ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 293, No. 2, March, pp. 333-341, 1992

A Microsomal Fatty Acid Synthetase from the Integument of Blattella Germanica Synthesizes Methyl- Branched Fatty Acids, Precursors to Hydrocarbon and Contact Sex Pheromone’

Patricia JuArez,2 Jody Chase, and Gary J. Blomquist3 Department of Biochemistry, University of Nevada, Reno, Nevada 89557-0014

Received September 16, 1991, and in revised form October 25, 1991

Methyl-branched fatty acids present in the integument of the German cockroach, Blattella germanica, were identified by gas chromatography-mass spectrometry of their methyl esters and reduction products (alkanes) as n-3-, n-4-, n-S-, n-7-, n-8-, and n-9-monomethyl fatty acids and as n-5,9-, n-3,9-, and n-3,11-dimethyl fatty acids with 16 to 20 total carbons. These fatty acids have the same branching patterns as do the major hydrocar- bons of this insect, including 3,11-dimethylnonacosane, the precursor to the major contact sex pheromone, and are presumed to be intermediates in hydrocarbon for- mation. A novel microsomal fatty acid synthetase (FAS) located in the integument of this insect incorporated [methyZ-‘4C]methylmalonyl-CoA into methyl-branched fatty acids as demonstrated by radio-high-performance liquid chromatography. A cytosolic FAS is also present in the integument. Both the microsomal and the soluble FAS incorporated [methyZ-14C]methylmalonyl-CoA into fatty acids, but only the microsomal FAS was able to ef- ficiently use methylmalonyl-CoA as the sole elongating agent. This is the first report of the characterization of methyl-branched fatty acids from the integument of an insect and of an integumental microsomal FAS that in- corporates methylmalonyl-Cob into branched fatty acids. 0 1992 Academic Press, Inc.

i This work was supported in part by the USDA-CSRS under Grant 91-37302-6192. We thank Dr. Murray Hackett for obtaining the mass spectra reported here and Dr. Charlotte Borgeson for help with the marker enzymes. A contribution of the Nevada Agricultural Experiment Station.

* Present address: Instituto de Investigaciones Bioquimicas de La Plats (INIBIOLP) Fac.Cs.Medicas, UNLP, calle 60 y 120-1900 La Plata, Ar- gentina

a To whom correspondence should be addressed.

0003-9861/92 $3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

Methyl-branched lipids predominate in insect waxes. Internally branched mono-, di-, and trimethylalkanes comprise the majority of the cuticular lipids of most in- sects (l-3). Mono-, di-, and trimethyl- fatty alcohols are major lipids produced by pupae in several lepidopteran species (4-6). In contrast, the waxes of plants and other organisms generally have much lower amounts of methyl- branched components (7).

Methyl-branched hydrocarbons constitute most of the cuticular lipid of the German cockroach, Blattella germanica (8, 9). The major components are 3,7-, 3,9-, and 3,11-dimethylnonacosane and a mixture of mono- methylnonacosanes. The major component of the fe- male contact sex pheromone is 3,11-dimethylnona- cosan-2-one (lo), which is derived from the parent hydrocarbon, 3,11-dimethylnonacosane (11; Chase, Touhara, Prestwich, Schal and Blomquist, unpublished data).

Hydrocarbons arise from the elongation of fatty acids to very-long-chain fatty acyl-CoAs (12-14) which are then reduced to aldehydes and converted to hydrocar- bons one carbon shorter (15, 16). Carbon-13 NMR studies have demonstrated that for those methyl- branched alkanes in insects which have the methyl branch near the end of the chain, the methyl-branching group is inserted early in chain synthesis (17-19). Thus, it was possible that methyl-branched fatty acids with the same methyl-branching pattern as the major al- kanes would be present in the integument tissue of the German cockroach, although such methyl-branched fatty acids have not been previously reported in insects (20). The data presented herein demonstrate the oc- currence of these methyl-branched fatty acids in the German cockroach.

333

334 JUAREZ, CHASE, AND BLOMQUIST

Many animal fatty acid synthetases (FAS)4 can use methyl-branched primers efficiently (21) and also can use methylmalonyl-CoA as the sole elongating agent (22) generating the same fatty acids as those synthesized by the uropygial gland of goose (23). When methylmalonyl- CoA replaces malonyl-CoA as the elongating group, the products synthesized are medium chain multimethyl- branched fatty acids (23,24). A mixture of malonyl-CoA and methylmalonyl-CoA generate longer aliphatic chains containing methyl branches.

The FAS from insects has not been extensively studied, and most of the work has involved FAS from whole in- sects, which mostly represents fat body FAS (20). de Re- nobales et al. (25) showed differences in the soluble FAS from whole Drosophilu melunogaster when compared to that of mammals. However, in the presence of both mal- onyl-CoA and methylmalonyl-CoA, Drosophila FAS syn- thesized what appeared to be methyl-branched fatty acids by radio-GLC analysis, but they were not further char- acterized.

