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Discovery of 5R-lipoxygenase activity in oocytes of the surf clam, Spisula solidissima
Biochimica et Biophysica Acta 1346 Ž1997. 109–119
Discovery of 5R-lipoxygenase activity in oocytes of the surf clam, Spisula solidissima Takahiko Hada 1, Larry L. Swift, Alan R. Brash
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Departments of Pharmacology and Pathology, Vanderbilt UniÕersity Medical Center, NashÕille, TN 37232-6602, USA Received 12 November 1996; accepted 26 November 1996
Abstract Arachidonic acid and 5-hydroxyeicosatetraenoic acid Ž5-HETE. are reported to induce reinitiation of meiosis in oocytes of the surf clam Spisula sachalinensis from the Sea of Japan ŽVaraksin et al., Comp. Biochem. Physiol. 101C, 627–630 Ž1992.. As the Atlantic surf clam Spisula solidissima is a commonly used model for the study of meiosis reinitiation, we examined these cells for the possible occurrence of lipoxygenases and for the bioactivity of the products. Incubation of w 14 Cxarachidonic acid with homogenates of S. solidissima oocytes led to the formation of two major metabolites: 5R-HETE, a novel lipoxygenase product, and 8R-HETE. The products were identified by HPLC, uv spectroscopy, and GC-MS. The corresponding hydroperoxy fatty acids, the primary lipoxygenase products, were isolated from incubations of ammonium sulfate fractionated oocyte cytosol. Arachidonic and eicosapentaenoic acids were identified as constituents of S. solidissima oocyte lipids and the free acids were equally good lipoxygenase substrates. We examined the activity of C18 and C20 polyunsaturated fatty acids and their lipoxygenase products on meiosis reinitiation in Spisula solidissima oocytes, using serotonin and ionophore A23187 as positive controls. The fatty acids and their derivatives were inactive. We conclude that in the surf clam, Žas in starfish., there are responding and non-responding species in regard to the maturation-inducing activity of the oocyte lipoxygenase products, and that the lipoxygenase has another, as yet uncharacterized, function in oocyte physiology. Keywords: Lipoxygenase; Arachidonic acid; Surf clam; Oocyte; Maturation
1. Introduction
Abbreviations: HŽP.ETE, hydroŽpero.xyeicosatetraenoic acid; HŽP.EPE, hydroŽpero.xyeicosapentaenoic acid; HPLC, high pressure liquid chromatography; RP-HPLC, reversed-phase HPLC; SP-HPLC, straight-phase HPLC; ED50 , 50% effective dose; GCMS, gas chromatography-mass spectrometry; El, electron impact. ) Corresponding author. Fax: q1 Ž615. 3224707. E-mail: [email protected] 1 Present address: Pharmaceutical Discovery Research Laboratories, Teijin Institute for Biomedical Research, 4-3-2 Agahi-gaoka, Hino, Tokyo 191, Japan.
Lipoxygenases are a class of dioxygenase that are widespread in plants, and in higher animals are best known for their occurrence in different types of blood cells and epithelial tissues w1,2x. Their conversion of polyunsaturated fatty acids to hydroperoxy derivatives is implicated in the production of different classes of signalling molecules, and in the synthesis of specific hydroxylated fatty acids w1,2x. One of the highest levels of expression of lipoxygenase activity in animal cells occurs in the eggs or
0005-2760r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 5 - 2 7 6 0 Ž 9 6 . 0 0 1 7 9 - 8
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T. Hada et al.r Biochimica et Biophysica Acta 1346 (1997) 109–119
oocytes of several species of marine invertebrate. Eggs of the sea urchin Strongylocentrotus purpuratus contain high levels of 11R- and 12R-lipoxygenase activity w3x. The eggs of several other sea urchin species do not have comparable activity w4x, and the function of the S. purpuratus egg lipoxygenaseŽs. has not been determined. Oocytes of starfish have a prominent 8R-lipoxygenase activity w5,6x. In many species, arachidonic acid and the 8R-lipoxygenase product 8R-hydroxy-eicosatetraenoic acid Ž 8R-HETE. are fully active agonists that mimic the activity of the hormone 1-methyadenine in the induction of starfish oocyte maturation Ž re-initiation of meiosis. w5x. In other starfish species, arachidonic acid and 8R-HETE have no maturation-inducing activity, although the oocytes still contain an 8R-lipoxygenase w6x. In these species there is some evidence that arachidonic acid or an arachidonic acid metabolite may have an inhibitory influence on 1-methyladenine-induced oocyte maturation w7,8x. The surf clam oocyte is another cell used for study of the reinitiation of meiosis. In the surf clam, serotonin has been identified as a natural hormone that induces oocyte maturation w9x. Recently it was reported by Varaksin and colleagues that in oocytes of Spisula sachalinensis from the Sea of Japan, the effects of serotonin are mimicked by certain polyunsaturated fatty acids including arachidonic acid, and by 5-HETE, the 5-hydroxy metabolite of arachidonic acid w10x. These results appear analogous to the activity of arachidonic acid and its 8R-hydroxy metabolite on oocyte maturation in starfish. To examine this system further, we investigated the metabolic fate and activity of arachidonic acid in oocytes of the most closely related animal that was available to us, the Atlantic surf clam Spisula solidissima.
