Insect Biochem. Molec. Biol. Vol. 22, No. 2, pp. 143-148, 1992 Printed in Great Britain. All rights reserved

0965-1748/92 $5.00 + 0.00 Copyright 0 1992 Pergamon Press plc

CHARACTERIZATION OF THE A”-PALMITOYL-CoA-DESATURASE FROM SPODOPTERA

LITTORALIS (LEPIDOPTERA:NOCTUIDAE)

FERRAN RODRIGUEZ,’ DAVID L. HALLAHAN,~ JOHN A. PICKETT~ and FRANCISCO CAMPS’* ‘Biological Organic Chemistry Department, C.I.D. (C.S.I.C.), Jordi Girona Salgado 18-26, 08034 Barcelona, Spain, *Biochemistry and Physiology Department and %secticides and Fungicides Department, AFRC Institute of Arable Crops Research, Rothamsted Experimental Station, Harpenden,

Herts AL5 2JQ, England

(Received 13 June 1991; revised and accepted 1 October 1991)

Abstract-One of the key enzymes involved in moth pheromone biosynthesis, palmitoyl-CoA A”-desat- urase, has been characterized in subcellular fractions from pheromonal glands of adult female Spodopteru littoralis. Desaturase activity was dependent on the presence of reduced pyridine nucleotides, and NADH was a better electron donor to the enzyme than NADPH. Incubation of gland microsomes with NADPH and [Vlpalmitoyl-CoA significantly increased one unidentified reaction product. Incubation with the corresponding free acids showed the highest activity for palmitic acid followed by myristic and stearic acids. (Z)-1 I-Hexadecenoic acid (as the methyl ester) was identified as the only radioactive product of the main pheromonal gland desaturase by radio-GC chromatography. The optimal pH for A”-desaturase activity was between 6.8 and 7.2, and 25°C was found to be the optimum temperature for this activity. BSA, EDTA and DTT were required for maximal desaturase activity. Cyanide and azide were inhibitory, and carbon monoxide had no effect.

Key Word Index: desaturase; sex pheromone biosynthesis; insect lipid biosynthesis; Spodoptera littoralis

INTRODUCTION

The oxidative introduction of a double bond into fatty acid carbon chains, or desaturation, has been demonstrated in many organisms, including mam- mals, birds, insects, fungi, plants and bacteria, and also in specific animal and plant tissues (Holloway, 1983; Harwood, 1988; Fulco, 1974). Unsaturated fatty acids are important components of cell mem- branes; in insects, however, they play an important additional role as precursors of pheromones, an essential group of chemical messengers or semio- chemicals (Stanley-Samuelson et al., 1988). Most of the lepidopteran sex pheromones are mixtures of fatty acid derivatives of chain length varying from 10 to 18 carbon atoms, with a primary alcohol, ester or aldehyde as a functional group, and generally one to three double bonds at a variety of positions and with different, but specific, Z or E configurations (Roelofs and Bjostad, 1984; Roelofs and Wolf, 1988; Bjostad et al., 1987).

Desaturase activity related to lipid biosynthesis in animals is membrane associated and is mediated by a flavoprotein, NADH-cytochrome b,-reductase; a hemoprotein, cytochrome b,; and a nonheme iron protein, the desaturase itself (Holloway, 1983). This electron transfer system uses electrons from NADH or NADPH to reduce the desaturase, which in the presence of molecular oxygen introduces a double bond in a specific position in the fatty acid molecule.

Whilst desaturase activity has been characterized in vitro and isolated from rat and chicken liver

*Author for correspondence.

(Strittmatter et al., 1974; Prasad and Joshi, 1979; Jeffcoat et al., 1977), fatty acid desaturase enzymes of insects are poorly understood. Stearate desaturation by microsomes on the locust fat body (Tietz and Stern, 1969) and fatty acid unsaturation by mito- chondria and microsomes during development of insects (Gonzalez-Buitrago et al., 1979) have been investigated. Studies on the A9-acylCoA desaturase in the housefly (Wang et al., 1982) and on the A”-desaturase in cabbage looper moth pheromone biosynthesis (Wolf and Roelofs, 1986) have also been carried out. More recently Ai2-desaturase activity has been reported in the fat body of the house cricket (Cripps et al., 1990) and in the american cockroach (Borgeson et al., 1990). This paucity of information has resulted from the problems inherent in studying the labile desaturase enzymes in subcellular fractions, and difficulties in obtaining sufficient material for study. We report here the preparation of sub- cellular membrane fractions from moth (Spodopteru littoralis) pheromonal glands, with retention of fattv acid desaturase activity. The properties of the * A”-palmitoyl-CoA desaiurase in preparations are described.