During development in the cabbage looper, Trichoplusiu ni, soluble FAS activity correlated closely with the rates of incorporation of labeled acetate and propionate into methyl-branched lipids during the larval stages, with high rates of synthesis and FAS activity observed during the feeding stages and low rates during the nonfeeding stages (4). In contrast, at specific points during the pupal stages, labeled acetate and propionate were efficiently incorpo- rated into methyl-branched lipid at times when soluble FAS activity was very low or undetectable. This suggested the possibility that a microsomal FAS was involved in forming methyl-branched fatty acid precursors to hydro- carbon in insects. This possibility was explored using in- tegument tissues of the German cockroach and the data presented herein provide support for this hypothesis.

MATERIALS AND METHODS

Insects. German cockroaches were reared in glass jars and fed dog chow and water ad lib&urn. They were kept at 27’C with a 12:12 light: dark cycle. Females were separated on the day of adult emergence (Day 0) and collected up to Day 8.

Labeled substrates. [methyl-“ClMethylmalonyl-CoA (53.5 mCi/ mmol), [2-‘4C]malonyl-CoA (50.3 mCi/mmol) and [l-‘4C]propionate (55 mCi/mmol) were purchased from New England Nuclear-DuPont, (Bos- ton, MA).

Chemicak. Unlabeled coenzyme A derivatives, reduced NADPH and NADH, dithioerythritol (DTE), EDTA, potassium phosphate, fatty acid standards, and silica gel type G were purchased from Sigma Chemical Co. (St. Louis, MO). Concentrated dye protein reagent and silicic acid (BioSil A) were obtained from Bio-Rad (Richmond, CA).

Tissue preparation. Integument tissue was obtained as follows. Cockroaches were chilled to 4’C and abdomens were excised from the

4 Abbreviations used: FAS, fatty acid synthetase; Me$O, dimethyl sulfoxide; DTE, dithioerythritol; FAME, fatty acid methyl ester; ECL, equivalent chain length; VLCFA, very long chain fatty acid; br, branched

thorax. A transverse cut near the end of the last somite pair and a lateral cut along one abdomen side freed the integument and internal abdominal organs. These were gently separated from the integument (for some experiments the fat body was also collected) with a razor blade and the integument tissue was immediately immersed in cold buffer. Before ho- mogenization, tissue was gently washed with fresh buffer. For the iso- lation of fatty acids for GC-MS analysis, the abdominal cuticle tissue was excised from approximately 120 adult virgin females which were l- 8 days postemergence.

Characterization of methyl-branched fatty acids. Lipid was extracted from the integument tissue by the procedure of Bligh and Dyer (26), saponified, and methylated as described by Jurenka et al. (27). The fatty acid methyl esters (FAME) were isolated from other lipids by TLC in hexane:diethyl ether (80:20). The unsaturated fatty acid methyl esters were partially removed by TLC on 10% AgNOB-silica gel plates developed in hexanediethyl ether (80:20). The saturated fatty acid methyl esters (ca. 9.5 mg), which contained both branched- and straight-chain com- ponents, were reduced to the corresponding alcohols by treatment with LiAlH,. The reaction was monitored by TLC and appeared to be quan- titative. The resulting alcohols were converted to the corresponding bromides according to the method described by Bjostad and Roelofs (28). The reaction was monitored by TLC. The NaBH, was placed in a 25-ml two-necked conical flask and dissolved in 4-5 ml of dimethyl sulfoxide (Me,SO). The alkyl bromide was added slowly in Me*SO to the reaction vessel, and heated to 85°C for 1.5 h in a N2 atmosphere. Upon cooling to room temperature, the solution was transferred to a separatory funnel, and washed with 3% HCl. The reaction mixture was partitioned three times between hexane and water. The combined hexane fractions were washed once with brine, and the hexane was removed under a stream of Nz. The hydrocarbon fraction was isolated on a BioSil- A column (6 cm X 0.5 cm) in hexane, and the methyl-branched com- ponents were isolated by inclusion of the straight chain components in 5A molecular sieve in isooctane.

GC analyses were performed on a Hewlett-Packard 5890A gas-liquid chromatograph with a 30 m X 0.32 mm DB-5 capillary column temper- ature programmed from 130 to 3OO’C at a rate of 3”C/min. GC-MS analyses were performed on a Finnigan 4023 mass spectrometer inter- faced with an INCOS data system. The GC-MS system operated at 70 eV (GC condition: 30 m X 0.32 mm DB-5 column temperature pro- grammed from 100 to 250°C at 4”C/min). The carrier gas was helium at 8 psi head pressure, linear velocity about 30 cm/s.

