BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 775-780
Vol. 176, No. 2, 1991 April 30, 1991
INHIBITION
OF
LIPOXYGENASE SYNTHASE
BY
AND PROSTAGLANDIN ANACARDIC ACIDS
ENDOPEROXIDE
• • .1 • • • 2 Rmhard Grazzml, Dawd Hesk,2 Ellen Helnlnger, George Hildenbrandt3, C. Channa Reddy3, Diana Cox-Foster2, June Medford4, Richard Craig5, and Ralph O. Mumma2.
1The Interdepartmental Graduate Program in Genetics, 2Department of Entomology, 3Department of Veterinary Science, 4Depamnent of Biology, 5Department of Horticulture The Pennsylvania State University University Park, PA 16802 Received
February
22,
1991
SUMMARY: C22:1 o)5-anacardic acid was found to be a good inhibitor of both potato lipoxygenase and ovine prostaglandin endoperoxide synthase with approximate IC50's of 6 and 27 ~tM, respectively. Very similar inhibition was seen with the crude exudate, rich in c05-anacardic acids, from glandular trichomes of an arthropod-resistant swain of geranium, Pelargonium xhortorum. The saturated anacardic acid (C22:0 sat), abundant in the trichome exudate of susceptible strains, was nearly as inhibitory toward both prostaglandin endoperoxide synthase and lipoxygenase as the o)5-unsatumted compound. However, the dimethyl derivative of C22:1 c05anacardic acid was a poor inhibitor of prostaglandin endoperoxide synthase and caused only moderate (32%) inhibition of lipoxygenase even at 135 ~tM. The possible role of prostaglandin endoperoxide synthase and lipoxygenase inhibition in the enhanced pest resistance of geraniums which produce the 0)5-AnAs is discussed. ©1991Academic P..... Inc. The zonal geranium (Pelargonium xhortorum) is naturally resistant to many arthropod and phytopathological pests. One resistance mechanism under investigation involves the production of anacardic acids (AnAs) by the tall glandular trichomes present on the epidermis of the geranium (1,2). AnAs are 6-alkyl salicylic acids in which the 6-alkyl chain may vary in length as well as in position and degree of unsaturation (Figure 1). Waiters et al. (1) demonstrated that resistant geraniums produce an exudate composed largely of C22:1 c05-AnA and C24:1 0~5-AnA, while susceptible inbred lines produce the corresponding saturated AnAs. The ability to make c05-AnA and to resist arthropod predation is inherited as a single, Mendelian dominant wait (unpublished data). Waiters proposed that the mechanism of c05-AnA-mediated arthropod resistance is a sticky trap action caused by the altered physical properties of the unsaturated AnAs (3,4). However, he also noted decreased fecundity of foxglove aphids reared on resistant geranium plants (3) supporting Stark's observations that the exudate from resistant geraniums decreased the fecundity of two-spotted spider mites and was toxic to mites, larval whiteflies, and mite eggs (5). To whomcorrespondenceshouldbe addressed. 0006-291X/91 $1.50 775
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
OH c°°"
id OH
~..~ cH3 COOCH 3
FIGURE 1 .
Structures
of
C22 a n a e a r d ± c
ae±ds
tested.
Prostaglandins (PGs) have been implicated in the reproduction of at least a dozen insect species, with potential roles in both male and female arthropods (6). Destephano and Brady observed prostaglandin endoperoxide synthase (PES) activity in the reproductive tract of adult male house crickets (Acheta domesticus) (7). The injection of PGE 2 into virgin female crickets mimicked the effects of insemination by inducing oviposition behavior followed by an increase in egg laying. Loher et ai. reported high PES activity in the genital tracts of mated females of A.