MATERIAIS AND METHODS

Insects Spodoptera littoralis pupae were supplied from cultures

maintained at the Department of Biological Organic Chem- istry (Barcelona, Spain). Pupae were express air mailed to Rothamsted Experimental Station. On arrival they were segregated by sex and kept under a 16:8 light:dark regime at 25°C. Adults were fed ad libitum with a 10% aqueous

n&rosomal

143

144 FERRAN RODRIGUEZ et al

ACTIVITY DISTRIBUTION

”

1,000 0 14,obo 0 105,doo 0

Fig. 1. Distribution of desaturase activity after differential centrifugation of S. littoralis glands (1023) extract. 100% activity corresponding to lOOOg, 14,000g and 105,OOOg fractions were 366,207 and 601 pmol product mini mg

protein-’ respectively.

three times with 0.7 ml of chloroform. The chloroform extracts were combined and evaporated to dryness under a stream of nitrogen. The residue was methylated by addition of 100~1 of 14% boron trifluoride in methanol. After 60min at room temperature, 100~1 of 1 M NaCl solution was added and fatty acid methyl esters were extracted three times with 0.7 ml hexane. Hexane extracts were combined and spotted on a 5% AgNO, silica gel TLC plae. The plate was developed with hexane/diethyl ether (9: 1, v/v). Products were detected by autoradiography or using a radioscanner (Isomess IM3000). Radioactivity was measured using a liquid scintillation counter (Kontron Betamatic).

Product identification The monoene fraction isolated from 5% AgNO, TLC was

analysed by combined gas chromatography-radioisotope detection, on a column containing 5% DEGS in a Hewlett Packard 5710 gas chromatograph interfaced with an ES1 nuclear radiodetector. Argon: CO, (95: 5) was used as car- rier at a flow of 30ml/min under isothermal conditions (165°C).

-

honey solution. Virgin females up to 3 days old were used in all experiments.

Chemicals [I-i4C]stearic acid (55.3 mCi/mmol), [l-‘4C]palmitic acid

(56.7 mCi/mmol), and [l-‘4C]myristic acid (55 mCi/mmol) were obtained from Amersham International plc, Amer- sham, England. [ I-“‘Clpalmitoyl-CoA (54 mCi/mmol) was obtained from the same supplier or prepared by the method of Bishop and Hajra (1980). Thin layer chromatography was performed on silica gel 60 Fzs4 TLC plates (Merck). All other chemicals were analytical grade or the purest quality available.

Cellular fractionation Insects were anaesthetized with CO, and kept on ice

during excision of the pheromonal glands, which were then pooled on dry ice and stored at -80°C until used. Glands were homogenized in a Potter Elvehjem homogenizer with 10 pi/gland of cold 0.1 M sodium phosphate buffer (pH 6.8), 0.35 M sucrose, 1 mM DTT, 1 mM EDTA, 1 mM PMSF and leupeptin (2.5pglml). Ten turns of the pestle were applied, and the homogenate was then centrifuged at 14,000g for 20 min (4°C). The mitochondrial pellet was resuspended to the original volume and centrifuged again. Both supernatants were pooled and centrifuged at 105,OOOg for 60 min (4°C). The microsomal pellet was resuspended in 10 mM phosphate buffer (pH 6.8), 0.35 M sucrose, 1 mM DTT, 1 mM EDTA, 20% glycerol and rehomogenized in a 2 ml Potter Elvehjem homogenizer before being centrifuged at 14,000g for 20 min (4°C). The supernatant fraction, termed washed microsomes, was used immediately or stored at -80°C until used.