Tissue slice incubations. Integuments obtained as described above were incubated in B. germunica saline solution A, usually seven integ- uments in 500 pl/vial, with 0.5 pCi [1-‘“Clpropionate. After a l-h in- cubation at 30°C in a shaking water bath, integuments were washed in the same incubation buffer and solvent extracted and converted to methyl esters as described below. Buffer solution was also extracted to recover fatty acids from the incubation medium.

Preparation of subcellular fractions. The integuments were homog- enized in the saline solution isoosmotic with B. germanica hemolymph, which contained 10.3 g of NaCl, 1.46 g of KCl, 0.36 g of NaHC03, 0.21 g of NaHPO, . HzO, 1.34 g of Na,HPO,, and 3 g of glucose per liter, pH 7.4 (29). The homogenate was filtered through three layers of cheesecloth. Organelles and cell debris were removed by centrifugation at 500g for 5 min and at 1200g for 10 min. The supernatant was centrifuged at 14,000g for 20 min and the resulting mitochondrial pellet was washed once and recentrifuged. The supernatant was centrifuged again at 105,OOOg for 60 min and the microsomal pellet was washed by resus- pension in the homogenizing buffer and recentrifuged. When this step was not performed, centrifugation was extended for 90 min. The purity of this fraction was analyzed with the enzyme markers Na+/K+-ATPase for endoplasmic reticulum (30, 31) and alcohol dehydrogenase (ADH) for cytoplasm (32). Protein concentrations were determined by the pro- cedure described by Bradford (33).

FAS assays. Unless otherwise specified, the reaction mixture con- tained 1 mM DTE, 1 mM EDTA, 200 pM NADPH, 30 pM acetyl-CoA,

METHYL-BRANCHED FATTY ACIDS IN COCKROACHES 335

and 60 pM malonyl-CoA plus methylmalonyl-CoA. [methyl- i4C]Methylmalonyl-CoA or [2-i4C]malonyl-CoA was included in the mixture ranging from 1.6 to 3.3 pM along with 10 or 50 pg of microsomal or soluble protein in a total volume of 250 or 500 pl of the homogenization buffer.

Product analysis. After appropriate incubation times, reactions were stopped by addition of 5% NaOH in methanol. Samples were saponified at 60°C for 30 min. Unsaponifiable material was extracted three times with hexane and the saponified fatty acids were extracted from the aqueous phase with hexane, after acidification with 1 N HCl. Esterifi- cation of fatty acids was carried out with 14% BF, in methanol. Methyl esters were purified by TLC on silica gel G plates, developed in hexane: ethyl ether:formic acid (30~20~2) and analyzed by radio-high-performance liquid chromatography (HPLC). A C-8 reverse-phase column (particle size 3 pm, 15 cm X 4.6 mm) coupled to a Spectra Physics SP 8700 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. FAME were detected by an ultraviolet spectrometer at 215 nm. Alter- natively, radioactive samples were analyzed by radio-GLC using a Hew- lett-Packard 5710A gas-liquid chromatograph with a thermal conduc- tivity detector interfaced with a Radiomatic Flo-One/Beta combustion flow-through proportional counter. The column was packed with 3% Dexsil400 on Supelcoport, temperature programmed from 140 to 23O“C at 2”C/min and held at the final temperature for 10 min. Radioactivity was also assayed by liquid scintillation counting on a Beckman LS 1701 with 3 ml diphenyloxazol(O.4%) in toluene, at about 90% efficiency.

RESULTS

The fatty acid methyl esters from the integument of the female German cockroach were analyzed by capillary gas-liquid chromatography and a representative chro- matogram is presented in Fig. 1A. In addition to the ex- pected major components (18:1, 16:0, l&O, 16:1, l&2, 14: 0, and 17:0), a number of minor peaks representing ap- proximately 7% of the total FAME were also present. GC-MS analysis of the total FAME gave data which in- dicated that some of the unidentified peaks were methyl- branched fatty acids, but did not yield clearly interpretable data for each component. To facilitate GC-MS analysis of the methyl-branched components, unsaturated material was removed by AgNO,-TLC, the FAME were reduced to the corresponding hydrocarbons, and the straight-chain components were removed by inclusion in 5 A molecular sieve. The branched alkanes obtained (Fig. lB), which correspond to the original branched fatty acids were an- alyzed by capillary GC-MS.

