Journal of Chemical Ecology, Vol. 12, No. 2, 1986
METABOLIC TRANSFORMATION OF TRITIUMLABELED PHEROMONE BY TISSUES OF Heliothis virescens MOTHS
Y U - S H I N D I N G and G L E N N
D. P R E S T W I C H 1
Department of Chemistry State University of New York Stony Brook, New York, 11794-3400 (Received April 26, 1985; accepted August 1, 1985) Abstract--Unsaturated aliphatic pheromones of H. virescens were prepared at high specific activity (3H, 58 Ci/mmol) and were employed to study tissue specificity of acetate esterase, alcohol oxidase, and aldehyde dehydrogenase in male and female Heliothis virescens. Thus, [9,10-3H~]Z9- 14:Ac was synthesized by partial tritiation of the corresponding alkyne and was converted to the labeled Z9-14:OH and Z9-14:AI for metabolic studies. Soluble and membrane-associated enzyme activities were determined by radio-TLC assays. Esterase activity is highest in legs of both sexes, but also occurs in antennal and glandular tissues. Oxidase activity requires 02 and is highest in female pheromone gland tissues, but it is also high in the male hairpencils. Aldehyde dehydrogenase activity was uniformly high in all tissues, but highest in antennal tissues of both males and females. Key Words--Pheromone metabolism, tritium-labeling, acetate esterase, alcohol oxidase, aldehyde dehydrogenase, Heliothis virescens, Lepidoptera, Noctuidae, sensory biochemistry.
INTRODUCTION P h e r o m o n e b i o s y n t h e s i s , p h e r o m o n e perception, and p h e r o m o n e c a t a b o l i s m are closely linked in terms o f m a t e - f i n d i n g b e h a v i o r s o f m o t h s (review: M a y e r and M a n k i n , 1985). In this paper, w e will present e x p e r i m e n t a l details on the phero m o n e - m e t a b o l i z i n g e n z y m e s o f Heliothis virescens, using h i g h - s p e c i f i c - a c t i v ity, t r i t i u m - l a b e l e d Z 9 - 1 4 : A 1 (and its c o r r e s p o n d i n g a l c o h o l and acetate). First, ~Fellow of the Alfred P. Sloan Foundation (1981-1985) and Camille and Henry Dreyfus TeacherScholar (1981-1986). 411 0098 0331/86/0200-0411505.00/0 9 1986 Plenum Publishing Corporation
412
DINGAND PRESTW1CH
however, we will survey pheromone biochemistry concisely (general review: Blomquist and Dillwith, 1983; catabolism: Ferkovich, 1981; biosynthesis: Roelofs and Bjostad, 1984). Additional recent results will be found in this symposium edition on pheromone biosynthesis (Bjostad and Roelofs, 1986; Teal and Tumlinson, 1986; Morse and Meighen, 1986) and pheromone catabolism (Lonergan, 1986; Vogt and Riddiford, 1986; Prestwich et al. 1986). Catabolism of pheromones has been examined in only seven insects: Bom-
byx mori, Lymantria dispar, Trichoplusia ni, Antheraea polyphemus, Choristoneura fumiferana, Heliothis virescens, and Reticulitermes flavipes. The silkworm moth, Bombyx mori, produces bombykol (El0, Z12-16:OH) which is converted to "ester" and " a c i d " metabolites by whole male antennae exposed to labeled bombykol (Kasang, 1974). The [12,13-3H]-labeled bombykol (30 Ci/ mmol) and its tetrahydroisomer were prepared by partial tritiation of the 12 triple bond. At exposures of 101~ molecules/antenna, the half-life for "uptake" was 1.6 min for male and 1.0 min for female antennae. Much slower uptake was found in legs, heads, and wing scales which lack the pore tubules. Conversion from the alcohol to the corresponding labeled bombic acid and an unidentified ester, isolated by preparative TLC, showed a 4-rain half-life. Chemical identifications were not performed, and no attempts to manipulate or purify the binding or catabolic proteins were reported. The Seeweisen group also did preliminary work on disparlure biosynthesis (Kasang et al., 1974a) from the (Z)-[7,8-3H]-2-methyl-7-octadecene (Sheads and Beroza, 1973) in female gypsy moth pupae and on racemic disparlure uptake and turnover (Kasang et al., 1974b) by male (and female) L. dispar antennae. In the former, aqueous ethanol suspensions of the labeled alkene were injected into female pupae two days preemergence and 34 % of [3H]disparlure (TLC) was recovered from female glands postemergence. There was no direct evidence of pheromone gland involvement in this conversion, however, and in view of the diel periodicity, life cycle and wild vs. lab variation of disparlure release and production (Charlton and Cardr, 1983; Richerson and Cameron, 1974), this work needs to be repeated on pheromone gland homogenates and with topical application of precursors to the gland surface. Such techniques have been used sucessfully to investigate the biosynthesis of Z11-14:Ac (Bjostad and Roelofs, 1981; Bjostad et al., 1981) in the red-banded leafroller moth and bombykol biosynthesis in silkworm moths (Yamaoka et al., 1984). The disparlure metabolic studies are also very sketchy. At 5 x 1011 molecules/antenna of racemic [7,8-3Hz]disparlure (40 Ci/mmol), about 2.5 min was required for disappearance of half the substrate and appearance of two uncharacterized polar metabolites (50%) as well as > 30% of non-TLC-mobile material. Claims for specificity vs. nonspecificity of uptake and turnover are based on small differences superimposed on an exponential decay curve and are not convincing. Also, it is difficult to evaluate these data since both the attractive
Heliothis PHEROMONEMETABOLISM
413
(+) and inhibitory ( - ) enantiomers were present. It is essential to distinguish binding and catabolism of the two enantiomeric forms, since the proteins involved reside in seperate sensory cells (Hansen, 1984). The in vivo and in vitro metabolism of Z7-12:Ac by Trichoplusia ni has been reported by Ferkovich, Mayer, and their coworkers in Gainesville (Ferkovich, 1981; Ferkovich et al., 1973; 1982a,b; Mayer et al., 1976). The acetate is converted to the corresponding alcohol by both specific and general esterases present in legs, wing scales, and antennae. The two primary roles of these esterases are to prevent sensory adaptation and to clear the body surface. The pheromone-specific catabolic proteins appear to show lower specificity for exact pheromone structures than do the receptors (measured behaviorally or by EAG or single cell), although they do show reduced susceptibility to general esterase inhibitors. This hydrolysis was demonstrated using (1) intact antennae and legs dipped in to a 10 -3 M emulsion of pheromone in buffer; (2) in vitro homogenates of antennae, legs, cuticle, wings, and fat body; and (3) soluble proteins from the sensillum liquor of sonicated antennae. The homogenates were assayed with [3H-acetate]Z7-12:Ac (200-800 mCi/mmol) at 10-6-10 -5 M, which is a much higher concentration ( > 1 0 0 0 • and a much lower specific activity (300 x) than for metabolic studies of bombykol, disparlure, and E6,Z 11-16:Ac (see below). Nonetheless, conversion rates in the order of 6 pmol/~g protein/ min in antennae were observed. Pheromone esterase activities decrease in the order of antennae > legs > wings in homogenates and wings > legs > antennae in eluates, consistent with both the postulated sensory deactivation and surface-cleaning functions. The most thorough biochemical analysis of a pheromone binding and inactivation system is reported for Antheraea polyphemus (Vogt, 1984; Vogt and Riddiford, 1980, 1981, 1985; Vogt et al., 1985; Prestwich et al., 1984, 1986). Using [11, 12-3Hz]E6,Zll-16:Ac (40 or 58 Ci/mmol), three electrophoretically distinct proteins were found which interact with pheromone: a nonenzymatic sensillar pheromone-binding protein (mol wt 15,000), a pheromone-hydrolyzing sensillar esterase (tool wt 55,000), and a second esterase in cuticular tissues (e.g., legs and wing scales) of males and females (Vogt and Riddiford, 1986). Oddly, an electrophoretically identical "binding protein" (mol wt 15,000) was also found in three other saturniids and a sphyngid with different pheromones, in unusually high concentrations in receptor lymph (approx. 