongation
products were detected together with microsomal
FAS products in crude microsomal preparations. A
labeled peak at 16 carbons plus a minor signal eluting at 14 carbons corresponded to microsomal FAS
activity and a major labeled peak at 18 carbon
chain length together with minor peaks co-eluting
with C20 and C22 fatty acids were produced by
elongating activity (Fig. 1A). Figure 1B shows the
Archives of Insect Biochemistry and Physiology
Fatty Acyl-CoA Elongation in B. germanica
Fig. 1. Radio-HPLC of fatty acid methyl esters (FAME) obtained by methylation of fatty acids derived after saponification of the incubation mixture containing B. germanica
integumental microsomes, [2-14C]malonyl-CoA, ATP, and
NADPH, in the presence (A) or absence (C) of 30 µM
methylmalonyl-CoA (endogenous primers). A mass trace (B)
indicates elution profile for 14C, 16C, 18C, 20C, and 22C.
elution pattern of even chain fatty acid standards
of 14 to 22 carbons. When methylmalonyl-CoA
(30 mM) was added to the incubation mixture, the
only product detected was a labeled peak eluting
between the 16 and 18 straight chain carbons (Fig.
1C), probably corresponding to a methyl-branched
16 carbon fatty acid produced by microsomal FAS
activity (Juárez et al., 1992). The incorporation of
radiolabeled exogenous primers was analyzed using [1-14C]palmitoyl-CoA and a major conversion
to a C18 acyl moietiy was detected; smaller but measurable amounts of labeled peaks co-eluting with
22, 24, 30, and 32 carbon fatty acid standards were
also produced, indicating that additional elongation
rounds were also occurring (Fig. 2A). When [114
C]stearoyl-CoA was used as primer, the major
product was detected as eicosanoic acid and minor
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Fig. 2. Radio-HPLC trace of the acyl-CoA elongase reaction products, FAME obtained as in Figure 1, after incubation of 10 µM acyl-CoA containing 90,000 cpm of [14C]
palmitoyl-CoA (A), or [14C] stearoyl-CoA (B) with 20 µM
malonyl-CoA, 0.2 mM NADPH, and 0.5 mg/ml of microsomal protein, as described in Materials and Methods.
amounts of C22 and C24 fatty acids (Fig. 2B). Elongation activity in partially purified microsomal
preparations, after solubilization of the microsomal
FAS as described in Materials and Methods, was
measured by spectrophotometry following NADPH
consumption at 340 nm with stearoyl-CoA used as
the elongating substrate (Fig. 3). The reaction velocity was linear with protein concentration up to
approximately 0.05 mg/ml and up to 3 min of reaction. The rate of acyl-CoA elongation increased
hyperbolically with an increasing concentration of
malonyl-CoA up to 20 µM (Fig. 3). The effect of
the chain length on the reaction velocity was compared for palmitoyl-CoA, stearoyl-CoA, and icosanoyl-CoA; the reaction reached to a maximum and
declined steadily for all substrates. The value of the
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Juárez
A
B
Fig. 4. Effect of acyl-CoA concentration and chain length
on NADPH consumption of partially purified integumental
microsome preparations from the german cockroach. Reaction conditions as described in Materials and Methods.
maximum depended on the chain length of the acylCoA. For C16-CoA, it was obtained at a substrate
concentration close to 10 µM, for C18-CoA it was
8–12 µM, and for C20-CoA it was close to 3 µM
(Fig. 4).
In Vivo Inhibition of [1-14C] Propionate Incorporation
C
Fig. 3. Effect of protein (A), time (B), and malonyl-CoA
concentration (C) on NADPH consumption for the elongation of stearoyl-CoA by partially purified microsomal
preparations. Incubation conditions as described in Materials and Methods.
The effect of 1 mM sodium trichloroacetate
(TCA), a known inhibitor of elongation reactions
(Juárez, 1994a,b), on [1-14C] propionate incorporation into cuticular lipids was analyzed 12 h after
injection of the labeled substrate. Radioactivity incorporated by control insects (1%) was detected
mostly in the cuticular hydrocarbons (data not
shown). When TCA was simultaneously injected
with the labeled precursor, the percentage of incorporation was inhibited to 72.5% of that in the
control insects, whereas for TCA-pretreated insects
(topical application 2 h prior to labeled precursor
injection), the incorporation percentage was reduced to 55.5% of that in the controls (Table 1).