Representative mass spectra from the GC-MS analysis of the alkanes obtained from the integument FAME are presented in Fig. 2. Peak number 3 (Fig. 1B) was identified as 5methylhexadecane based on its equivalent chain length (ECL) of 16.5 and its mass spectrum. Fragmen- tation on either side of the methyl-branching group at position 5 with retention of the charge on the fragment containing the methyl-branch group gave fragments with m/z at 84/85 and 182/183. The M+ was at m/z 240. Peak 5 (Fig. 1B) was identified as 3-methylhexadecane based on its ECL of 16.7 and the strong M-29 (M+ = 240) and somewhat weaker M-57 fragment ions (Fig. 2B). GC peaks

7 and 8 were identified as n-5,9- and n-3,11dimethyl- hexadecane based on their ECL of about one carbon less than a straight chain alkane of the same number of car- bons and the fragmentation patterns (Figs. 3A and 3B) with cleavage on either side of the branching methyl groups. Interpretation of the spectra was as described in Jurenka et al. (9) for the longer chain homologs present as major cuticular hydrocarbon components. Examination of the mass spectra from the FAME, while not yielding an unambiguous structure for each component, supported the assignments based on the spectra of the alkanes de- rived from them and allowed the assignment of the methyl branches on the methyl end of some of the FAME. The mass spectra from the other alkanes derived from fatty acids were interpreted in a similar manner and the results are summarized in Table I.

GC traces of the fatty acids from the microsomal and soluble fractions from B. germanica integument showed a major difference from those of whole tissue extracts in that a very small amount of unsaturated fatty acids was present in the microsomal and soluble fractions. In ad- dition, methyl-branched fatty acids, while still comprising a low percentage of the total fatty acids, are present in much higher amounts in the microsomal fraction than in the soluble fraction (data not shown). Also, the micro- somal fraction contains significantly higher amounts of very-long-chain fatty acids (VLCFA), mainly n-C20 and n-C22, than the soluble fraction. The branched-chain fatty acids and the VLCFA are the expected intermediates in the synthesis of cuticular alkanes, and are usually present in very small amounts or are undetectable in fatty analyses from whole insects or most other organs (20).

Initial studies were performed to determine the tissue and subcellular location of the enzyme (enzymes) involved in the synthesis of the methyl-branched fatty acids. After incubation of integument tissue slices with [l- “C]propionate for 1 h, lipid was isolated and the fatty acids were converted to their methyl ester derivatives and analyzed by radio-HPLC. The retention times of the la- beled fatty acids were compared to that obtained from the mass trace. [1-14C]Propionate specifically labeled the fatty acids which eluted in the positions corresponding to methyl-branched fatty acids. Two main radioactive peaks were observed which eluted after n-Cl6 and n-C18, at positions corresponding to brC16 and brC18 with minor amounts of shorter and longer chain fatty acids also ob- served (Fig. 4). The radio-HPLC does not resolve each branched fatty acid as does capillary GC (Fig. 1). Thus, it is not known which branched fatty acids are formed from propionate by integument tissue, but the data clearly show that they are methyl-branched. The assignment of a branching pattern is based on its elution times being intermediate between those of normal chains. The other possible components eluting in that position, unsaturated FAME, were ruled out by the fact that the elution of the

336 JUAREZ, CHASE, AND BLOMQUIST

-

FIG. 1. Capillary gas-chromatqraphic trace of integumental fatty acid methyl esters (A) and the methyl-branched hydrocarbons (B) derived from them, obtained from B. germanica females. Extraction, reduction, and chromatographic conditions are described under Materials and Methods.

samples through AgN03-impregnated BioSil-A columns (which removes unsaturated FAME) did not affect the radioactive trace.

To determine tissue and subcellular location of the en- zymes which synthesize methyl-branched fatty acids, sol- uble and microsomal FAS activity was assayed in both integument and fat body tissue. In order to make accurate comparisons between the crude soluble and the micro- somal FAS, radiochemical assays were routinely used for all further studies reported here, although preliminary experiments showed that a spectrophotometric assay worked well with the soluble FAS. Using assay conditions of 30 PM malonyl-CoA and 30 PM methylmalonyl-CoA, both microsomal and soluble preparations from integu- ment incorporated labeled methylmalonyl-CoA into fatty acids much more efficiently than did similar preparations from fat body tissue (Table II). The possibility of con- tamination of the microsomal fraction with the soluble synthetase was ruled out by two observations. First, the incorporation of label by crude or washed microsomal preparations was essentially the same and secondly, the purity of each fraction was confirmed with enzyme mark- ers as shown by Na+/K+-ATPase specific activity of 96 nmolPJmg * min for the microsomal fraction and unde- tectable activity for the cytosol. An assay for alcohol de- hydrogenase gave a cytoplasmic activity of 25 pmol NAD/ mg - min, while the microsomal preparation had only 0.9 pmol NAD/mg- min, which indicated a high degree of

purity for each fraction. The total radioactivity from [methyl-‘4C]methylmalonyl-CoA incorporated into fatty acids by the microsomal fraction was higher or equal to that of the cytoplasmic fraction.