105 molecules/sensillum) (Vogt and Riddiford, 1980). The sensillar esterase shows considerable specificity for acetates with Z-olefinic linkages and a very high Vma• over a large concentration range. The studies on Choristoneura fumiferana (Morse and Meighen, 1984a,b, 1986) show the importance of studying multiple enzyme systems in different tissues of males and females. Three activities--an acetate esterase (E/Z1114:Ac ~ E/Zll-14:OH), and O2-requiring alcohol oxidase (E/Z11-14:OH
414
DING AND PRESTWICH
E/Z11-14:A1), and an NAD+-requiring aldehyde dehydrogenase (E/Z11-14:A1 --, E/Z11-14:Acid)--were all found in glandular and other tissues using a novel bioluminescence assay (Meighen et al., 1981) and confirmed using labeled saturated analogs. Finally, the dodecatrienol trail pheromone of Reticulitermes undergoes an co-oxidation to the 1,12-diol as determined for monoene and diene analogs (Prestwich et al., 1985; G. Prestwich, C. Sack, and J.J. Brown, unpublished results). This conversion results in defluorination of (3Z, 6Z)-12-ftuorododecadien-l-ol, and the monooxygenase system is currently under investigation in our laboratories. METHODS AND MATERIALS
Animals and Enzyme Sources Heliothis virescens were purchased from the Cotton Foundation (Memphis, Tennessee) as pupae, sexed, and reared to adult eclosion in separate 8-oz containers in a 16:8 light-dark photoperiod. At 2 hr after lights off, adults were anesthetized with CO2 and the antennae (male and female), legs, (male and female), extruded pheromone gland (female) and extruded hairpencil-genital organ complex (male) were excised and stored in separate microfuge tubes (tissue of 5-10 animals per tube) at - 9 0 ~ until required for enzyme assays. Tissue homogenates were prepared following the procedure illustrated for antennal tissue. Twelve pairs of male (female) antennae were placed in a conical ground-glass homogenizer containing a few milligrams of glass beads (0.15 mm diameter); 100txl of pH 7.4 phosphate buffer (76 mM sodium phosphate) was added, and a motor driven pestle was used to homogenize tissue at 4~ for 5 rain. Another 100 tzl buffer was added, and the mixture was homogenized again. The solution and rinsings were combined in a microfuge tube to give about 12 pairs of antenna per 300/xl of buffer, and then centrifuged at 12,000 g for 10 min at 4~ The soluble fraction (S) was removed and 300 txl of a detergent buffer solution (76 mM sodium phosphate, pH 7.4, 1 mM dithiothreitol (DDT), 1 mM EDTA, and 0.1% w/v Triton X-100) was added to the first pellet and sonicated for 7 rain at 4~ using a Sonifer cell disrupter (model 185) operating at 50-60 W. After centrifugation at 12,000g for 10 rain at 4 ~ C, the supernatant (D) was removed. Finally, 300 /~1 of detergent buffer was added, and the pellet was resuspended by vortexing (P). Bioassays for esterase, oxidase, and aldehyde dehydrogenase were performed on each of the three protein samples (S, D, P) to account for all enzyme activity present. Protein solutions were prepared fresh for each day's experiments. Ultrasonic hair-fracture as described by Ferkovich (1981) was also performed to obtain soluble enzymes. Thus, 50 pairs of antennae were placed in
Heliothis PHEROMONEMETABOLISM
415
200 ~1 of phosphate buffer containing 0.5 M sucrose in an ice bath and then mildly sonicated at 4~ using a Heat Systems ultrasonic cleaning bath for 60 rain. These sonicates contained only soluble enzymes at 10,000g and were compared with homogenates for esterase and aldehyde dehydrogenase activities.
Synthesis of pheromone and metabolites [9,10-3Hz](Z)-9-Tetradecenyl Acetate. This was prepared by reductive tritiation of the corresponding 9-alkyne, prepared in turn by alkylation of the acetylide of 1-tetrahydropyranyloxy-8-bromooctane with 1-hexyne in THF/ HMPA (Henrick, 1977). Acid hydrolysis of the THP ether (CH3CO2H-THFH20, 4:2:1), acetylation (Ac20 , Py), and flash chromatography provided the necessary starting material. The tritiation was carried out at the NIH Tritiation Facility at Lawrence Berkeley Laboratory. To a sample of 10 mg of 9-tetradecyn-l-yl acetate in 5 ml of hexane was added 3 mg of 5 % Pd/BaSO 4 and 10 ixl of a 100 #g/#l hexane solution of quinoline. The reaction vessel was freeze-degassed, and the reaction was stirred 20-30 min under 1 atm of carrier-free T2 gas. The reaction was stopped at ca. 60 % conversion. The tritium and volatiles were removed in vacuo to reduce the volume by half, and the crude product mixture was centrifuged to remove the catalyst. The supernatant was concentrated under N 2 and chromatographed on 40-m ("flash") silica gel with 3% ethyl acetate-hexane and then on 20% AgNO3-coated flash silica gel with 6% ethyl actate-hexane to give complete separation of the desired [9,10-3H2](Z)-9-tetradecenyl acetate (58 Ci/mmol) from the alkyne precursor. [9,10-3H2](Z)-9-Tetradecenyl Alcohol. A solution of [9,10-3H2](Z)-9-te tradecenyl acetate (approx. 7 mCi) in 150 ~1 of 4:1 CH3OH-3N NaOH was stirred 3 hr at 20~ Then, 300 txl of H20 was added, the mixture was extracted with three 200 #1 portions of 1:1 ether-hexane. The extracts were dried (MgSO4) and concentrated in vacuo to give radiochemically homogeneous [thin-layer chromatography (TLC) scraping, liquid scintillation counting (LSC)] alcohol (approx. 6.3 mCi, 90% yield) which was used without further purification. [9,10-3Hz](Z)-9-Tetradecenal. A sample of [9,10-3Hz](Z)-9-tetradecen-1 ol of approx. 3 mCi was used for this oxidation. A total of 500 ~1 of CH2C12 containing the labeled alcohol was added to a solution of a small crystal of pyridinium dichromate (PDC) in 100 ~1 of CHzC12. The reaction mixture was stirred at room temperature under N 2 for 1-2 hr. The mixture was diluted with 5 vol of ether. This material was first chromatographed to remove the oxidant on flash silica gel with ether in a disposable pipette. After concentration in vacuo, the residue was rechromatographed on flash silica with gradually increasing percentages of ethyl acetate in hexane in a column to give radiochemically homogeneous aldehyde, using a TLC solvent system of hexane-ether-
416
D1NG AND PRESTWICH
acetic acid (90:10:2). The labeled aldehyde was particularly susceptible to chemical decomposition and was used for experiments within four days of preparation.