DISCUSSION
Elongation reactions in subcellular fractions of
insects have been studied to date only in the houseArchives of Insect Biochemistry and Physiology
Fatty Acyl-CoA Elongation in B. germanica
TABLE 1. Effect of 1 mM Sodium Trichloroacetate (TCA) on [1-14C]
Propionate Incorporation Into Cuticular Lipids
Treatmenta
Control
TCA-injected
TCA-topically pretreated
% Incorporationb
100.0
72.5
55.5
a
B. germanica females were injected with 50,000 cpm/insect of [1-14C] propionate
for each condition; the vehicle was 2 µl saline (Control), 2 µl saline containing 1
mM TCA (TCA-injected), or 1 mM TCA was topically applied 2 h prior to label
injection.
b
Insects were killed 12 h after injection, lipids extracted and radioactivity determined by liquid scintillation, as described in Materials and Methods. Numbers
represent the mean value of duplicates of 5 insects each.
fly and in the American cockroach. Whole insect
microsomal preparations incubated with [14C]malonyl-CoA and NADPH resulted in the production
of 20–30 carbon acyl moieties (Vaz et al., 1989).
Specificity related to degree of unsaturation was
demonstrated in the American cockroach (Vaz et
al., 1988), but it is not yet known whether different elongases are involved. Tissue specificity has
also been proposed, with higher amounts of verylong-chain length products found in epidermalenriched compared to fat body preparations.
Significant amounts of VLCFA are known to be
present in a variety of plants or animal tissues
(Cassagne et al., 1994b; Kolattukudy, 1980) although the lack of detectable amounts of VLCFA in
most insects was proposed to be accounted for a
tight coupling between insect elongating and decarboxylating activities involved in hydrocarbon production (Blomquist et al., 1989). However, after
dissection of integumental tissues of the kissing bug
T. infestans, significant amounts of VLCFA were detected, with hexacosanoic acid as the major component (Juárez and Brenner, 1989). Strong evidence
is available now for the microsomal fraction of the
integument, probably from the oenocyte cells, as
the most responsible for all the steps involved in
hydrocarbon synthesis. Both in B. germanica and T.
infestans, hydrocarbons are synthesized in the oenocyte-containing integument (Juárez and Brenner,
1989; Fan et al., 2003). In both systems, a microsomal integumental FAS was shown to be the most
responsible for methyl-branched fatty acid synthesis (Juárez et al., 1992, 1996). The final conversion of very long chain fatty acyl-CoA into
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175
hydrocarbon was shown to occur in the integumental microsomal fraction of the housefly (Reed et
al., 1996).
The multifunctional enzyme FAS uses malonylCoA, acetyl-CoA, and NADPH to synthesize fatty
acids in 2-carbon increments. The principal end
product of FAS in most systems is palmitic acid
(16:0); a high proportion of this palmitic acid is
then converted to stearic acid (18:0) through elongation systems that have been mostly localized to
the endoplasmic reticulum. Two different elongating activities exist that use malonyl-CoA as the
elongating agent: the ATP-dependent (type I) that
uses endogenous primers and another elongating
system (type II) capable of elongating exogenous
acyl-CoA primers without using ATP (Domergue
et al., 1999). In Blatella, both systems are active;
C18:0 is the major product both for ATP-dependent elongation as well as for exogenous [14C]C16CoA elongation (Figs.1A, 2A). In B. germanica,
crude microsomal FAS preparations incorporated
[14C]malonyl-CoA into palmitic and stearic acid in
a 4:1 ratio (Juárez et al., 1992). The present assays
for endogenous primer elongation showed label
in C18:0 > C16:0, with very little amounts in C20:0
and C22:0. While C16:0 is certainly formed by the
microsomal FAS, the precise role of each enzyme
system for the in vivo elongation of C16:0 to C18:0
will require further analysis. In the presence of 30
µM methylmalonyl-CoA under similar incubation
conditions, [14C]malonyl-CoA was incorporated in
a single peak eluting between C16:0 and C18:0 standard (Fig. 1B), very probably a methyl-branched
fatty acid of 16 carbon skeleton, due to FAS activity (Juárez et al., 1992). Lack of elongated products might be due to competitive inhibition of
methylmalonyl-CoA on malonyl-CoA incorporation (Gu et al., 1993). Incubation of exogenous
primers showed [14C]palmitoyl-CoA elongation in
a variety of acyl chains up to C32, in addition to
the main product of 18 carbons (Fig. 2A).The elongation of [14C]stearoyl-CoA gave mostly a C20 fatty