Radio-HPLC analysis of the products from the soluble and microsomal preparations from integument tissue (Fig. 5) showed that the major products formed in both prep- arations were fatty acids that eluted at different retention times than the straight chain moieties, thus indicating that they were methyl-branched fatty acids. The main peak is found in a position corresponding to a methyl- branched fatty acid with 16 carbons in the backbone of the molecule. Smaller radioactive peaks were estimated as methyl-branched fatty acids with 14 and 15 carbons in the backbone with some longer chain moieties also present. The resolution on radio-HPLC is such that closely related isomers do not separate and it is not pos- sible to assign exact methyl branch positions.

When methylmalonyl-CoA and malonyl-CoA were in- cubated with fat body FAS, the activity was very low and the only products detected were small amounts of medium chain fatty acids with carbon chain lengths of less than 14 carbons (data not shown).

Similar assays which contained only malonyl-CoA as the elongating unit and [2-14C]malonyl-CoA as the radio- active tracer were performed. Under those conditions, both the microsomal and the soluble integumental FAS produced only straight chain fatty acids of 16 and 16 car-

METHYL-BRANCHED FATTY ACIDS IN COCKROACHES 337

5-methylhexadecane A

182

3methylhexadecane B

-1 I 26592

S712Ml

FIG. 2. Mass spectra of monomethyl-branched alkanes derived from monomethyl-branched fatty acids from the integument of female B. germanica. The spectra were obtained as described under Materials and Methods. Peak 3 (Fig. 1) is interpreted as 5-methylhexadecane (A), peak 5.as 3-methylhexadecane (B).

bons at a ratio of about 4/l (data not shown), as is usually found for soluble FAS of animal origin (21).

When malonyl-CoA was omitted from the incubation media and only methylmalonyl-CoA was used as the elongating agent, the microsomal FAS from the integu- ment showed a much higher activity than the soluble FAS (Fig. 6A). At 60 PM methylmalonyl-CoA, the soluble FAS showed no activity whereas the microsomal FAS gave maximum activity. However, [methyl-‘*C]methylmalonyl- CoA incorporation increased with increasing methylmal- onyl-CoA concentration for both microsomal and cyto- plasmic FAS in the presence of variable amounts of mal- onyl-CoA (Fig. 6B).

Further characterization of the microsomal FAS from the integument showed that the reaction was essentially linear for 60 min (Fig. 7A). A linear relationship between FAS activity and protein concentration was observed up

to 80 ng protein/p1 (Fig. 7B). NADPH supported the mi- crosomal FAS activity and increased activity was observed up to 200 PM NADPH (Fig. 7C). NADH by itself did not support FAS activity and was not synergistic with NADPH (data not shown).

DISCUSSION

The methyl-branching groups of the terminally branched hydrocarbons have been shown to be inserted early in.chain synthesis by both carbon-13 NMR and mass spectral techniques using carbon-13-labeled precursors in the housefly (18), the American cockroach (17), and the German cockroach (19). Similarly, Nelson et al. (34) showed that in multimethyl-branched very-long-chain alcohols, the methyl branch group is inserted early in chain synthesis. Thus, expected intermediates in the for-

m/z

1OOS

50s

m/z

7

1 73600

5,9-dimethylhrxndecane A

3,11-dimethylhexadecane B

-1 7664

FIG. 3. Mass spectra of dimethyl-branched alkanes derived from di- methyl-branched fatty acids from the integument of female I?. germunim. The spectra were obtained as described under Materials and Methods. Peak 6 (Fig. 1) is interpreted as 5,9-dimethylhexadecane (A) and peak 7 as 3,11-dimethylhexadecane (B).

338 JUAREZ, CHASE, AND BLOMQUIST

TABLE I

Structural Identification of the Methyl-Branched Fatty Acids from the Abdominal Integument of Female B. germanica

Peak Hydrocarbon Derived from Equivalent

chain length Characteristic ion

fragments

9 5-Methylheptadecane 10 I-Methylheptadecane 12 9-, lo-, 11-Methyloctadecane

13 5-Methyloctadecane 14 4-Methyloctadecane 15 3-Methyloctadecane 16 5,9-Dimethyloctadecane 17 3,9-Dimethyloctadecane

4-Methylpentadecane n-4-Methylpentadecanoic 14.6 3-Methylpentadecane n-3-Methylpentadecanoic 14.7 5-Methylhexadecane n-5-Methylhexadecanoic 16.5 I-Methylhexadecane n-4-Methylhexadecanoic 16.6 3-Methylhexadecane n-3-Methylhexadecanoic 16.7 5,9-Dimethylhexadecane n-5,9-Dimethylhexadecanoic 16.9 3,11-Dimethylhexadecane n-3,11-Dimethylhexadecanoic 17.0 7-, 8-, 9-Methylheptadecane n-7-, 8-, 9-Methylheptadecanoic 17.4

n-5-Methylheptadecanoic 17.4 n-4-Methylheptadecanoic 17.5 n-9-, lo-, 11-Methyloctadecanoic 18.3 n-5-Methyloctadecanoic 18.4 n-4-Methyloctadecanoic 18.5 n-3-Methyloctadecanoic 18.6 n-5,9-Dimethyloctadecanoic 18.8 n-3,9-Dimethyloctadecanoic 19.0