domesticus and the Australian field cricket (Teleogryllus commodus) but not in those of virgin females (8). The copulatory transfer of active PES in the spermatophore of the male T. commodus has been demonstrated, with subsequent conversion of arachidonic acid into PGs in the female (9). Murtaugh and Denlinger report that feeding aspirin or indomethacin, known inhibitors of the cyclooxygenase activity of PES (10), to house crickets reduced prostaglandin levels in both male and female reproductive organs (11). With the Australian field cricket, injections of aspirin reduced PES activity in the spermatophore (12). The structure of AriA (Figure 1) has a strong similarity to that of aspirin (acetylsalicylic acid). Bhattacharya et ai.(13) investigated the pharmacology of a sodium salt and an acetate derivative of a saturated (C22:0) AnA isolated from a Himalayan sumac species (Rims semialata) and determined that the saturated AnA functioned as a PES inhibitor. Kubo et al. (14) and Yamagiwa et al. (15) indicate that AnAs isolated from cashew nut oil and Ginkgo biloba leaves are active as PES inhibitors; however, neither group provides analytical methods or chemical characterization of the isolates. Based on the structural similarity between AnA and aspirin, and the involvement of PES in insect reproduction, the study reported here was initiated to examine the possible effect of AnAs and trichome exudate on pure PES. The related enzyme lipoxygenase (LOX) which is not known to be inhibited by aspirin was also examined. MATERIALS AND METHODS Preoaration of crude glandular trichome exudate: Trichome exudate was harvested from an inbred geranium line maintained clonally and previously characterized as arthropod-resistant by Waiters (3). Crude trichome exudate was prepared from flower pedicels using a minor 776
Vol. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
modification of the microscope slide collection method of Gerhold et al. (2). A glass microscope slide (washed in CH2C12 and air-dried) was gently pressed against thepedicel, allowing the sticky trichome exudate to collect on the glass surface. The exudate was washed from the slides with CH2C12, pooled and evaporated under a stream of nitrogen (ca. 40°C). The residue was redissotved in CH2C12. Previously published observations indicate that crude trichome exudate from resistant plants contain C22:1 ~5 and C24:1 ¢05-AnAs in a ratio of 40:60 (1, 2). A weighted average molecular weight based on this ratio (approx. 360 MW) was used to estimate molar concentration of the crude exudate. Isolation and purification of anacardic acids: Purification of AnAs from the bulk crude extract was accomplished by a combination of TLC and HPLC methods. Flowers were gently soaked in CH2C12 for about 15 minutes. Two CH2CI2 extracts were pooled, dried over anhydrous Na2SO4, filtered, and concentrated by rotary-evaporation (30°C) to about 50 ml. Semipreparative (2 mm thick) silica gel G plates (E. Merck, Darmstadt, FRG) were streaked with approximately 500 txl of the crude extract and the chromatogram developed in a solvent system of benzene: ether: ethanol: acetic acid (100:80:4:2, v/v/v/v). The AnA band which absorbed strongly at 254nm was detected under UV illumination, scraped off the plate, eluted with CH2C12, evaporated under a stream of N 2 (40°C), and redissolved in the HPLC solvent system. The TLC-purified extract was applied to a 25 cm x 10 mm reversed-phase C8 semiprepamtive HPLC column (SPLC 8-DB), Supelco, Bellefonte, PA) and eluted with a mobile phase consisting of isopropanol: acetonitrile: glacial acetic acid: water (500:150:10:290, v/v/v/v), at a flow rate of 3 ml.min -1. Detection was by UV absorption at 254 nm using a Waters Lambda-Max LC Spectrometer (Waters Associates, Milford, MA, USA). Confirmation of purity was accomplished by dimethylating an aliquot of the collected fractions with diazomethane (ether/methanol), and subsequently analyzing by HPLC, using an analytical column (25 c m x 4.6 mm) of the same stationary phase as above but with a