Desaturase assay The reaction mixture consisted, unless otherwise indi-

cated, of 20 PM [1-i4C]palmitoyl-CoA (200 nCi), 0.1 M sodium phosphate buffer (PH 6.8), 0.35 M sucrose, 0.1% BSA (fatty acid free), 1 mM DTT, 1 mM EDTA, 1 mM NADH, 0.1 mg protein/ml, in a total volume of 200 ~1. The reaction was normally carried out at 25°C for 10 min. After this time 0.5 ml of 0.5 M methanolic KOH solution was added and the mixture left for 30 min. After acidifying with 200 ~1 of 2 M HCl in 1 M NaCl, the mixture was extracted

2

~

rd Fig. 2. Radio-GC trace of the mono-unsaturated fraction isolated from 5% AgNO,-TLC after incubation of phero- monal gland washed microsomes with [l-‘4C]pahnitoyl- CoA. The upper trace is the FID output, the lower is the radiodetector trace. The methylated mono-unsaturated frac- tion was injected together with unlabelled; 1: (Z)-1 l-hexade- cenoate and 2: (Z)d-hexadecenoate, retention time 1.78 and

1.48 min respectively.

Spodoptera littoralis A”-desaturase 145

i I

Y--+

f rL

10

I 01

0 5 10 16 20 25

Reaction time (mid

Fig. 3. Desaturase activity vs reaction time and protein con- centration. 50 /Al aliquots were removed from a single reac- tion for extraction and quantification of desaturase product. (0) 0.025 mg protein ml-‘, (0) 0.050 mg protein ml-‘,

(A) 0.1 mg protein ml-‘, (0) 0.225 mg protein ml-‘.

Protein determination Protein concentration was estimated by the method

of Lowry er al. (1951) as modified by Bensadouin and Weinstein (1976); and Markwell et al. (1978), utilizing bovine serum albumin as standard.

RESULTS

Desaturase activity of S. littoralis glands and sub- cellular localization of activity

Extracts of S. littoralis pheromonal glands actively catalyzed the A”-desaturation of added [1-14C] palmitoyl-CoA to 1 1-hexadecenoate, as determined by analysis of methylated reaction products on TLC and comparison of their mobility with authentic compounds. The specific activity distributed in fractions after differential centrifugation of a crude gland homogenate (after 1000 g for 10 min, 14,000 g for 20 min, and 105,OOOg for 60 min), is displayed in Fig. 1. It is clear from the results that desaturase activity was mainly located in the microsomal frac-

1800

” 1400 z

g 1200

P 7 .g 1000

z 600

s

z 600

L .g s 400

200

0

Ok 0 10 20 30 40 60 60 70 80 QO tO0

Palmltoyl-CoA hM)

Fig. 4. Variation in desaturase activity with substrate con- centration. Desaturase activity of S, littoralis gland washed microsomes was determined in the presence of varying

concentrations of [l-14C]palmitoyl-CoA.

1100

F L ; 1000

‘; .g 900

z 600 S

z-. .% ,z 700

2 600

600 1-T i 5.6 6 6.4 6.6 7.2 7.6

PH

90

60

Fig. 5. pH dependence of desaturase activity. S. littoralis gland washed microsomes were assayed for desaturase activity in 0.1 M sodium phosphate buffers at the pH indicated. Other conditions were as described in Materials

and Methods.

tion (105,OOOg pellet), with a specific activity of 600 pmol product mini mg-’ protein.

The identity of the desaturase product as (Z)-1 l- hexadecenoate was confirmed using combined GC- radioisotope detection. The only radioactive compound in the extracted monoene fraction after incubation of washed microsomes with [l- i4C]palmitoyl-CoA and TLC analysis was found to co-chromatograph with standard methyl (Z)-1 l- hexadecenoate on radio-GC (Fig. 2).

Properties of S. littoralis A “-desaturase; optimization of assay conditions

Desaturase activity was initially linear with time during assay, and dependent on the amount of micro- somal protein (Fig. 3). Activity was linear with protein concentration within the range tested, up to 1 mgprotein ml-‘. Based on these data, a 10 min assay period was chosen for routine use. With sub- strate concentrations up to 50 p M, a linear increase in enzyme activity was observed, but an inhibition

90

60 20 22 24 26 28 30 32 34 36 30

Temperature (“C)

Fig. 6. Effect of temperature on desaturase activity. The desaturase activity of S. littoralis gland washed microsomes was determined under standard assay conditions at different temperatures. 100% activity represents 61 pmol product

min-’ mg protein-‘.