155;182/183 165 (M-57); 182/183 85;154/155;182/183 168/169;196/197;240 (M+) 56/57;181/182;210/211 85;126/127;155;197 98/99;155;183;225 111/112; 125/126; 140/141;

154/155;168/169 85;168/169;196/197 182/183;210/211 140/141;154/155;168/169 85;210/211 224/225;253 (M-15) 210/211;238/239 85;154/155;225;267 (M-15) 154/155;253 (M-29)

Note. Interpretations were based on hydrocarbons which were obtained by chemical reduction of the naturally occurring fatty acids.

mation of methyl-branched hydrocarbons would be methyl-branched fatty acids with the same positional iso- mers as the major hydrocarbons. To our knowledge, such methyl-branched fatty acids have not been previously identified in any insect. It has been shown that in addition to being incorporated into branched hydrocarbons, [l- 14C]propionate is incorporated at very low levels into what appeared to be branched fatty acids in the German cock- roach (19) and other insects (P. Juarez, unpublished), al- though the methyl-branch positions were not determined. The similarity in the positional isomers of the methyl- branched fatty acids present in the integument of the German cockroach to those of the major hydrocarbons (8, 9) strongly suggests that they are intermediates in hydrocarbon formation.

Consistent with many observations that integument- related tissue is the site of hydrocarbon formation (2,35, 36), the methyl-branched fatty acids were observed in in-

tegument tissue but not fat body tissue. As few studies have concentrated on the fatty acids of the integument of insects, it is not surprising that methyl-branched fatty acids with the same positional isomers as the major hy- drocarbons have not been previously observed in insects. In analyses of whole insect fatty acids, the large amounts of straight chain fatty acids present in the fat body triac- ylglycerols made detection of the very small amounts of methyl-branched fatty acids difficult or impossible. Re- cent studies in the American cockroach and the Southern armyworm have also demonstrated the presence of small amounts of methyl-branched fatty acids in the integument tissue (Chase, Guo, and Blomquist, unpublished data), suggesting that they may be common to insects.

It has been shown that in addition to hydrocarbon syn- thesis, the integument is responsible for the synthesis of most of the cuticular fatty acids in Triatoma infestuns, by both de nouo and elongation reactions (36)) suggesting

r

FIG. 4. Radio-HPLC analysis of the FAME of B. germaniea fatty acids isolated from integument tissues incubated with [1-‘“Clpropionate. Incubation, extraction, and chromatographic conditions are given under Materials and Methods.

METHYL-BRANCHED FATTY ACIDS IN COCKROACHES 339

TABLE II

FAS Activity in Integument and Fat Body Soluble and Microsomal Fractions

% Radioactivity incorporated

pmol incorporated/ mg protein

Microsomes Cytoplasm

Integument 50 2.6 0.6 Fat body 4 0.2 0.1

Note. Enzyme activity was assayed radiochemically in both integument and fat body subcellular fractions as described under Materials and Methods with 30 pM of malonyl-CoA and 30 pM of methylmalonyl-CoA incubated for 1 h. For the integument assay, 0.41 nmol of [methyl- ‘“C]methylmalonyl-CoA were used while 0.82 nmol was employed for the fat body assay.

the presence of an integumental FAS. Though there is no strict correlation among fatty acids synthesized by in- ternal tissues and epicuticular hydrocarbons, data ob- tained from pupa of the cabbage looper indicated that large amounts of methyl-branched lipid were produced at times when the total soluble FAS activity was very low or not detectable (4). This suggested that a soluble FAS might not be involved in the synthesis of hydrocarbon precursors. The much higher amount of methyl-branched fatty acids associated with the microsomal fraction of the German cockroach integument compared to the soluble fraction further supports this concept.

Direct evidence that a microsomal FAS is involved in forming the precursors to methyl-branched hydrocar-

I dL, 0 10 20 30 40 50

nMEtMmlJTEs)

FIG. 5. Radio-HPLC analysis of the FAME obtained after integument subcellular fractions were incubated with [methyl-“C]methylmalonyl- CoA. The upper trace (A) is a mass trace. B, soluble fraction; C, micro- somal fraction. Incubations were performed, and lipid was extracted, derivatized, and analyzed as described under Materials and Methods. The malonyl-CoA/methylmalonyl-CoA ratio = 1. A protein concentra- tion of 0.2 pg/pl was used.