slightly different mobile phase (isopropanol: acetonitrile: 0.01 M acetic acid, 490:147:385, v/v/v) and a flow rate of 0.9 ml.min -1. The identity of each purified dimethyl (di-Me) AriA was confirmed by GC-MS analysis as described by Hesk et al. (16). Preparation of LOX and PES enzymes: ElectrophoreticaUy pure potato tuber LOX was prepared according to Reddanna et al. (17). In brief, tissue homogenates were subjected to ammonium sulfate fractionation and anion-exchange chromatography to yield a preparation with a specific activity of 5 ~tmoles of 02 consumed-min-l.mg protein-1. PES was prepared from sheep vesicles as described by van der Ouderaa et al. (18) and modified by Burgess et al. (19). In brief, the microsome fraction of the tissue was solubilized with Triton X-100 and subjected to anionexchange chromatography. The enzyme preparation had a specific activity of 7 ~tmoles of 02 consumed°min-l"mg protein -I. Activities of both enzymes were assayed with arachidonic acid as the substrate under the conditions described below. Protein was determined by the Hess et al. (20) method, a modification of the Lowry et al. method (21). Assay for inhibition of LOX and PES activities by AnAs: For LOX assays, the incubation medium was 3 ml of air-saturated sodium phosphate buffer, 0.15 M, pH 6.3, and 100 Ixg of enzyme protein. For PES assays, the incubation medium was 3 ml of air-saturated Tris-HC1 buffer, 0.1 M, pH 7.6 containing 3 mM EDTA, 5 mM tryptophan, 16 ~tM Mn protoporphyrin, and 30 Ixg of enzyme protein. For both assays, 10 ~tl of ethanol containing any test compound was added to the reaction mixture about 2 min prior to initiating the reaction with 10 ~tl of ethanol containing 30 mM arachidonic acid (100 ~tM f'mal concentration). Ten 111ethanol blanks were used as controls. The assays were performed at 37°C and [02] was monitored polarigraphically with a Clark oxygen electrode (Yellow Springs Instrument Co., Yellow Springs, OH). RESULTS The effect of AnAs on LOX activity: Crude resistant trichome exudate and purified AnAs were very effective inhibitors of LOX, being active at concentrations as low as 1.1 IxM (Table 1). Complete inhibition of LOX activity was observed at a 12 IxM concentration of the crude extract. Of the three purified compounds examined, both the C22:1 o~5-AnA and the C22:0 sat AriA were 777
Vol. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 1. Inhibition of LOX by crude trichome exudate end pure C22 anacardic acids
Compound
Concentration (llM)
Percent inhibition*
Crude exudate
1.2 12.0
22.4 (3.0) 100.0 (0.0)
C22:1 ¢5 anacardic acid
2.8 5.6 11.1 33.8
18.0 (4.8) 49.4 (7.9) 97.8 (1.1)
lOO.O (o.o)
C22:0 sat anacardic acid
1.1 5.6 11.1
9.4 (1.4) 29.8 (2.5) 90.4 (2,8)
C22:1 ox5 di-Me anacardic acid
11.1 135,0
20.9 (3.2) 32.2 (3.2)
* Each value is an averageof at least three experiments. Standard error of the mean reportedwithin parentheses.
potent inhibitors of LOX, showing 50% and 30% inhibition, respectively, at a concentration of -5.6 paM. The di-Me C22:1 t05-AnA was much less inhibitory than the C22:1 m5-AnA, suggesting that a free carboxyl and/or a free phenol may be necessary for optimal LOX inhibition. At a concentration of 135 ~VI, the di-Me C22:1 m5-AnA inhibited LOX by 32 percent, in comparison to the C22:1 m5-AnA which completely inhibited LOX activity at l 1.1 I.tM. The effect of AnA~ on PES activity: As with LOX, crude trichome exudate and the purified AnAs all inhibited PES activity (Table 2). However, unlike the inhibition of IX)X, in which TABLE 2. Inhibition of PES by crude trlchome exudate and pure C22 anacardic acids
Compound
Concentration (I~M)
Percent inhibition*
Crude exudate
37.0 67.5 122.0
77.8 (5.4) 77.3 (8,7) 95.9 (1,1)
C22:1 (o5 anacardic acid
1.4 13.5 27.0 67.5 135.0
6.2 31.8 50.1 85.7 99.2
C22:0 sat anacardic acid
135.0
91.2 (0,2)
C22:1 0)5 di-Me anacardic acid
11.1
4.9 (3.2)
indomethacin
10.0
82.5 (1,3)
(2.4) (2,8) (4,9) (2,2) (0,7)
* Each value is an averageof at least three experiments. Standard error of the mean reportedwithin parentheses.