146 FERRAN RODRIGUEZ et al.

Table 1. Effect of BSA on desaturase activity

BSA (mg/ml) Activity (%)

0 28 1 100 2 a4

Desaturase activity of S. lirtoralis gland washed microsomes was assayed as described in Materials and Methods, in the presence of different concentrations of BSA. 100% ac- tivity represents 566 pmol product min-’ mg protein-’

by substrate was observed at higher concentrations (Fig. 4). The effect of pH on activity was studied in the range 5.8-7.6, using 0.1 M sodium phosphate buffers (Fig. 5). Desaturase activity exhibited a broad pH optimum between pH 6.4 and 7.2. The effect of temperature on desaturase activity was also examined (Fig. 6). The optimal activity was obtained at 25°C in the standard assay mixture. The presence of BSA in the assay mixture was required for maximal activity (Table 1).

Eflects of dIrerent electron donors on desaturase activity

The different exogenous electron donors, NADH and NADPH, were examined for their ability to support desaturase activity. As shown in Table 2, NADH was a more effective electron donor than NADPH at all concentrations tested. Surprisingly, activity was less when either NADH or NADPH were supplied at concentrations above 1 .O mM. Incubation of washed microsomes with [l-14C]palmitoyl-CoA and NADPH yielded methyl (Z)- 1 1-hexadecenoate as the principal product but, in the presence of NADPH, a significant increase of an unidentified reaction product was observed.

Substrate specijicity The substrate specificity of the desaturase was

investigated using the free acid form of myristic, pal- mitic and steak acids. These were added to washed microsomes with 10 mM ATP, 1 mM CoASH and 4mM MgCl,, and activity assayed under standard conditions. Activity was greatest with palmitic acid (100%) followed by myristic (50%) and steak (18%).

EfSect of insect age on microsomal protein content and desaturase activity

The specific activity of S. littoralis microsomal A”-desaturase decreased with the age of the animals,

Table 2. Efficiency of NADH and NADPH as electron donors to the desaturase

Activity (%)

Concentration (mM) NADH NADPH

1.0 100 60 3.0 93 48 5.0 80 43

Desaturase activity of S. littoralis gland microsomes was assayed as described in Materials and Methods, in the presence of NADH or NADPH. 100% activity represents 143 pm01 product min-’ mg protein-‘.

0 24 48 72 AQE (hour.)

Fig. 7. Effect of age on A”-desaturase activity and protein content of pheromone glands of S. littoralis. Emerging moths were selected during the last 2 h before onset of scotophase (f = 0), and glknds removed during the maxi- mum pheromone release period (2 h into the scotophase).

(0) desaturase activity, (0) protein content.

being greatest a few hours before eclosion, and decreasing to 35% of this value 74 h post-eclosion (Fig. 7). The protein content @gproteingland-‘) displayed a different pattern, being least 2 h post-eclo- sion (Fig. 7).

Enzyme stability The stability of insect desaturase systems has been

found to be low, with considerable losses of activity following freeze-thawing of membrane preparations. Enzyme stability in washed microsomes of S. littoralis was also found to be low under the conditions of assay, with DTT and EDTA required for maximal activity (Table 3). When washed microsomes were incubated at 25°C prior to assay, the activity was drastically reduced. A delay of 30 min at the assay temperature reduced desaturase activity to 18% of its original value. However, microsomal desaturase activity was stable during storage at -80°C for at least 1 yr without significant loss of activity.

Eflect of inhibitors The effect of CO, sodium azide or potassium

cyanide on desaturase activity was studied; the results are summarized in Table 4. Surprisingly, CO had a

Table 3. Effect of EDTA and DTT on desaturase activity

Addition (mM) Activity (%)

2.6

2 $ f

1.6 i

lf

0.6

.O

DTT 0.0 100. 1.0 142

EDTA 0.0 1OOt 0.1 151 0.5 168 1.0 181

Desaturase activity of S. littoralis gland microsomes was assayed as de- scribed in Materials and Methods, with the addition of DTT or EDTA.

100% activity in the experiment with DTT represents 746 pmol product min-’ mg protein-‘.

tlOO% activity in the experiment with EDTA represents 436 pmol product min-’ mg protein-‘.

Spodoptera littoralis A”-desaturase 147

Table 4. Effect of azide and cyanide on deaaturase activitv

Addition (mM) Activity (%) KCN 0.5 100

1.0 17 5.0

NaN, 0.5 : 1.0 9

Desaturasc activity of S. tinor& gland microsomes was assayed as de- scribed in Materials and Methods, in the presence of potassium cyanide or sodium aride. 100% activity rep resents 159 pmol product min-’ mg protein-‘.

slight activating effect on the desaturase (not shown), whilst, as might be expected potassium cyanide and sodium azide were effective inhibitors (Oshino et al., 1971).