acyl moiety together with small amounts of C22
and C24 fatty acids (Fig. 2B). Most of the products were detected as free fatty acids (90%) whereas
radioactivity in acyl-CoAs was less than 10% (data
176
Juárez
not shown). C18:0 and C20:0 are minor components in the B. germanica fatty acid pool. However,
a strong competition for the acyl-CoA derivatives
by the acyltransferases and acyl-CoA hydrolases
very probably prevented further C2-elongations to
proceed to significant extents in vitro. At low substrate concentrations (<5 µM), more close to that
of physiological conditions and well below the estimated critical micellar concentration (cmc) for
palmitoyl-CoA (60–70 µM) and stearoyl-CoA (12
µM) (Constantinides and Stein, 1985), specific activity was higher for eicosanoyl-CoA (no published data available). At increasing acyl-CoA
concentration, substrate inhibition was evident for
the longer acyl-CoA. Maximal specific activity was
higher for shorter chains (C16 ~ C18 > C20),
whereas the optimal substrate concentration diminished significantly at increasing chain length
above 18 carbons, from 10 µM for C16-CoA and
C18-CoA to 3 µM for C20-CoA. The long chain
acyl-CoAs cmc ranges from 5 to 200 µmol/L depending on chain length, number of double bonds
in the acyl chain, and salt concentration (Færgeman
and Knudsen, 1997). The actual free concentration
is, therefore, unknown if the critical micellar concentration has not been determined under the experimental conditions used. Enzyme activity is
markedly diminished at growing acyl-CoA concentrations. However, the effect is more evident for
C20 and C16-CoA, whereas C18-CoA retained
about 40% activity even at >30 µM. Selective partitioning of acyl-CoA esters into membranes, specific or non-specific binding to proteins, could also
result in very different final concentrations. Further experiments should be carried out in order to
determine substrate specificity and metabolites produced using methyl-branched fatty acyl-CoAs, the
putative precursors to 3,11-dimethylnonacosane
and the contact sex pheromone 3,11-dimethyl-2nonacosanone, and to determine whether one or
more elongases are involved in this reaction.
VLCFA-CoA conversion to hydrocarbon (Reed et
al., 1996) and further conversion into the epoxide
and ketone sex recognition factors by another integumental enzyme, a cytochrome P-450 monooxygenase, were shown to be involved in contact
sex pheromone metabolism in the housefly (Ahmad
et al., 1987). Chase et al. (1992) showed that the
contact sex pheromone of the german cockroach
is formed by hydroxylation and oxidation of the
major alkane component at the 2-position; the
conversion of 3,11-dimethylnonacosane to 3,11dimethyl-2-nonacosanone is age and sex specific.
Recent evidence indicated that the integument is
the site of synthesis of the B. germanica contact sex
pheromone and that a sex-specific cytochrome P450 enzyme system is involved (Juárez and Schal,
unpublished data).
Chloroacetamides, oxyacetamides, oxyhalogenoacids, and thiocarbamates are known to inhibit
the synthesis of long-chain fatty acids. However, it
was not shown whether this takes place by direct
inhibition of, or interaction with, fatty acid elongases (Abulnaja and Harwood, 1991). Topical application of halofatty acids were shown to inhibit
pheromone production in lepidopteran species
presumably by inhibition of acylreductases, in
agreement with bioassays indicating diminished
sexual attractiveness of treated females (Hernanz
et al., 1997). Inhibition of cuticular lipid synthesis and its effect on insect survival and egg hatchability was shown in T. infestans after Na TCA
treatment (Juárez, 1994a,b). In order to evaluate
the potential disruption of methyl-branched hydrocarbon and pheromone production, we tested the
effect of NaTCA on [14C]propionate metabolism.
Topical application showed a larger inhibitory effect than injection (Table 1), suggesting that a direct contact with the integument, the site of
elongation reactions, prevents dilution or detoxification of the xenobiotic. Further work is needed
to examine whether KCS, the rate limiting elongating enzyme, is the actual target for inhibition
and to characterize integumental enzymes in order to provide an understanding of the regulation
and production of the contact sex pheromone.
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Archives of Insect Biochemistry and Physiology
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coa, blatella, germanica, elongation, fatty, microsomal, acyl, integumental
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