778
Vol. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
complete inhibition was observed at a crude exudate concentration of 11.1 }aM, a concentration in excess of 122 ~tM was required for nearly complete inhibition of PES. For C22:1 c05-AnA, a 50% inhibition was observed at 27~Vl while a concentration in excess of 135 laM was required for complete PES inhibition. At equivalent concentrations (135 ~tM), C22:1 to5-AnA was only slightly more active as a PES inhibitor than C22:0 sat AnA (99.2% vs. 91.2%). Di-Me C22:1 0xS-AnA displayed virtually no PES inhibition at a concentration of 135 I.tM. This is in contrast to its effect on LOX activity in which a concentration of 135 ~tM inhibited LOX activity by 32%. These observations suggest that the mechanism of AnA inhibition on PES requires the participation of the carboxyl and/or the phenol, and that the mechanism of AnA inhibition may differ between LOX and PES.
DISCUSSION C22 AnAs exhibited a novel ability to inhibit both LOX and PES with the C22:1 to5-AnA being a slightly better inhibitor than its saturated counterpart. It inhibited LOX and PES with estimated ICso'S of 6 and 30 ~Vl respectively. Further investigation of the inhibitory mechanism of AnAs on these two enzymes may provide some new insight into similarities in the catalytic mechanisms of these oxygenases. This work establishes the clear possibility that one mechanism of C22:1 o5-AnA-mediated arthropod resistance is an inhibition of prostagiandin production leading to reproductive failure or other physiological dysfunction. Indeed, additional experiments will be necessary to establish the actual in vivo concentrations of AnAs when insects are exposed to susceptible versus resistant geraniums. This will establish whether, as expected, the sticky nature of the unsaturated AnA allows it to adhere to the cuticle and subsequently penetrate it due to more prolonged and intimate contact. The stickiness may thereby allow great differences in effective concentration that may be more important than modest enhancement of inhibitory strength for conferring arthropod resistance. Each trichome carries sufficient AnA, as determined by individual trichome extraction (3), to provide an insect the size of an aphid (ca.1 ~tl) with 300X the 1 ~tM dose needed to marginally impair PES or LOX function. In arthropod species with short reproductive cycles, even a slight reduction in fecundity could result in a significant reduction in the pest population in subsequent generations. The unexpectedly strong inhibition of LOX provides new incentive to look for lipoxygenase-derived mediators in physiological processes of insects. LOX-products appear to play a prostaglandin-like role in a primitive insect species, (the silverfish, Thermobia domestica) in which there are no endogenous prostaglandins (22). REFERENCES .
2.
Waiters, D. S., Craig, R., and Mumma, R. O. (1988) J. Chem. Ecol. 14, 743-751. Gerhold, D. L., Craig, R., and Mumma, R. O. (1984) J. Chem. Ecol. 10, 713-722. 779
Vol. 176, No. 2, 1991
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Waiters, Donald S. (1988) Biochemical and morphological characteristics involved in the pest resistance mechanisms of geraniums. Ph.D. thesis, Pennsylvania State University, University Park, PA. Waiters, D. S., Craig, R., and Mumma, R. O. (1989) Entomol. exp. appl. 53, 105-109. Stark, Richard S. (1975) Morphological and biochemical factors relating to spider mite resistance in the geranium. Ph.D. thesis, Pennsylvania State University, University Park, PA. Brady, U. E. (1983) Insect Biochem. 13, 443-451. Destephano, D. B., and Brady, U. E. (1977) J. Insect Physiol. 23, 905-911. Loher, W., Ganjian, I., Kubo, I., Stanley-Samuelson, D., and Tobe, S. S. (1981) Proc. NatL Acad. Sci. U.S.A. 78, 7835-7838. Stanley-Samuelson, D. W., Jurenka, R. A., Blomquist, G. J., and Loher, W. (1987) Physiol. Entomol. 12, 347-354. Mizuno, K., Yamamoto, S., and Lands, W. E. M. (1982) Prostaglandins 23, 743-757. Murtaugh, M. P. and Denlinger, D.L. (1982) Insect Biochem. 12, 599-603. Tobe, S. S., and Loher, W. (1983) Insect Biochem. 