0 10 20 30 40 50 60 70

Methylrnalonyl-CoA (ph4)

0 IO 20 30 40 50 60

Methylmalonyl-CoA (PM)

I - I., . I.,. , . ,

60 50 40 30 20 10 0

Malonyl-CoA (PM)

FIG. 6. Effect of methylmalonyl-CoA concentration on microsomal and soluble FAS activity with methylmalonyl-CoA as the only elongating agent (A) and with methylmalonyl-CoA in the presence of varying con- centrations of malonyl-CoA (B). FAS activity was assayed by deter- mining the incorporation of [methyl-l’C]methylmalonyl-CoA into fatty acids. Preparation of subcellular fractions, incubation conditions, ex- tractions, and product assays was performed as described under Materials and Methods.

bon was obtained by observing the incorporation of la- beled methylmalonyl-CoA into methyl-branched fatty acids in microsomal preparations. Furthermore, when these labeled methyl-branched fatty acids were extracted and incubated with fresh integument tissue, 10% of the radioactivity was incorporated into branched hydrocar- bons (data not shown). That a soluble FAS may also be involved is indicated by the synthesis of methyl-branched fatty acids by this subcellular fraction as well. Taken to- gether these data are the first evidence of FAS activity in insect integument. Some insects exhibit very high specificity in the placement of the methyl groups in the hydrocarbon chain, and the mechanism by which this is accomplished is not known. The discovery of a microsomal

340 JUAREZ, CHASE, AND BLOMQUIST

50 100

Time (minutes)

, . 1 . I ,

0.0 0.1 0.2 0.3 0.4 0.5 0.6 Protein (Pgg/~.Il)

C

0 100 200 300 400 NADPH (1tM)

FIG. 7. Effect of time (A), protein (B), and NADPH (C) concentration on the microsomal incorporation of [methyl-‘4C]methylmalonyl-CoA into fatty acids. Assays for A and C were carried out with 10 pg protein and a malonyl-CoA/methylmalonyl-CoA ratio = 1. Incubation conditions, extractions, and product determination were performed as described under Materials and Methods.

FAS that incorporates methylmalonyl-CoA into the methyl-branched fatty acid precursors to hydrocarbons will allow this phenomenon to be studied.

Since the enzyme activities that elongate fatty acyl- CoAs to very-long-chain fatty acyl-CoAs and those that convert these very-long-chain acyl-CoAs to hydrocarbon are in the microsomal fraction (12-14), it is tempting to speculate that the reason that very small amounts of methyl-branched fatty acids are present in insects and why they are associated with the microsomal fraction is that they are both formed in the microsomes and then elongated and converted to hydrocarbons by the same subcellular fraction, It is possible that the enzymes which

channel methyl-branched fatty acids into the elongation pathway toward hydrocarbon formation rather than es- terifying them as acylglycerols or phospholipids may ex- hibit the necessary specificity to ensure that they do not accumulate in tissues. Further work is needed to deter- mine the specificities in these processes.

Although fatty acyl-CoA elongation systems are located in the microsomal fraction of many organisms (7), very few reports of a microsomal FAS are available. Khan and Kolattukudy (37) presented evidence that a microsomal FAS in Euglenu gracilis is involved in producing the acyl chains of wax esters. Bolton and Harwood (38, 39) also found both particulate and soluble FAS in germinating seeds.

The discovery of a microsomal FAS that appears to synthesize the methyl-branched fatty acid precursors to hydrocarbons should allow rapid advances in our under- standing of hydrocarbon formation in insects. Work is needed to determine what controls the specificity of methylmalonyl-CoA insertion in the growing acyl chain, the specificity which controls whether one, two, or three methylmalonyl-CoA units are inserted, and to examine the elongation reactions to determine the role of methyl branches in regulating the chain length of the hydrocar- bon product.

REFERENCES

1. Nelson, D. R. (1978) Adv. Insect Physiol. 13. l-33.

2. Blomquist, G. J., Nelson, D. R., and de Renobales, M. (1987) Arch. Insect Biochem. Physiol. 6, 227-265.

3. Lackey, M. (1988) Comp. Biochem. Physiol. B. 89, 595-645. 4. de Renobales, M., Nelson, D. R., Zamboni, A. C., Mackay, M. E.,

Dwyer, L. A., Theisen, M. O., and Blomquist, G. J. (1989) Insect Biochem. 19,209-214.

5. Nelson, D. R., Fatland, C. L., Buckner, J. S., de Renobales, M., and Blomquist, G. J. (1990) Insect Biochem. 20,809-819.

6. Nelson, D. R., Fatland, C. L., Buckner, J. S., de Renobales, M., and Blomquist, G. J. (1990) Insect Biochem. 20,821-827.

7. Kolattukudy, P. E. (1976) in Chemistry and Biochemistry of Natural Waxes (Kolattukudy, P. E., Ed.), pp. 1-15, Elsevier, Amsterdam.