Enzyme Assays Pheromone Solutions. The radiolabeled substrates used were: [9,t03H2]Z9-14:OH for oxidase assay, [9,10-3H2]Z9-14:A1 for aldehyde dehydrogenase assay, and [9,10-3H2]Z9-14:Ac for esterase assay. Hexane stock solutions were checked for purity by scraping TLC plates and counting by LSC. When the radiochemical purity was < 96 %, repurification by flash chromatography in a Pasteur pipet was performed. Pheromone buffer solutions were prepared fresh for a day's experiments as follows. A hexane solution (200/zl containing 30-60 t~Ci) of labeled substrate was evaporated under N 2 and redissolved in 500/zl of pH 7.4 phosphate buffer, which had been throughly degassed by sonication under reduced pressure followed by sparging with helium. Degassing was crucial to prevent oxidation of the labeled aldehyde to labeled acid in the pheromone buffer. Cofactor Solutions. The oxidized cofactor nicotinamide adenine dinucleotide (oxidized form, NAD +) (19.9 mg) was dissolved in 10 ml of pH 7.4 phosphate buffer to give 3 mM NAD + for use in the oxidase and dehydrogenase assays. Prior experimentation demonstrated a requirement for an oxidized cofactor as well as little difference between NAD + to NADP + (also at 3 raM) as the cofactor for this pheromone dehydrogenase. Aldehyde Dehydrogenase (ALDH) Assay. Fifty microliters of [3H]Z9-14:AI buffer (9 x 10 -8 M) and 50/zl of 3 mM NAD + were added to each of eight 10 x 75-mm borosilicate tubes. At 2-min intervals, 50 tzt of protein solutions (S, D, or P from different tissues) or 50 tA buffer (blank) was added. Each incubation was for 1 hr at 20~ At t = 1 hr, 150/zl of ethyl acetate was added and the tube was vortexed vigorously for 15 sec. A 3-/zl aliquot of the upper layer was removed to determine recovery (by LSC) and another 6 /zl aliquot was spotted on 4 x 8-cm Machery-Nagel Polygram Sil G/UV 254 plates. Plates were divided into four vertical stripes and were prespotted with unlabeled Z914:A1, Z9-14:OH, and Z9-14:Acid. The plates were developed in hexaneether-acetic acid (90:10:2). Spots were visualized with I2, the TLC plate was cut into 1-cm sections, the sections were placed into 7-ml scintillation vials, and the sections counted in 4 ml of Aquasol (New England Nuclear) or Scintiverse II (Fisher). Scintillation counting was performed on a Packard TriCarb Model 3300 instrument operating at 37-45% efficiency for tritium, as determined by automatic external standardization. Alcohol Oxidase Assay. Using [3H]Z9-14:OH as substrate, the same procedure for the dehydrogenase was followed. To test for the dependence of the alcohol oxidase on the presence of oxygen, a modified assay was performed as
Heliothis PHEROMONEMETABOLISM
417
follows. Male legs (22 pairs) were homogenized in pH 7.4 phosphate buffer to get 2 ml of the soluble (S) fraction. An aqueous solution of [sH]Z9-14:OH was made by evaporation of 100 ~tl ( - 0.15 mCi) of the hexane solution of labeled alcohol under N2 and redissolving the residue in 2 ml of pH 7.4 phosphate buffer. Two sets of ten duplicates were prepared: one set of ten was degassed and held under N2, and one set was exposed to air. Into each test tube, 100/~1 of [3H]Z9-14:OH buffer solution and 100 #1 of protein solution were added at 4~ The tubes were transferred to 30~ bath, shaken vigorously for 30 sec, and then incubated for periods of 5, 10, 15, 20, 25, 30, 35, 40, and 45 min. After 15 rain, those tubes which had been flushed with N 2 were exposed to air, vortexed vigorously, and then incubation was continued. At each interval, duplicates of each set were quenched with 200 ~1 of ethyl acetate. A 6-~1 aliquot of upper layer was spotted on TLC, developed, cut, and counted as described above. Acetate Esterase Assay. The assays were the same as described above for the aldehyde dehydrogenase (ALDH), but using [3H]Z9-14:Ac as substrate and replacing the 50 ~1 of NAD + buffer with phosphate buffer. Autoradiography. TLC plates spotted with 6 ~1 of ethyl acetate layers of blank, S, D, and P enzyme incubations were sprayed with En3Hance (New England Nuclear) spray, placed in contact with preflashed Kodak X-Omat XAR5 X-ray film for 4 hr to 4 days at - 8 0 ~ and developed as usual with Kodak GBX chemicals. Protein Concentrations. These were determined by the dye-binding method (Bradford, 1976) using bovine serum albumin as the standard. RESULTS AND DISCUSSION Pheromone and derivatives (Figure 1) were synthesized at high specific activity by partial reductive tritiation of 9-tetradecyn-l-yl acetate on poisoned Pd/BaSO4, affording the [9,10-3H2] (Z) -9 -tetradecen-1-yl acetate ([3H] Z9-14:Ac) at a nominal specific activity of 58 Ci/mmol. The location and extent of the tritium labels is assumed, although [3H]NMR examination is in progress. Hydrolysis with aqueous methanolic sodium hydroxide on a micromole scale gave corresponding [3H]Z9-14:OH. Oxidation of this alcohol to [3H]Z9-14:A1 was generally performed on a 3-mCi (52-nmol) scale and required careful monitoring to avoid overoxidation or conversion to other products. Several attempts with pyridinium chlorochromate (PCC) in dichloromethane led to undesired labeled materials intermediate in TLC polarity between the alcohol and aldehyde. It was not readily controlled by decreasing the amount of oxidant. However, use of pyridinium dichromate (PDC) in CH2C12 under N2, even in > 100fold excess, gave a controlled conversion to the labeled aldehyde in 2 hr at 20~
418
DING A N D
PRESTWrCH
OAc
T2iPd/BaSO 4
T
~
~
~
PDC,o~H2CI 2 02, oxldase
0
T
T
T
[ aq. Na0H/Clt30Heseerase~
. / N . / " x ~ . X ~ Y
T
T
0X
Y
Aldehyde dehydrogenase
[ FDClD.~
OH
Flo. 1. Synthesis of labeled substrates and enzymic interconversions (T
=
3H).