13, 137-141. Bhattacharya, S. K., Mukhopadhyay, M., Rao, P. J. R. M., Bagchi, A., and Ray, A. B. (1987) Phytotherapy Research 1,127-134. Kubo, I., Kim, M., Naya, K., Komatsu, S., Yamagira, Y., Ohashi, K., Sakamoti, Y., Hirakawa, S., and Kamikawa, T. (1987) Chem. Lett. 1101-1104. Yamagiwa, Y., K. Ohashi, Y. Sakamoto, S. Hirikawa, and T. Kamikawa (1987) Tetrahedron 43, 3387-3394. Hesk, D., Collins, L., Minard, R., Craig, R., and Mumma, R.O. (1990) Arthropodresistant and -susceptible geraniums: Comparison of chemistry. In P. A. Hedin, ed., Naturally Occurring Pest Bioregulators. Amer. Chem. Soc. Sym. Series 449, 224-250. Reddanna, P., Whelan, J., Maddipati, K. R., and Reddy, C.C. (1990) Methods Enzymol. 187, 268-277. Van der Ouderaa, F. J., Buytenhek, M. and van Dorp, D. A. (1980) in "Adv. Prostaglandin and Thromboxane Res." 6, 139-144. Burgess, J. R., Yang, H., Chang, M., Rao, M. K., Tu, C. P.-D., and Reddy, C. C. (1987) Biochem. Biophys. Res. Commun. 142, 441-447. Hess, H.H., M.B. Lees, and J.E. Derr. (1978) Anal. Biochem. 85: 295-300. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. Ragab, A., Bitsch, C., Thomas, J. M. F., Bitsch, J. and Chap, H. (1987) Insect Biochem. 17, 863-870.
780
Vol. 176, No. 2, 1991 April 30, 1991
INHIBITION
OF
LIPOXYGENASE SYNTHASE
BY
AND PROSTAGLANDIN ANACARDIC ACIDS
ENDOPEROXIDE
• • .1 • • • 2 Rmhard Grazzml, Dawd Hesk,2 Ellen Helnlnger, George Hildenbrandt3, C. Channa Reddy3, Diana Cox-Foster2, June Medford4, Richard Craig5, and Ralph O. Mumma2.
1The Interdepartmental Graduate Program in Genetics, 2Department of Entomology, 3Department of Veterinary Science, 4Depamnent of Biology, 5Department of Horticulture The Pennsylvania State University University Park, PA 16802 Received
February
22,
1991
SUMMARY: C22:1 o)5-anacardic acid was found to be a good inhibitor of both potato lipoxygenase and ovine prostaglandin endoperoxide synthase with approximate IC50's of 6 and 27 ~tM, respectively. Very similar inhibition was seen with the crude exudate, rich in c05-anacardic acids, from glandular trichomes of an arthropod-resistant swain of geranium, Pelargonium xhortorum. The saturated anacardic acid (C22:0 sat), abundant in the trichome exudate of susceptible strains, was nearly as inhibitory toward both prostaglandin endoperoxide synthase and lipoxygenase as the o)5-unsatumted compound. However, the dimethyl derivative of C22:1 c05anacardic acid was a poor inhibitor of prostaglandin endoperoxide synthase and caused only moderate (32%) inhibition of lipoxygenase even at 135 ~tM. The possible role of prostaglandin endoperoxide synthase and lipoxygenase inhibition in the enhanced pest resistance of geraniums which produce the 0)5-AnAs is discussed. ©1991Academic P..... Inc. The zonal geranium (Pelargonium xhortorum) is naturally resistant to many arthropod and phytopathological pests. One resistance mechanism under investigation involves the production of anacardic acids (AnAs) by the tall glandular trichomes present on the epidermis of the geranium (1,2). AnAs are 6-alkyl salicylic acids in which the 6-alkyl chain may vary in length as well as in position and degree of unsaturation (Figure 1). Waiters et al. (1) demonstrated that resistant geraniums produce an exudate composed largely of C22:1 c05-AnA and C24:1 0~5-AnA, while susceptible inbred lines produce the corresponding saturated AnAs. The ability to make c05-AnA and to resist arthropod predation is inherited as a single, Mendelian dominant wait (unpublished data). Waiters proposed that the mechanism of c05-AnA-mediated arthropod resistance is a sticky trap action caused by the altered physical properties of the unsaturated AnAs (3,4). However, he also noted decreased fecundity of foxglove aphids reared on resistant geranium plants (3) supporting Stark's observations that the exudate from resistant geraniums decreased the fecundity of two-spotted spider mites and was toxic to mites, larval whiteflies, and mite eggs (5). To whomcorrespondenceshouldbe addressed. 0006-291X/91 $1.50 775
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
OH c°°"
id OH
~..~ cH3 COOCH 3
FIGURE 1 .