2. Materials and methods 2.1. Materials and reagents Unlabeled arachidonic acid was purchased from Nu Chek Prep Inc. ŽElysian, MN., and eicosapentaenoic acid from Cayman Chemical Ž Ann Arbor, MI. . w1- 14 CxArachidonic and w1- 14 Cxeicosapentaenoic acids were obtained from Dupont-New England Nuclear.
Racemic HŽP.ETE and HŽP.EPE standards were prepared by vitamin E-controlled autoxidation w11x. The 8R-HETE and 8R-HEPE were prepared by incubation of arachidonic and eicosapentaenoic acids respectively with acetone powder of the coral Plexaura homomalla w12x. Authentic 5R- and 5S-HETEs were prepared by resolution of racemic 5-HETE as the methyl ester, dehydroabietylamine derivative w13x. Ketoeicosatetraenoic acid standards were isolated from the mixture of products formed on treatment of the corresponding HPETE with hematin w14x. Surf clams were supplied by Ocean Resources ŽIsle Allhaut, ME. . 2.2. Isolation of cells and enzyme preparation Ovaries were removed and placed in ice-cold Shapiro’s calcium-free artificial sea water w452 mM NaCl Ž 26.43 grl. , 10 mM KCl Ž0.75 grl., 29.8 mM MgCl 2 P 7H 2 O Ž4.24 grl., 10 mM Tris base Ž1.21 grl.x adjusted to pH 7.9 with HCl. Oocytes were freed from the ovary by tearing the tissue with fine forceps, followed quickly by filtration through three layers of cheesecloth to remove tissue fragments w15x. The oocytes were allowed to settle to the bottom and the supernatant fluid was discarded. This washing process was repeated at least three times. The washed oocytes were disrupted using a Potter-Elvehjem homogenizer in 5 volumes of ice-cold 20 mM Tris-HCl ŽpH 7.4. containing 1 mM EDTA and 1 mM dithiothreitol. Fractionation of the enzyme activity was carried out by ammonium sulfate precipitation and DEAE column chromatography. The homogenate was initially centrifuged at 10 000= g for 10 min and then 105 000 = g for 60 min at 48C. The cytosol was brought to 50% saturation with solid ammonium sulfate, stirred for 1 h and then centrifuged at 10 000 = g for 20 min. The pellet was resuspended in 20 mM Tris ŽpH 7.4. containing 1 mM EDTA and 1 mM dithiothreitol. DEAE column chromatography was carried out at room temperature. The ammonium sulfate fraction Ž 2 ml. was applied to a DEAE Sephacel column Ž 11 = 50 mm. equilibrated with 20 mM Tris-HCl Ž pH 7.4. containing 1 mM EDTA and 1 mM dithiothreitol. After the column was washed with 12 ml of the equilibration buffer, 0.4 M NaCl in
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Incubations with homogenate or enzyme preparation were carried out in 50 mM Tris-HCl buffer Ž pH 7.4., with addition of calcium Ž 2 mM. . Arachidonic or eicosapentaenoic acids Ž 25 m M., including the corresponding 14C fatty acid Ž 0.1–0.2 m Ci. , were added in ethanol solution. Incubation were conducted at room temperature for 1–30 min. After the incubation, the products were acidified to pH 3–4 with 1 N HCl and extracted with diethyl ether, or the incubation was extracted directly using the Bligh and Dyer procedure w16x. Incubations of intact oocytes used a 10% Ž by volume. suspension of cells in artificial sea water, with gentle rocking of the cell suspension during the incubation. Subsequently the cells and sea water were extracted together using the Bligh and Dyer procedure w16x.