8. Augustynowicz, M., Malinski, E., Warnke, Z., Szafranek, J., and Nawrot, J. (1987) Comp. Biochem. Physiol. B 86,519-523.

9. Jurenka, R. A., Schal, C., Burns, E., Chase, J., and Blomquist, G. J. (1989) J. Chem. Ecol. 16,939-949.

10. Nishida, R., and Fukami, H. (1983) Mem. Coil. Agric. Kyoto Univ. 122,1-24.

11. Schal, C., Bums, E., Gadot, M., Chase, J., and Blomquist, G. J. (1991) Insect B&hem. 21,73-79.

12. Vaz, A. H., Blomquist, G. J., and Reitz, R. C. (1988) Insect Biochem. 18.177-184.

13. Vaz, A. H., Jurenka, R. A., Blomquist, G. J., and Reitz R. C. (1988) Arch. B&hem. Biophys. 267,551-557.

14. Blomquist, G. J., Vaz, A. H., Jurenka, R. A., and Reitz, R. C. (1989) in Biocatalyst in Agricultural Biotechnology (Whitaker, J. R., and Sonnet, P. E., Eds.), pp. 314-322. ACS Symposium Series 389, Washington, D.C.

15. Bognar, A. L., Paliyath, G., Rogers, L., and Kolattukudy, P. E. (1984) Arch. B&hem. Biophys. 236,8-l&

METHYL-BRANCHED FATTY ACIDS IN COCKROACHES 341

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

Dennis, M. W., and Kolattukudy, P. E. (1991) Arch. Biochem. Eio- phys. 287,268-275.

Dwyer, L. A., Blomquist, G. J., Nelson, J. H., and Pomonis, J. G. (1981) Eiochim. Biophys. Acta 663,536-544.

Dillwith, J. W., Nelson, J. H., Pomonis, J. G., Nelson, D. R., and Blomquist, G. J. (1982) J. Biol. Chem. 267, 11,305-11,314.

Chase, J., Jurenka, R. A., Schal, C., HaIamkar, P. P., and Blomquist, G. J. (1990) Znsect &o&cm. 20,149-156.

Stanley-Samuelson, D. W., Jurenka, R. C., Cripps, C., Blomquist, G. J., and de Renobales, M. (1988) Arch. Insect Biochem. Physiol. 9, l-33.

Wakil, S. J., and Stoops, J. K. (1983) in The Enzymes (Boyer, A. D., Ed.), Vol. 16 pp. 3-61. Academic Press, New York.

Buckner, J. S., Kolattukudy, P. E., and Rogers, L. (1978) Arch. Eiochem. Eiophys. 186,152-163. Buckner, J. S., and Kolattukudy, P. E. (1975) Biochemistry 14, 1774-1782. Buckner, J. S., Kolattukudy, P. E., and Poulose, A. J. (1976) Arch. Eiochem. Eiophys. 177, 539-545. de Renobales, M., Woodin, T. S., and Blomquist, G. J. (1986) Insect Eiochem. l&887-894. Bligh, E. G., and Dyer, W. J. (1959) Can. J. Eiochem. Physiol. 37, 911-917.

27.

28.

29. 30.

31.

32.

33. 34. 35.

36.

37.

38.

39.

Jurenka, R. A., de Renobales, M., and Blomquist, G. J. (1987) Arch. Eiochem. Eiophys. 255, 184-193.

Bjostad, L. B., Wolf, W. A., and Roelofs, W. L. (1987) in Pheromone Biochemistry (Prestwich, G. D., and Blomquist, G. J., Eds.), pp. 77-120, Academic Press, New York. Kurtti, T. J., and Brooks, M. A. (1976) In Vitro 2, 141-146. Lord, J. M., Kagawa, T., Moore, T. S., and Beevers, H. (1972) Proc. Natl. Acad. Sci. USA 69,2429-2432. Borgeson, C. E., and Bowman, B. J. (1983) J. Bacterial. 156, 362- 368. Elliot, J. I., and Knopp, J. A. (1981) in Methods of Enzymology (Wood, W. A., Ed.), Vol. 41, pp. 374-379, Academic Press, New York.

Bradford, M. M. (1976) Anal. Biochem. 72, 248-254.

Nelson, D. R. (1969) Nature 221,854-855. Juirez, M. P., and Brenner, R. R. (1987) Comp. Eiochem. Physiol. E 87,233-239.

Juirez, M. P., and Brenner, R. R. (1989) Comp. Biochem. Physiol. E 93,763-772. Kahn, A. A., and Kolattukudy, P. E. (1973) Arch. Eiochem. Biophys. 170,400-408. Bolton, P., and Harwood, J. L. (1977) Eiochim. Eiophys. Acta 489, 15-24. Bolton, P., and Harwood, J. L. (1977) Eiochem. J. 168, 261-269.