Structures
of
C22 a n a e a r d ± c
ae±ds
tested.
Prostaglandins (PGs) have been implicated in the reproduction of at least a dozen insect species, with potential roles in both male and female arthropods (6). Destephano and Brady observed prostaglandin endoperoxide synthase (PES) activity in the reproductive tract of adult male house crickets (Acheta domesticus) (7). The injection of PGE 2 into virgin female crickets mimicked the effects of insemination by inducing oviposition behavior followed by an increase in egg laying. Loher et ai. reported high PES activity in the genital tracts of mated females of A.
domesticus and the Australian field cricket (Teleogryllus commodus) but not in those of virgin females (8). The copulatory transfer of active PES in the spermatophore of the male T. commodus has been demonstrated, with subsequent conversion of arachidonic acid into PGs in the female (9). Murtaugh and Denlinger report that feeding aspirin or indomethacin, known inhibitors of the cyclooxygenase activity of PES (10), to house crickets reduced prostaglandin levels in both male and female reproductive organs (11). With the Australian field cricket, injections of aspirin reduced PES activity in the spermatophore (12). The structure of AriA (Figure 1) has a strong similarity to that of aspirin (acetylsalicylic acid). Bhattacharya et ai.(13) investigated the pharmacology of a sodium salt and an acetate derivative of a saturated (C22:0) AnA isolated from a Himalayan sumac species (Rims semialata) and determined that the saturated AnA functioned as a PES inhibitor. Kubo et al. (14) and Yamagiwa et al. (15) indicate that AnAs isolated from cashew nut oil and Ginkgo biloba leaves are active as PES inhibitors; however, neither group provides analytical methods or chemical characterization of the isolates. Based on the structural similarity between AnA and aspirin, and the involvement of PES in insect reproduction, the study reported here was initiated to examine the possible effect of AnAs and trichome exudate on pure PES. The related enzyme lipoxygenase (LOX) which is not known to be inhibited by aspirin was also examined. MATERIALS AND METHODS Preoaration of crude glandular trichome exudate: Trichome exudate was harvested from an inbred geranium line maintained clonally and previously characterized as arthropod-resistant by Waiters (3). Crude trichome exudate was prepared from flower pedicels using a minor 776
Vol. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
modification of the microscope slide collection method of Gerhold et al. (2). A glass microscope slide (washed in CH2C12 and air-dried) was gently pressed against thepedicel, allowing the sticky trichome exudate to collect on the glass surface. The exudate was washed from the slides with CH2C12, pooled and evaporated under a stream of nitrogen (ca. 40°C). The residue was redissotved in CH2C12. Previously published observations indicate that crude trichome exudate from resistant plants contain C22:1 ~5 and C24:1 ¢05-AnAs in a ratio of 40:60 (1, 2). A weighted average molecular weight based on this ratio (approx. 360 MW) was used to estimate molar concentration of the crude exudate. Isolation and purification of anacardic acids: Purification of AnAs from the bulk crude extract was accomplished by a combination of TLC and HPLC methods. Flowers were gently soaked in CH2C12 for about 15 minutes. Two CH2CI2 extracts were pooled, dried over anhydrous Na2SO4, filtered, and concentrated by rotary-evaporation (30°C) to about 50 ml. Semipreparative (2 mm thick) silica gel G plates (E. Merck, Darmstadt, FRG) were streaked with approximately 500 txl of the crude extract and the chromatogram developed in a solvent system of benzene: ether: ethanol: acetic acid (100:80:4:2, v/v/v/v). The AnA