80:20:0.01 Ž by volume. at a flow rate of 1 mlrmin. UV spectra and the profiles at 205 nm, 220 nm, 235 nm and 270 nm were recorded using a HewlettPackard 1040A diode array detector, and radioactivity was monitored on-line using a Radiomatic Instruments Flo-One detector. The main radiolabeled peaks were further purified by SP-HPLC using a Beckman Ultrasphere 5 m silica column Ž 25 = 0.46 cm. and a solvent of hexanerisopropyl alcoholrglacial acetic acid 100:2:0.1 Ž by volume. . Steric analysis of the hydroxy products was carried out on the methyl ester derivatives by chiral phase HPLC using a Chiralcel OB column Ž 250 = 4.6 mm, Daicel, Japan. and a solvent mixture of hexane:isopropyl alcohol, 100:5 Ž vrv. for 5-HETE methyl ester and 100:2 Ž vrv. for 8-HETE methyl ester w17x. The order of elution of R and S enantiomers was established using chiral standards prepared as described in Section 2.1. In chiral analysis of the 5-hydroxyeicosapentaenoates, we assumed that the order of elution of R and S on the chiral column is identical to the order observed for the HETEs.
2.4. Transmethylation
2.6. Gas chromatography-mass spectrometry
Transesterification was carried out by dissolving an aliquot of the lipid extract Ž equivalent to 0.5 ml of the original cell incubation. in dry methanol Ž 100 m l. plus 10 m l dichloromethane to help solubilize the extract; the sample was treated with 100 m l of 0.5 M sodium methoxide in methanol Ž a 10-fold dilution of the 5M Aldrich product.. After 10 min at room temperature, 600 m l of 0.1 N HCl was added together with 100 m l of 1 M KH 2 PO4 to help stabilize the pH, and the solution was extracted using the Bligh and Dyer method w16x. The organic phase was washed with Bligh and Dyer upper phase Ž prepared with water. and then taken to dryness with a stream of nitrogen and stored in 200 m l methanol under argon at y708C.
Products were derivatized to the methyl ester using ethereal diazomethane:methanol Žf 4:1 vrv., and trimethylsilyl derivatives were prepared by treatment with BSTFArpyridine Ž 2:1 vrv. for 30 min at room temperature. Mass spectra were recorded in the positive ion electron impact mode using a Finnegan Incos 50 instrument operated at 70 eV and coupled to a Hewlett-Packard 5890 gas chromatograph equipped with a SPB-1 fused silica capillary column Ž 5 m= 0.25 mm inner diameter. . Samples dissolved in hexane were injected on column at an oven temperature of 1908; after 1 min the temperature was programmed to 3008C at 108C or 208Crmin.
2.5. HPLC analysis
Lipids were extracted from oocytes using the method of Folch et al. w18x. Fatty acids in the total lipid extract were methylated using boron trifluoride-methanol w19x and analyzed by gas chromatography using a Hewlett-Packard 5890A gas chromatograph equipped with a 6 ft = 2 mm i.d.
equilibration buffer was used to elute enzyme activity. 2.3. Incubation conditions
Typically, the extracted materials were analyzed initially by reversed-phase HPLC using a Beckman Ultrasphere 5 m m ODS column Ž 25 = 0.46 cm. with a solvent of methanolrwaterrglacial acetic acid
2.7. Analysis of fatty acid content
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glass column packed with 10% SP 2330 on 100r120 Chromosorb W Ž Supelco, Bellefonte, PA. . Fatty acid methyl esters were identifed by comparison of retention times to those of known standards.
3. Results 3.1. Fatty acid composition of S. solidissima oocytes Both arachidonic acid and eicosapentaenoic acid are constituents of surf clam oocyte lipids Ž Table 1. . Eicosapentaenoate predominates by a ratio of ; 15:1 over arachidonate. For comparison, the reported content of arachidonic and eicosapentaenoic acids in a North Atlantic species of starfish Ž Asterias Õulgaris. is about 4:1 in favor of the eicosapentaenoate w20x. Our studies on lipoxygenase activity focussed mainly on arachidonic acid, as this fatty acid is active in inducing re-initiation of meiosis in Spisula sachalinensis, whereas eicosapentaenoic acid is without ef-
fect w10x. The work described below indicates that the two polyunsaturated fatty acids are equally good substrates for the lipoxygenase activity in S. solidissima oocytes. Several GC peaks in our fatty acid analyses Žapprox. 13% by area. did not correspond to available standards. Some of these may comprise fatty acids with branched chains or non-methylene-interrupted double bonds are reported to occur in other marine invertebrates w20x. 3.2. Major metabolites in oocyte homogenates Incubation of w 14 Cxarachidonic acid with homogenates of S. solidissima oocytes led to the formation mainly of HETE products. As illustrated in the reversed-phase HPLC analysis in Fig. 1, the typical radio-chromatogram shows two major peaks, designated 1 Ž17.5 min. and 2 Ž 20.5 min. . The retention times of peaks 1 and 2 correspond to authentic 8-HETE and 5-HETE, respectively, and identification was further substantiated by their co-chromatography
Table 1 Fatty acid composition of S. solidissima oocytes Fatty acid
Retention time Žmin.