All labeled materials were purified by flash chromatography over silica gel in a disposable pipet using hexane-ethyl acetate eluents. The labeled aldehyde was never more than one week old, and never more than 4 hr in aqueous working solutions. Failure to degas aqueous buffers used for pheromone stocks or incubations led to high "background," i.e., nonenzymic conversion to the carboxylic acid. Tissue homogenates were prepared fresh each day from tissues stored at - 9 0 ~ and used completely. This eliminated problems due to using enzyme solutions of variable age and subject to freeze-thaw sequences. The tissues were homogenized first in a phosphate buffer lacking additives, and the pellet was rehomogenized and ultrasonicated in a detergent buffer containing EDTA, dithiothreitol (DDT), and Triton X-100 to solubilize membrane-associated proteins. As discussed below, from the tissue distributions of esterase, oxidase, and dehydrogenase activities, it appears that essentially all enzyme activities are readily solubilized in buffer or in a very mild nonionic detergent. Only a small (but reproducible) residual activity of aldehyde dehydrogenase is found in the pellet fraction from female antennae. All enzyme assays were performed with no-carrier-added substrate solutions at 10-8-10 -9 M, i.e., using a physiologically meaningful concentration of substrate. At least three repetitions per enzyme assay have been performed with separate tissue preparations and reproducible results have been obtained. The data presented in the tables and figures often illustrate a representative result as the average of duplicate subsamples for a given tissue preparation. Using the TLC assay, we could readily detect differences of 1-2% conversion (100-200 cpm out of 10,000 cpm). Figure 2 shows a set of autoradiograms of TLC plates to allow visualization of three enzyme activities for each of three extraction methods from both male and female leg tissue. Autoradiograms for antennal and glandular tissues appear quite similar and are omitted here; the quantative data for enzyme activ-
Heliothis PHEROMONEMETABOLISM
419
ity appear in Figure 3 below. The autoradiograms provide evidence for: (1) conversion of [3H]Z9-14:AI to [3H]Z9_ 14:Acid by the aldehyde dehydrogenase (ALDH), (2) conversion of [3H]Z9-14:OH to the aldehyde by the alcohol oxidase (OX) with further transformation to the carboxylic acid by ALDH, and (3) hydrolysis of [3H]Z9-14:Ac to the labeled alcohol by the esterase (EST) with subsequent transformations by OX and ALDH to the labeled acid. The autoradiograms demonstrate clearly the high level of radiochemical purity for each starting substrate (see blank B lanes). It also appears that the soluble aldehyde dehydrogenase is the most active enzyme in both sexes (documented below), giving nearly quantitative conversion of aldehyde to carboxylic acid. This occurs whether the aldehyde is added as substrate or is produced in vitro from oxidase on Z9-14:OH or combined esterase-oxidase action on Z9-14:Ac. Only in the detergent fraction for the esterase assay do we observe simple conversion of Z9-14:Ac to Z9-14:OH, since the OX activity has been removed and the ALDH activity is low. As seen in Table 1, the enzymatic hydrolysis of [3H]Z9-14:Ac to the alcohol is rapidly lost by dilution below 100 antennal equivalents (AE) per milliliter of homogenate. However, the aldehyde dehydrogenase activity was still detectable as low as 0.8 AE/ml. The oxidase activity appeared to be lower than the aldehyde dehydrogenase activity. Thus, in both male and female antennal tissues, the aldehyde debydrogenase is the most active enzyme, consistent with its postulated importance in pheromone removal. Any aldehyde produced via oxidase action is thus rapidly converted to carboxylic acid as seen in Figure 2. Comparison of the homogenization method and hair-fracture sonic bath method (Ferkovich, 1981) for obtaining antennal enzymes (Table 2) showed that essentially all the esterase and, indeed, a greater level of aldehyde dehydrogenase could be obtained by this milder treatment. Moreover, the quantity of cuticular material is considerably reduced. Mechanical fracture of lyophilized sensory hairs from the antennal branches used in collecting the larger (100-300 ~m long) sensilla of Antheraea polyphemus gave protein solutions free of hemolymph and antennal branch-cuticle contamination (Vogt and Riddiford, 1981; Prestwich et al., 1986). However, this mechanical fracture/hair separation method was not readily transferred to the filiform antennae with 10 to 30-#m-long sensory hairs, such as those of male and female H. virescens moths. Thus, our assay results show total enzyme activity present in cuticular, hemolymph, and sensillar proteins of the antennae. The use of leg tissue is meant to control for the presence of general enzyme activities found in the cuticle and hemolymph. Figure 3 illustrates the distribution of enzyme activities present in Heliothis moth tissues. The key features are : (1) Aldehyde dehydrogenase is the primary enzyme activity found in both leg and antennal tissues of males and females. (2) Oxidase activity is low in antennae, but higher in male legs and glandular
420
DING AND PREs'rwicri
la.i ._1
ALD H
0X
EST
F~c5. 2. Autoradiograms of TLC plates illustrating interconversions of [3H]Z9-14:Ac (Ac) and the corresponding alcohol (OH), aldehyde (A1), and carboxylic acid (CA) by male (top) and female (bottom) leg tissues. The letters B, S, D, and P denote blanks, soluble, detergent, and pellet activities, respectively (see text lbr details); ALDH = aldehyde dehydrogenase, OX = alcohol oxidase, EST = acetate esterase.
FEMALE
4a b3
9
rd
9 Z
9
422
DING AND PRESTWICH TABLE 1. DILUTION SERIES FOR SOLUBLE ANTENNAL ENZYMES a
Aldehyde dehydrogenase (%)d
Esterase ( % y Antennal equivalent per milliliter solutionb
Male
Female
Male
Female
360 120 80 26.67 8.0 2.67 0.8 0.267
27 10 0 ------
30 5 0 ------
--62 50 50 31 10 0
--53 33 33 15 15 0
Percent conversion to product, expressed as means of duplicate assays. bA homogenate of male or female antennae of 350 AE/ml has 0.34 mg/ml total protein for the esterase. For the 80 AE/ml used for aldehyde dehydrogenase assays, males had 0.087 mg/ml and females 0.074 mg/ml total protein. c One-hour incubation. d Fifty-minute incubation.
tissues of both sexes. (3) Esterase activity is low in antennae, higher in legs, and highest glandular tissue of both sexes. (4) Males and females show all three enzymic activities in all tissues. Striking male-female differences include (a) a pellet-associated aldehyde dehydrogenase in female antennae; (b) relatively lower oxidase activity in female legs and glandular tissue, and (c) relatively higher esterase and dehydrogenase activity in female glandular tissues. Figure 4 illustrates the results of the experiment which demonstrated that the oxidase from male legs (the highest activity source) is an O2-requiring enTABLE 2. COMPARISON OF HOMOGENIZATION AND ULTRASONIC AGITATION FOR OBTAINING ENZYMES a
Aldehyde dehydrogenase"
Esteraseb
Homogenate Ultrasonicate
Male
Female
Male
Female
28 33
30 35
22 59
35 61
aConversions expressed as percent product from duplicate assays. hFifty pair of antennae to get 200 ~1 of soluble fraction. Protein concentrations for homogenate: males, 0.39 mg/ml; females, 0.45 mg/ml. Protein concentrations for ultrasonicate: males, 0.47 mg/ml; females, 0.47 mg/ml. "Twelve pair of antennae to get 300/zl of soluble fraction. Protein concentrations: males, 0.022 mg/ml; females, 0.035 mg/ml.
Heliothis
PHEROMONE
423
METABOLISM
Hel/oth/s v/rescens moths: Pheromone processing by tissues MALES 80' '
Antennae
Legs
Hair Pencils
t-
.s
60
> cO
4.0
~O_
20
(D
-
I
S
E
0
AD
E
0
AD
E
0
AD
FEMALES Antenna,
80--
P Legs
c o
60--
Glands
D
O9 (33 >
c o (D
40
o
20-
I I
I
S
~D
o
;
E
0
AD
E Enzyme
0
AD
E
0
AD
Activity
FIG. 3. Morphological distribution of acetate esterase (E), alcohol oxidase (O), and aldehyde dehydrogenase (AD) activities in adult moths (Heliothis virescens). All incubations were run for 1 hr at 20~ and values shown are means of percent conversion for duplicates. Homogenates of two pairs of antennae and legs per 50/xl were used and two pheromone glands or hairpencils per 50/~1 were used. Enzyme activities are shown for soluble (S, open bars), detergent-solubilized (D, solid bars), and insoluble pellet (P, cross-hatched) fractions.