band which absorbed strongly at 254nm was detected under UV illumination, scraped off the plate, eluted with CH2C12, evaporated under a stream of N 2 (40°C), and redissolved in the HPLC solvent system. The TLC-purified extract was applied to a 25 cm x 10 mm reversed-phase C8 semiprepamtive HPLC column (SPLC 8-DB), Supelco, Bellefonte, PA) and eluted with a mobile phase consisting of isopropanol: acetonitrile: glacial acetic acid: water (500:150:10:290, v/v/v/v), at a flow rate of 3 ml.min -1. Detection was by UV absorption at 254 nm using a Waters Lambda-Max LC Spectrometer (Waters Associates, Milford, MA, USA). Confirmation of purity was accomplished by dimethylating an aliquot of the collected fractions with diazomethane (ether/methanol), and subsequently analyzing by HPLC, using an analytical column (25 c m x 4.6 mm) of the same stationary phase as above but with a slightly different mobile phase (isopropanol: acetonitrile: 0.01 M acetic acid, 490:147:385, v/v/v) and a flow rate of 0.9 ml.min -1. The identity of each purified dimethyl (di-Me) AriA was confirmed by GC-MS analysis as described by Hesk et al. (16). Preparation of LOX and PES enzymes: ElectrophoreticaUy pure potato tuber LOX was prepared according to Reddanna et al. (17). In brief, tissue homogenates were subjected to ammonium sulfate fractionation and anion-exchange chromatography to yield a preparation with a specific activity of 5 ~tmoles of 02 consumed-min-l.mg protein-1. PES was prepared from sheep vesicles as described by van der Ouderaa et al. (18) and modified by Burgess et al. (19). In brief, the microsome fraction of the tissue was solubilized with Triton X-100 and subjected to anionexchange chromatography. The enzyme preparation had a specific activity of 7 ~tmoles of 02 consumed°min-l"mg protein -I. Activities of both enzymes were assayed with arachidonic acid as the substrate under the conditions described below. Protein was determined by the Hess et al. (20) method, a modification of the Lowry et al. method (21). Assay for inhibition of LOX and PES activities by AnAs: For LOX assays, the incubation medium was 3 ml of air-saturated sodium phosphate buffer, 0.15 M, pH 6.3, and 100 Ixg of enzyme protein. For PES assays, the incubation medium was 3 ml of air-saturated Tris-HC1 buffer, 0.1 M, pH 7.6 containing 3 mM EDTA, 5 mM tryptophan, 16 ~tM Mn protoporphyrin, and 30 Ixg of enzyme protein. For both assays, 10 ~tl of ethanol containing any test compound was added to the reaction mixture about 2 min prior to initiating the reaction with 10 ~tl of ethanol containing 30 mM arachidonic acid (100 ~tM f'mal concentration). Ten 111ethanol blanks were used as controls. The assays were performed at 37°C and [02] was monitored polarigraphically with a Clark oxygen electrode (Yellow Springs Instrument Co., Yellow Springs, OH). RESULTS The effect of AnAs on LOX activity: Crude resistant trichome exudate and purified AnAs were very effective inhibitors of LOX, being active at concentrations as low as 1.1 IxM (Table 1). Complete inhibition of LOX activity was observed at a 12 IxM concentration of the crude extract. Of the three purified compounds examined, both the C22:1 o~5-AnA and the C22:0 sat AriA were 777
Vol. 176, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 1. Inhibition of LOX by crude trichome exudate end pure C22 anacardic acids
Compound
Concentration (llM)
Percent inhibition*
Crude exudate
1.2 12.0
22.4 (3.0) 100.0 (0.0)
C22:1 ¢5 anacardic acid
2.8 5.6 11.1 33.8
18.0 (4.8) 49.4 (7.9) 97.8 (1.1)
lOO.O (o.o)
C22:0 sat anacardic acid
1.1 5.6 11.1
9.4 (1.4) 29.8 (2.5) 90.4 (2,8)
C22:1 ox5 di-Me anacardic acid
11.1 135,0
20.9 (3.2) 32.2 (3.2)
* Each value is an averageof at least three experiments. Standard error of the mean reportedwithin parentheses.