DISCUSSION

Although microsomal desaturation of fatty acids has been well characterized and isolated in mammals (Strittmatter et al., 1974; Jeffcoat et al., 1977) and birds (Prasad and Joshi, 1979), these studies have been carried out on A9-stearoyl-CoA-desaturase which produces oleic acid, the major unsaturated fatty acid cell membrane component. Desaturation at the 11 position has been described in bacteria by Stern et al. (1969), in Leptospira canicula and by Wood and Lee (1980), in hepatoma cultures. Like- wise, desaturation at this particular position is a common step in the biosynthesis of lepidopteran pheromones (Roelofs and Bjostad, 1984). It has been shown that in S. littoralis (Z)-1 I-tetradecadienyl acetate is synthesized from palmitic acid by the action of a A*‘-desaturase enzyme, followed by B-oxidation (Martinez et al., 1990). The data presented in this paper demonstrate the presence of an active A”-de- saturase in washed microsomes prepared from phero- monal glands of this moth. It would appear that some aspects of the A”-desaturase of S. littoralis are, however, different to the enzyme previously studied from the cabbage looper moth Trichoplusia ni (Wolf and Roelofs, 1986). The A”-desaturase from S. lit- toralis was active with either NADH or NADPH as source of reducing equivalents, with NADH as more efficient source of reducing power, as has been ob- served for Locusta migratoria A9-desaturase (Tietz and Stern, 1969), and T. ni A”-desaturase (Wolf and Roelofs, 1986) as well as the majority of animal desaturases (Holloway, 1983) however, it was differ- ent from Musca domestica A9-desaturase (Wang et al., 1982) and Achaeta domesticus An-desaturase (Cripps et al., 1990) that preferred NADPH as electron donor. Significant activity of the Spodoptera enzyme was also observed at a lower concentration of NADH, 1 mM instead of 5 mM, than required by the enzyme from T. ni. Under our assay conditions the S. littoralis desaturase accepted 14, 16- and 18-carbon fatty acids as substrates, showing highest activity with palmitic, followed by myristic and then stearic acid. This order of preference might explain the lack of Cn A”-unsaturated derivatives in the insect pheromone blend (Dunkelblum and Kehat, 1987; Martinez et al.,

1990). The S. littoralis enzyme displayed a broad optimum pH range of 6.4-7.2 (S.A. >90%) which is more similar to the A9-desaturase of IU. abmestica, than to the enzyme from T. ni. The S. littoralis A”-desaturase displayed inhibition at high substrate concentration plausibly due to an unspecific deter- gent effect of fatty acids at concentrations over 50pM (Wilson et al., 1980). The enzyme was also insensitive to CO, with an increase in desaturase activity observed in the presence of CO. This might be due to inhibition of microsomal cytochrome P-450 enzymes, which would be expected to compete with the desaturase for electrons from cytochrome bJ . The S. littoralis enzyme displayed the same sensitivity to cyanide as that of T. ni and L. migratoria, and was almost completely inhibited by azide at levels of 1 mM. The variation of specific activity with insect age was also different from that exhibited by the T. ni enzyme, in which activity followed the pheromone release pattern, being highest on the second day after emergence and leveling off by the fifth day. In S. littoralis the highest activity appeared during the first hours of adult life, whilst the maximum of phero- mone release was found in the second and third scotophase. On the other hand the amount of protein in isolated glands followed the pheromone release pattern. This change in gland protein content might explain the different profiles in enzyme activity.

As with the liver A9-desaturase, the S. littoralis enzyme was quite unstable, and needed the addition of reducing agents (DTT) and the divalent metal ion chelator, EDTA, to maintain its activity during the assay. However, gland microsomes could be stored deeply frozen for extended periods of time with retention of activity. The development of successful methods for solubilization, purification, and reconsti- tution of the desaturase enzyme system from isolated microsomes will enable further study of these en- zymes and their involvement in pheromone bio- synthesis.

Acknowledgements-The authors wish to thank Dr L. J. Wadhams, Insecticides and Fungicides Department, Rothamsthed Experimental Station, for his helpful com- ments and encouragement during this study. Financial support from CICYT (grant AGR89-532) and CEE (con- tract SCl-0076-C) is gratefully acknowledged. Ferran Rodriguez also thanks Spanish M.E.C. for a pre-doctoral fellowship.

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