0
4q
6
10
12
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30 35 40 45
Incubation Time,
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0
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dependence
leg alcohol
v/'rescens moths
I 10
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30
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Time,
l_____L ~ 15 20 25
Incubation
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FIG. 4. Dependence of alcohol oxidase of legs of male Heliothis virescens moths on the presence of oxygen, Samples were incubated in air (circles) or under N: for 15 rain followed by air (triangles). The left panel shows only conversion of [3H]Z9 14:OH to the aldehyde, while the right panel shows conversion to the aldehyde plus carboxlic acid.
el_
METABOLIC TRANSFORMATION OF TRITIUMLABELED PHEROMONE BY TISSUES OF Heliothis virescens MOTHS
Y U - S H I N D I N G and G L E N N
D. P R E S T W I C H 1
Department of Chemistry State University of New York Stony Brook, New York, 11794-3400 (Received April 26, 1985; accepted August 1, 1985) Abstract--Unsaturated aliphatic pheromones of H. virescens were prepared at high specific activity (3H, 58 Ci/mmol) and were employed to study tissue specificity of acetate esterase, alcohol oxidase, and aldehyde dehydrogenase in male and female Heliothis virescens. Thus, [9,10-3H~]Z9- 14:Ac was synthesized by partial tritiation of the corresponding alkyne and was converted to the labeled Z9-14:OH and Z9-14:AI for metabolic studies. Soluble and membrane-associated enzyme activities were determined by radio-TLC assays. Esterase activity is highest in legs of both sexes, but also occurs in antennal and glandular tissues. Oxidase activity requires 02 and is highest in female pheromone gland tissues, but it is also high in the male hairpencils. Aldehyde dehydrogenase activity was uniformly high in all tissues, but highest in antennal tissues of both males and females. Key Words--Pheromone metabolism, tritium-labeling, acetate esterase, alcohol oxidase, aldehyde dehydrogenase, Heliothis virescens, Lepidoptera, Noctuidae, sensory biochemistry.
INTRODUCTION P h e r o m o n e b i o s y n t h e s i s , p h e r o m o n e perception, and p h e r o m o n e c a t a b o l i s m are closely linked in terms o f m a t e - f i n d i n g b e h a v i o r s o f m o t h s (review: M a y e r and M a n k i n , 1985). In this paper, w e will present e x p e r i m e n t a l details on the phero m o n e - m e t a b o l i z i n g e n z y m e s o f Heliothis virescens, using h i g h - s p e c i f i c - a c t i v ity, t r i t i u m - l a b e l e d Z 9 - 1 4 : A 1 (and its c o r r e s p o n d i n g a l c o h o l and acetate). First, ~Fellow of the Alfred P. Sloan Foundation (1981-1985) and Camille and Henry Dreyfus TeacherScholar (1981-1986). 411 0098 0331/86/0200-0411505.00/0 9 1986 Plenum Publishing Corporation
412
DINGAND PRESTW1CH
however, we will survey pheromone biochemistry concisely (general review: Blomquist and Dillwith, 1983; catabolism: Ferkovich, 1981; biosynthesis: Roelofs and Bjostad, 1984). Additional recent results will be found in this symposium edition on pheromone biosynthesis (Bjostad and Roelofs, 1986; Teal and Tumlinson, 1986; Morse and Meighen, 1986) and pheromone catabolism (Lonergan, 1986; Vogt and Riddiford, 1986; Prestwich et al. 1986). Catabolism of pheromones has been examined in only seven insects: Bom-
byx mori, Lymantria dispar, Trichoplusia ni, Antheraea polyphemus, Choristoneura fumiferana, Heliothis virescens, and Reticulitermes flavipes. The silkworm moth, Bombyx mori, produces bombykol (El0, Z12-16:OH) which is converted to "ester" and " a c i d " metabolites by whole male antennae exposed to labeled bombykol (Kasang, 1974). The [12,13-3H]-labeled bombykol (30 Ci/ mmol) and its tetrahydroisomer were prepared by partial tritiation of the 12 triple bond. At exposures of 101~ molecules/antenna, the half-life for "uptake" was 1.6 min for male and 1.0 min for female antennae. Much slower uptake was found in legs, heads, and wing scales which lack the pore tubules. Conversion from the alcohol to the corresponding labeled bombic acid and an unidentified ester, isolated by preparative TLC, showed a 4-rain half-life. Chemical identifications were not performed, and no attempts to manipulate or purify the binding or catabolic proteins were reported. The Seeweisen group also did preliminary work on disparlure biosynthesis (Kasang et al., 1974a) from the (Z)-[7,8-3H]-2-methyl-7-octadecene (Sheads and Beroza, 1973) in female gypsy moth pupae and on racemic disparlure uptake and turnover (Kasang et al., 1974b) by male (and female) L. dispar antennae. In the former, aqueous ethanol suspensions of the labeled alkene were injected into female pupae two days preemergence and 34 % of [3H]disparlure (TLC) was recovered from female glands postemergence. There was no direct evidence of pheromone gland involvement in this conversion, however, and in view of the diel periodicity, life cycle and wild vs. lab variation of disparlure release and production (Charlton and Cardr, 1983; Richerson and Cameron, 1974), this work needs to be repeated on pheromone gland homogenates and with topical application of precursors to the gland surface. Such techniques have been used sucessfully to investigate the biosynthesis of Z11-14:Ac (Bjostad and Roelofs, 1981; Bjostad et al., 1981) in the red-banded leafroller moth and bombykol biosynthesis in silkworm moths (Yamaoka et al., 1984). The disparlure metabolic studies are also very sketchy. At 5 x 1011 molecules/antenna of racemic [7,8-3Hz]disparlure (40 Ci/mmol), about 2.5 min was required for disappearance of half the substrate and appearance of two uncharacterized polar metabolites (50%) as well as > 30% of non-TLC-mobile material. Claims for specificity vs. nonspecificity of uptake and turnover are based on small differences superimposed on an exponential decay curve and are not convincing. Also, it is difficult to evaluate these data since both the attractive
Heliothis PHEROMONEMETABOLISM
413
(+) and inhibitory ( - ) enantiomers were present. It is essential to distinguish binding and catabolism of the two enantiomeric forms, since the proteins involved reside in seperate sensory cells (Hansen, 1984). The in vivo and in vitro metabolism of Z7-12:Ac by Trichoplusia ni has been reported by Ferkovich, Mayer, and their coworkers in Gainesville (Ferkovich, 1981; Ferkovich et al., 1973; 1982a,b; Mayer et al., 1976). The acetate is converted to the corresponding alcohol by both specific and general esterases present in legs, wing scales, and antennae. The two primary roles of these esterases are to prevent sensory adaptation and to clear the body surface. The pheromone-specific catabolic proteins appear to show lower specificity for exact pheromone structures than do the receptors (measured behaviorally or by EAG or single cell), although they do show reduced susceptibility to general esterase inhibitors. This hydrolysis was demonstrated using (1) intact antennae and legs dipped in to a 10 -3 M emulsion of pheromone in buffer; (2) in vitro homogenates of antennae, legs, cuticle, wings, and fat body; and (3) soluble proteins from the sensillum liquor of sonicated antennae. The homogenates were assayed with [3H-acetate]Z7-12:Ac (200-800 mCi/mmol) at 10-6-10 -5 M, which is a much higher concentration ( > 1 0 0 0 • and a much lower specific activity (300 x) than for metabolic studies of bombykol, disparlure, and