potent inhibitors of LOX, showing 50% and 30% inhibition, respectively, at a concentration of -5.6 paM. The di-Me C22:1 t05-AnA was much less inhibitory than the C22:1 m5-AnA, suggesting that a free carboxyl and/or a free phenol may be necessary for optimal LOX inhibition. At a concentration of 135 ~VI, the di-Me C22:1 m5-AnA inhibited LOX by 32 percent, in comparison to the C22:1 m5-AnA which completely inhibited LOX activity at l 1.1 I.tM. The effect of AnA~ on PES activity: As with LOX, crude trichome exudate and the purified AnAs all inhibited PES activity (Table 2). However, unlike the inhibition of IX)X, in which TABLE 2. Inhibition of PES by crude trlchome exudate and pure C22 anacardic acids
Compound
Concentration (I~M)
Percent inhibition*
Crude exudate
37.0 67.5 122.0
77.8 (5.4) 77.3 (8,7) 95.9 (1,1)
C22:1 (o5 anacardic acid
1.4 13.5 27.0 67.5 135.0
6.2 31.8 50.1 85.7 99.2
C22:0 sat anacardic acid
135.0
91.2 (0,2)
C22:1 0)5 di-Me anacardic acid
11.1
4.9 (3.2)
indomethacin
10.0
82.5 (1,3)
(2.4) (2,8) (4,9) (2,2) (0,7)
* Each value is an averageof at least three experiments. Standard error of the mean reportedwithin parentheses.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
complete inhibition was observed at a crude exudate concentration of 11.1 }aM, a concentration in excess of 122 ~tM was required for nearly complete inhibition of PES. For C22:1 c05-AnA, a 50% inhibition was observed at 27~Vl while a concentration in excess of 135 laM was required for complete PES inhibition. At equivalent concentrations (135 ~tM), C22:1 to5-AnA was only slightly more active as a PES inhibitor than C22:0 sat AnA (99.2% vs. 91.2%). Di-Me C22:1 0xS-AnA displayed virtually no PES inhibition at a concentration of 135 I.tM. This is in contrast to its effect on LOX activity in which a concentration of 135 ~tM inhibited LOX activity by 32%. These observations suggest that the mechanism of AnA inhibition on PES requires the participation of the carboxyl and/or the phenol, and that the mechanism of AnA inhibition may differ between LOX and PES.
DISCUSSION C22 AnAs exhibited a novel ability to inhibit both LOX and PES with the C22:1 to5-AnA being a slightly better inhibitor than its saturated counterpart. It inhibited LOX and PES with estimated ICso'S of 6 and 30 ~Vl respectively. Further investigation of the inhibitory mechanism of AnAs on these two enzymes may provide some new insight into similarities in the catalytic mechanisms of these oxygenases. This work establishes the clear possibility that one mechanism of C22:1 o5-AnA-mediated arthropod resistance is an inhibition of prostagiandin production leading to reproductive failure or other physiological dysfunction. Indeed, additional experiments will be necessary to establish the actual in vivo concentrations of AnAs when insects are exposed to susceptible versus resistant geraniums. This will establish whether, as expected, the sticky nature of the unsaturated AnA allows it to adhere to the cuticle and subsequently penetrate it due to more prolonged and intimate contact. The stickiness may thereby allow great differences in effective concentration that may be more important than modest enhancement of inhibitory strength for conferring arthropod resistance. Each trichome carries sufficient AnA, as determined by individual trichome extraction (3), to provide an insect the size of an aphid (ca.1 ~tl) with 300X the 1 ~tM dose needed to marginally impair PES or LOX function. In arthropod species with short reproductive cycles, even a slight reduction in fecundity could result in a significant reduction in the pest population in subsequent generations. The unexpectedly strong inhibition of LOX provides new incentive to look for lipoxygenase-derived mediators in physiological processes of insects. LOX-products appear to play a prostaglandin-like role in a primitive insect species, (the silverfish, Thermobia domestica) in which there are no endogenous prostaglandins (22). REFERENCES .
2.
Waiters, D. S., Craig, R., and Mumma, R. O. (1988) J. Chem. Ecol. 14, 743-751. Gerhold, D. L., Craig, R., and Mumma, R. O. (1984) J. Chem. Ecol. 10, 713-722. 779
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