E6,Z 11-16:Ac (see below). Nonetheless, conversion rates in the order of 6 pmol/~g protein/ min in antennae were observed. Pheromone esterase activities decrease in the order of antennae > legs > wings in homogenates and wings > legs > antennae in eluates, consistent with both the postulated sensory deactivation and surface-cleaning functions. The most thorough biochemical analysis of a pheromone binding and inactivation system is reported for Antheraea polyphemus (Vogt, 1984; Vogt and Riddiford, 1980, 1981, 1985; Vogt et al., 1985; Prestwich et al., 1984, 1986). Using [11, 12-3Hz]E6,Zll-16:Ac (40 or 58 Ci/mmol), three electrophoretically distinct proteins were found which interact with pheromone: a nonenzymatic sensillar pheromone-binding protein (mol wt 15,000), a pheromone-hydrolyzing sensillar esterase (tool wt 55,000), and a second esterase in cuticular tissues (e.g., legs and wing scales) of males and females (Vogt and Riddiford, 1986). Oddly, an electrophoretically identical "binding protein" (mol wt 15,000) was also found in three other saturniids and a sphyngid with different pheromones, in unusually high concentrations in receptor lymph (approx. 105 molecules/sensillum) (Vogt and Riddiford, 1980). The sensillar esterase shows considerable specificity for acetates with Z-olefinic linkages and a very high Vma• over a large concentration range. The studies on Choristoneura fumiferana (Morse and Meighen, 1984a,b, 1986) show the importance of studying multiple enzyme systems in different tissues of males and females. Three activities--an acetate esterase (E/Z1114:Ac ~ E/Zll-14:OH), and O2-requiring alcohol oxidase (E/Z11-14:OH
414
DING AND PRESTWICH
E/Z11-14:A1), and an NAD+-requiring aldehyde dehydrogenase (E/Z11-14:A1 --, E/Z11-14:Acid)--were all found in glandular and other tissues using a novel bioluminescence assay (Meighen et al., 1981) and confirmed using labeled saturated analogs. Finally, the dodecatrienol trail pheromone of Reticulitermes undergoes an co-oxidation to the 1,12-diol as determined for monoene and diene analogs (Prestwich et al., 1985; G. Prestwich, C. Sack, and J.J. Brown, unpublished results). This conversion results in defluorination of (3Z, 6Z)-12-ftuorododecadien-l-ol, and the monooxygenase system is currently under investigation in our laboratories. METHODS AND MATERIALS
Animals and Enzyme Sources Heliothis virescens were purchased from the Cotton Foundation (Memphis, Tennessee) as pupae, sexed, and reared to adult eclosion in separate 8-oz containers in a 16:8 light-dark photoperiod. At 2 hr after lights off, adults were anesthetized with CO2 and the antennae (male and female), legs, (male and female), extruded pheromone gland (female) and extruded hairpencil-genital organ complex (male) were excised and stored in separate microfuge tubes (tissue of 5-10 animals per tube) at - 9 0 ~ until required for enzyme assays. Tissue homogenates were prepared following the procedure illustrated for antennal tissue. Twelve pairs of male (female) antennae were placed in a conical ground-glass homogenizer containing a few milligrams of glass beads (0.15 mm diameter); 100txl of pH 7.4 phosphate buffer (76 mM sodium phosphate) was added, and a motor driven pestle was used to homogenize tissue at 4~ for 5 rain. Another 100 tzl buffer was added, and the mixture was homogenized again. The solution and rinsings were combined in a microfuge tube to give about 12 pairs of antenna per 300/xl of buffer, and then centrifuged at 12,000 g for 10 min at 4~ The soluble fraction (S) was removed and 300 txl of a detergent buffer solution (76 mM sodium phosphate, pH 7.4, 1 mM dithiothreitol (DDT), 1 mM EDTA, and 0.1% w/v Triton X-100) was added to the first pellet and sonicated for 7 rain at 4~ using a Sonifer cell disrupter (model 185) operating at 50-60 W. After centrifugation at 12,000g for 10 rain at 4 ~ C, the supernatant (D) was removed. Finally, 300 /~1 of detergent buffer was added, and the pellet was resuspended by vortexing (P). Bioassays for esterase, oxidase, and aldehyde dehydrogenase were performed on each of the three protein samples (S, D, P) to account for all enzyme activity present. Protein solutions were prepared fresh for each day's experiments. Ultrasonic hair-fracture as described by Ferkovich (1981) was also performed to obtain soluble enzymes. Thus, 50 pairs of antennae were placed in
Heliothis PHEROMONEMETABOLISM
415
200 ~1 of phosphate buffer containing 0.5 M sucrose in an ice bath and then mildly sonicated at 4~ using a Heat Systems ultrasonic cleaning bath for 60 rain. These sonicates contained only soluble enzymes at 10,000g and were compared with homogenates for esterase and aldehyde dehydrogenase activities.
Synthesis of pheromone and metabolites [9,10-3Hz](Z)-9-Tetradecenyl Acetate. This was prepared by reductive tritiation of the corresponding 9-alkyne, prepared in turn by alkylation of the acetylide of 1-tetrahydropyranyloxy-8-bromooctane with 1-hexyne in THF/ HMPA (Henrick, 1977). Acid hydrolysis of the THP ether (CH3CO2H-THFH20, 4:2:1), acetylation (Ac20 , Py), and flash chromatography provided the necessary starting material. The tritiation was carried out at the NIH Tritiation Facility at Lawrence Berkeley Laboratory. To a sample of 10 mg of 9-tetradecyn-l-yl acetate in 5 ml of hexane was added 3 mg of 5 % Pd/BaSO 4 and 10 ixl of a 100 #g/#l hexane solution of quinoline. The reaction vessel was freeze-degassed, and the reaction was stirred 20-30 min under 1 atm of carrier-free T2 gas. The reaction was stopped at ca. 60 % conversion. The tritium and volatiles were removed in vacuo to reduce the volume by half, and the crude product mixture was centrifuged to remove the catalyst. The supernatant was concentrated under N 2 and chromatographed on 40-m ("flash") silica gel with 3% ethyl acetate-hexane and then on 20% AgNO3-coated flash silica gel with 6% ethyl actate-hexane to give complete separation of the desired [9,10-3H2](Z)-9-tetradecenyl acetate (58 Ci/mmol) from the alkyne precursor. [9,10-3H2](Z)-9-Tetradecenyl Alcohol. A solution of [9,10-3H2](Z)-9-te tradecenyl acetate (approx. 7 mCi) in 150 ~1 of 4:1 CH3OH-3N NaOH was stirred 3 hr at 20~ Then, 300 txl of H20 was added, the mixture was extracted with three 200 #1 portions of 1:1 ether-hexane. The extracts were dried (MgSO4) and concentrated in vacuo to give radiochemically homogeneous [thin-layer chromatography (TLC) scraping, liquid scintillation counting (LSC)] alcohol (approx. 6.3 mCi, 90% yield) which was used without further purification. [9,10-3Hz](Z)-9-Tetradecenal. A sample of [9,10-3Hz](Z)-9-tetradecen-1 ol of approx. 3 mCi was used for this oxidation. A total of 500 ~1 of CH2C12 containing the labeled alcohol was added to a solution of a small crystal of pyridinium dichromate (PDC) in 100 ~1 of CHzC12. The reaction mixture was stirred at room temperature under N 2 for 1-2 hr. The mixture was diluted with 5 vol of ether. This material was first chromatographed to remove the oxidant on flash silica gel with ether in a disposable pipette. After concentration in vacuo, the residue was rechromatographed on flash silica with gradually increasing percentages of ethyl acetate in hexane in a column to give radiochemically homogeneous aldehyde, using a TLC solvent system of hexane-ether-
416
D1NG AND PRESTWICH
acetic acid (90:10:2). The labeled aldehyde was particularly susceptible to chemical decomposition and was used for experiments within four days of preparation.
Enzyme Assays Pheromone Solutions. The radiolabeled substrates used were: [9,t03H2]Z9-14:OH for oxidase assay, [9,10-3H2]Z9-14:A1 for aldehyde dehydrogenase assay, and [9,10-3H2]Z9-14:Ac for esterase assay. Hexane stock solutions were checked for purity by scraping TLC plates and counting by LSC. When the radiochemical purity was < 96 %, repurification by flash chromatography in a Pasteur pipet was performed. Pheromone buffer solutions were prepared fresh for a day's experiments as follows. A hexane solution (200/zl containing 30-60 t~Ci) of labeled substrate was evaporated under N 2 and redissolved in 500/zl of pH 7.4 phosphate buffer, which had been throughly degassed by sonication under reduced pressure followed by sparging with helium. Degassing was crucial to prevent oxidation of the labeled aldehyde to labeled acid in the pheromone buffer. Cofactor Solutions. The oxidized cofactor nicotinamide adenine dinucleotide (oxidized form, NAD +) (19.9 mg) was dissolved in 10 ml of pH 7.4 phosphate buffer to give 3 mM NAD + for use in the oxidase and dehydrogenase assays. Prior experimentation demonstrated a requirement for an oxidized cofactor as well as little difference between NAD + to NADP + (also at 3 raM) as the cofactor for this pheromone dehydrogenase. Aldehyde Dehydrogenase (ALDH) Assay. Fifty microliters of [3H]Z9-14:AI buffer (9 x 10 -8 M) and 50/zl of 3 mM NAD + were added to each of eight 10 x 75-mm borosilicate tubes. At 2-min intervals, 50 tzt of protein solutions (S, D, or P from different tissues) or 50 tA buffer (blank) was added. Each incubation was for 1 hr at 20~ At t = 1 hr, 150/zl of ethyl acetate was added and the tube was vortexed vigorously for 15 sec. A 3-/zl aliquot of the upper layer was removed to determine recovery (by LSC) and another 6 /zl aliquot was spotted on 4 x 8-cm Machery-Nagel Polygram Sil G/UV 254 plates. Plates were divided into four vertical stripes and were prespotted with unlabeled Z914:A1, Z9-14:OH, and Z9-14:Acid. The plates were developed in hexaneether-acetic acid (90:10:2). Spots were visualized with I2, the TLC plate was cut into 1-cm sections, the sections were placed into 7-ml scintillation vials, and the sections counted in 4 ml of Aquasol (New England Nuclear) or Scintiverse II (Fisher). Scintillation counting was performed on a Packard TriCarb Model 3300 instrument operating at 37-45% efficiency for tritium, as determined by automatic external standardization. Alcohol Oxidase Assay. Using [3H]Z9-14:OH as substrate, the same procedure for the dehydrogenase was followed. To test for the dependence of the alcohol oxidase on the presence of oxygen, a modified assay was performed as
Heliothis PHEROMONEMETABOLISM
417
follows. Male legs (22 pairs) were homogenized in pH 7.4 phosphate buffer to get 2 ml of the soluble (S) fraction. An aqueous solution of [sH]Z9-14:OH was made by evaporation of 100 ~tl ( - 0.15 mCi) of the hexane solution of labeled alcohol under N2 and redissolving the residue in 2 ml of pH 7.4 phosphate buffer. Two sets of ten duplicates were prepared: one set of ten was degassed and held under N2, and one set was exposed to air. Into each test tube, 100/~1 of [3H]Z9-14:OH buffer solution and 100 #1 of protein solution were added at 4~ The tubes were transferred to 30~ bath, shaken vigorously for 30 sec, and then incubated for periods of 5, 10, 15, 20, 25, 30, 35, 40, and 45 min. After 15 rain, those tubes which had been flushed with N 2 were exposed to air, vortexed vigorously, and then incubation was continued. At each interval, duplicates of each set were quenched with 200 ~1 of ethyl acetate. A 6-~1 aliquot of upper layer was spotted on TLC, developed, cut, and counted as described above. Acetate Esterase Assay. The assays were the same as described above for the aldehyde dehydrogenase (ALDH), but using [3H]Z9-14:Ac as substrate and replacing the 50 ~1 of NAD + buffer with phosphate buffer. Autoradiography. TLC plates spotted with 6 ~1 of ethyl acetate layers of blank, S, D, and P enzyme incubations were sprayed with En3Hance (New England Nuclear) spray, placed in contact with preflashed Kodak X-Omat XAR5 X-ray film for 4 hr to 4 days at - 8 0 ~ and developed as usual with Kodak GBX chemicals. Protein Concentrations. These were determined by the dye-binding method (Bradford, 1976) using bovine serum albumin as the standard. RESULTS AND DISCUSSION Pheromone and derivatives (Figure 1) were synthesized at high specific activity by partial reductive tritiation of 9-tetradecyn-l-yl acetate on poisoned Pd/BaSO4, affording the [9,10-3H2] (Z) -9 -tetradecen-1-yl acetate ([3H] Z9-14:Ac) at a nominal specific activity of 58 Ci/mmol. The location and extent of the tritium labels is assumed, although [3H]NMR examination is in progress. Hydrolysis with aqueous methanolic sodium hydroxide on a micromole scale gave corresponding [3H]Z9-14:OH. Oxidation of this alcohol to [3H]Z9-14:A1 was generally performed on a 3-mCi (52-nmol) scale and required careful monitoring to avoid overoxidation or conversion to other products. Several attempts with pyridinium chlorochromate (PCC) in dichloromethane led to undesired labeled materials intermediate in TLC polarity between the alcohol and aldehyde. It was not readily controlled by decreasing the amount of oxidant. However, use of pyridinium dichromate (PDC) in CH2C12 under N2, even in > 100fold excess, gave a controlled conversion to the labeled aldehyde in 2 hr at 20~
418
DING A N D
PRESTWrCH
OAc
T2iPd/BaSO 4
T
~
~
~
PDC,o~H2CI 2 02, oxldase
0
T
T
T
[ aq. Na0H/Clt30Heseerase~
. / N . / " x ~ . X ~ Y
T
T
0X
Y
Aldehyde dehydrogenase
[ FDClD.~
OH
Flo. 1. Synthesis of labeled substrates and enzymic interconversions (T
=
3H).
All labeled materials were purified by flash chromatography over silica gel in a disposable pipet using hexane-ethyl acetate eluents. The labeled aldehyde was never more than one week old, and never more than 4 hr in aqueous working solutions. Failure to degas aqueous buffers used for pheromone stocks or incubations led to high "background," i.e., nonenzymic conversion to the carboxylic acid. Tissue homogenates were prepared fresh each day from tissues stored at - 9 0 ~ and used completely. This eliminated problems due to using enzyme solutions of variable age and subject to freeze-thaw sequences. The tissues were homogenized first in a phosphate buffer lacking additives, and the pellet was rehomogenized and ultrasonicated in a detergent buffer containing EDTA, dithiothreitol (DDT), and Triton X-100 to solubilize membrane-associated proteins. As discussed below, from the tissue distributions of esterase, oxidase, and dehydrogenase activities, it appears that essentially all enzyme activities are readily solubilized in buffer or in a very mild nonionic detergent. Only a small (but reproducible) residual activity of aldehyde dehydrogenase is found in the pellet fraction from female antennae. All enzyme assays were performed with no-carrier-added substrate solutions at 10-8-10 -9 M, i.e., using a physiologically meaningful concentration of substrate. At least three repetitions per enzyme assay have been performed with separate tissue preparations and reproducible results have been obtained. The data presented in the tables and figures often illustrate a representative result as the average of duplicate subsamples for a given tissue preparation. Using the TLC assay, we could readily detect differences of 1-2% conversion (100-200 cpm out of 10,000 cpm). Figure 2 shows a set of autoradiograms of TLC plates to allow visualization of three enzyme activities for each of three extraction methods from both male and female leg tissue. Autoradiograms for antennal and glandular tissues appear quite similar and are omitted here; the quantative data for enzyme activ-
Heliothis PHEROMONEMETABOLISM
419
ity appear in Figure 3 below. The autoradiograms provide evidence for: (1) conversion of [3H]Z9-14:AI to [3H]Z9_ 14:Acid by the aldehyde dehydrogenase (ALDH), (2) conversion of [3H]Z9-14:OH to the aldehyde by the alcohol oxidase (OX) with further transformation to the carboxylic acid by ALDH, and (3) hydrolysis of [3H]Z9-14:Ac to the labeled alcohol by the esterase (EST) with subsequent transformations by OX and ALDH to the labeled acid. The autoradiograms demonstrate clearly the high level of radiochemical purity for each starting substrate (see blank B lanes). It also appears that the soluble aldehyde dehydrogenase is the most active enzyme in both sexes (documented below), giving nearly quantitative conversion of aldehyde to carboxylic acid. This occurs whether the aldehyde is added as substrate or is produced in vitro from oxidase on Z9-14:OH or combined esterase-oxidase action on Z9-14:Ac. Only in the detergent fraction for the esterase assay do we observe simple conversion of Z9-14:Ac to Z9-14:OH, since the OX activity has been removed and the ALDH activity is low. As seen in Table 1, the enzymatic hydrolysis of [3H]Z9-14:Ac to the alcohol is rapidly lost by dilution below 100 antennal equivalents (AE) per milliliter of homogenate. However, the aldehyde dehydrogenase activity was still detectable as low as 0.8 AE/ml. The oxidase activity appeared to be lower than the aldehyde dehydrogenase activity. Thus, in both male and female antennal tissues, the aldehyde debydrogenase is the most active enzyme, consistent with its postulated importance in pheromone removal. Any aldehyde produced via oxidase action is thus rapidly converted to carboxylic acid as seen in Figure 2. Comparison of the homogenization method and hair-fracture sonic bath method (Ferkovich, 1981) for obtaining antennal enzymes (Table 2) showed that essentially all the esterase and, indeed, a greater level of aldehyde dehydrogenase could be obtained by this milder treatment. Moreover, the quantity of cuticular material is considerably reduced. Mechanical fracture of lyophilized sensory hairs from the antennal branches used in collecting the larger (100-300 ~m long) sensilla of Antheraea polyphemus gave protein solutions free of hemolymph and antennal branch-cuticle contamination (Vogt and Riddiford, 1981; Prestwich et al., 1986). However, this mechanical fracture/hair separation method was not readily transferred to the filiform antennae with 10 to 30-#m-long sensory hairs, such as those of male and female H. virescens moths. Thus, our assay results show total enzyme activity present in cuticular, hemolymph, and sensillar proteins of the antennae. The use of leg tissue is meant to control for the presence of general enzyme activities found in the cuticle and hemolymph. Figure 3 illustrates the distribution of enzyme activities present in Heliothis moth tissues. The key features are : (1) Aldehyde dehydrogenase is the primary enzyme activity found in both leg and antennal tissues of males and females. (2) Oxidase activity is low in antennae, but higher in male legs and glandular
420
DING AND PREs'rwicri
la.i ._1
ALD H
0X
EST
F~c5. 2. Autoradiograms of TLC plates illustrating interconversions of [3H]Z9-14:Ac (Ac) and the corresponding alcohol (OH), aldehyde (A1), and carboxylic acid (CA) by male (top) and female (bottom) leg tissues. The letters B, S, D, and P denote blanks, soluble, detergent, and pellet activities, respectively (see text lbr details); ALDH = aldehyde dehydrogenase, OX = alcohol oxidase, EST = acetate esterase.
FEMALE
4a b3
9
rd
9 Z
9
422
DING AND PRESTWICH TABLE 1. DILUTION SERIES FOR SOLUBLE ANTENNAL ENZYMES a
Aldehyde dehydrogenase (%)d
Esterase ( % y Antennal equivalent per milliliter solutionb
Male
Female
Male
Female
360 120 80 26.67 8.0 2.67 0.8 0.267
27 10 0 ------
30 5 0 ------
--62 50 50 31 10 0
--53 33 33 15 15 0
Percent conversion to product, expressed as means of duplicate assays. bA homogenate of male or female antennae of 350 AE/ml has 0.34 mg/ml total protein for the esterase. For the 80 AE/ml used for aldehyde dehydrogenase assays, males had 0.087 mg/ml and females 0.074 mg/ml total protein. c One-hour incubation. d Fifty-minute incubation.
tissues of both sexes. (3) Esterase activity is low in antennae, higher in legs, and highest glandular tissue of both sexes. (4) Males and females show all three enzymic activities in all tissues. Striking male-female differences include (a) a pellet-associated aldehyde dehydrogenase in female antennae; (b) relatively lower oxidase activity in female legs and glandular tissue, and (c) relatively higher esterase and dehydrogenase activity in female glandular tissues. Figure 4 illustrates the results of the experiment which demonstrated that the oxidase from male legs (the highest activity source) is an O2-requiring enTABLE 2. COMPARISON OF HOMOGENIZATION AND ULTRASONIC AGITATION FOR OBTAINING ENZYMES a
Aldehyde dehydrogenase"
Esteraseb
Homogenate Ultrasonicate
Male
Female
Male
Female
28 33
30 35
22 59
35 61
aConversions expressed as percent product from duplicate assays. hFifty pair of antennae to get 200 ~1 of soluble fraction. Protein concentrations for homogenate: males, 0.39 mg/ml; females, 0.45 mg/ml. Protein concentrations for ultrasonicate: males, 0.47 mg/ml; females, 0.47 mg/ml. "Twelve pair of antennae to get 300/zl of soluble fraction. Protein concentrations: males, 0.022 mg/ml; females, 0.035 mg/ml.
Heliothis
PHEROMONE
423
METABOLISM
Hel/oth/s v/rescens moths: Pheromone processing by tissues MALES 80' '
Antennae
Legs
Hair Pencils
t-
.s
60
> cO
4.0
~O_
20
(D
-
I
S
E
0
AD
E
0
AD
E
0
AD
FEMALES Antenna,
80--
P Legs
c o
60--
Glands
D
O9 (33 >
c o (D
40
o
20-
I I
I
S
~D
o
;
E
0
AD
E Enzyme
0
AD
E
0
AD
Activity
FIG. 3. Morphological distribution of acetate esterase (E), alcohol oxidase (O), and aldehyde dehydrogenase (AD) activities in adult moths (Heliothis virescens). All incubations were run for 1 hr at 20~ and values shown are means of percent conversion for duplicates. Homogenates of two pairs of antennae and legs per 50/xl were used and two pheromone glands or hairpencils per 50/~1 were used. Enzyme activities are shown for soluble (S, open bars), detergent-solubilized (D, solid bars), and insoluble pellet (P, cross-hatched) fractions.
0
4q
6
10
12
/
/
5
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15 zo 25
min
30 35 40 45
Incubation Time,
Io
02
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X
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Eq.) ~
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0
I0
20
30
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60
7O
0
A" /
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oxidase
dependence
leg alcohol
v/'rescens moths
I 10
/
30
-
-
1
min
1 35
1
I 40
~
~ A ~ ---,A- _A..-
Time,
l_____L ~ 15 20 25
Incubation
/
v
A
I 45
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FIG. 4. Dependence of alcohol oxidase of legs of male Heliothis virescens moths on the presence of oxygen, Samples were incubated in air (circles) or under N: for 15 rain followed by air (triangles). The left panel shows only conversion of [3H]Z9 14:OH to the aldehyde, while the right panel shows conversion to the aldehyde plus carboxlic acid.
el_