Percent composition 3 individuals
14.0 16.0 16.1 ? ? 18.0 18.1 v 9 18.2 v 6 ? 18.3 v 3 20.1 ? ? 20.4v 6 ? 20.5 v 3 22.4v 6 22.5 v 3 22.6 v 3 Total identified Unidentified
Mean value
a1
a2
a3
4.4 25.0 6.4 0.5 0.3 6.9 11.1 0.4 0.6 0.8 5.7 8.0 2.1 1.1 1.3 14.9 1.6 0.7 8.2
4.3 23.4 7.0 0.5 0.3 5.6 9.6 0.8 0.6 0.9 5.9 7.8 2.5 1.3 1.2 16.3 1.9 0.8 9.3
4.1 22.0 8.0 0.5 0.3 5.4 11.2 0.5 0.7 0.8 5.6 6.9 2.1 1.2 1.1 18.7 1.8 0.8 8.3
4.3 23.5 7.1 0.5 0.3 6.0 10.6 0.6 0.6 0.8 5.7 7.6 2.2 1.2 1.2 16.6 1.8 0.8 8.6 87.6% 12.4%
1.72 2.78 3.24 3.53 4.02 4.47 5.05 5.98 6.24 7.26 7.60 8.14 8.88 10.50 11.50 12.30 14.54 16.56 17.35
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with the respective standards on SP-HPLC. The UV spectrum of each product was typical of a conjugated diene chromophore. Subtle differences in the lmax of the two chromophores Žobserved at 237 nm and 235 nm respectively for peaks 1 and 2. also matched precisely the spectra of the standards w21x. The product identities were confirmed as 8-HETE Ž peak 1. and 5-HETE Ž peak 2. by gas chromatography-mass spectrometry of the methyl ester trimethylsilyl ether derivatives Ž data not shown. w22x. The absolute configuration of the products was determined by chiralphase HPLC of the methyl ester derivatives w17x.
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Fig. 2. Steric analysis of surf clam oocyte products by chiral column HPLC. The HETEs were converted to the methyl ester derivatives, repurified by SP-HPLC, and the enantiomers resolved using a Chiralcel OB column Ž25=0.46 cm., a flow rate of 1 mlrmin, and with UV detection at 235 nm. A solvent of hexanerisopropanol 100:5 Žvrv. was used for 5-HETE methyl ester analysis, and hexanerisopropanol 100:2 Žvrv. for 8-HETE methyl ester.
These steric analyses revealed that both products were greater than 97% of the ‘R’ configuration Ž Fig. 2.. In all incubations of oocyte homogenates, the HEPE metabolites of eicosapentaenoic acid were formed from endogenously released substrate and were evident on HPLC analysis with UV detection at 235 nm. The ratio of 5- to 8-hydroxy was always very similar for the 20.5 metabolites and the HETEs formed from added 20.4 substrate. Study of the metabolism of w 14 Cxeicosapentaenoic acid confirmed that the metabolic fate is essentially the same as for arachidonic acid. The 20.5 v 3 substrate was converted to its 5R-HEPE and 8R-HEPE metabolites ŽFig. 1B..
Fig. 1. RP-HPLC analysis of products from surf clam oocytes incubated with w1- 14 Cxarachidonic acid or w1- 14 Cxeicosapentaenoic acid. Panel A: w1- 14 CxArachidonic acid Ž25 m M. was incubated with a homogenate of surf clam oocytes for 30 min at room temperature. Products were extracted using the Bligh and Dyer procedure w16x. HPLC was carried out on a Beckman 5 m Ultrasphere ODS column at 1 mlrmin with a solvent of methanolrwaterrglacial acetic acid Ž80:20:0.01, by volume.; the solvent was changed to 100% methanol to elute the arachidonic acid. Panel B: An oocyte homogenate was incubated as above with radiolabeled eicosapentaenoic acid and the products were analyzed by RP-HPLC using the solvent methanolrwaterrglacial acetic acid Ž85:15:0.01, by volume..
Table 2 Subcellular distribution of the lipoxygenase activity in surf clam oocytes Fraction
Total activity Ž%.
Specific activity Žnmolr30 minr mg protein.
Homogenate 10,000=g pellet 10,000=g supernatant 100,000=g pellet 100,000=g supernatant
100 70 67 18 59
1.9 4.3 1.8 3.0 2.6
Each fraction was allowed to react with w1- 14 Cxarachidonic acid Ž25 m M. at room temperature for 30 min. The products were extracted and analyzed by RP-HPLC.
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3.3. Fractionation of enzymatic actiÕity The specific enzyme activity of the oocyte homogenate was 1.9 nmolr30 minrmg protein at room temperature. As shown in Table 2, enzymatic activity was detected in all subcellular fractions although it was most abundant in the 10 000 = g pellet. When the 10 000 = g pellet was resuspended and centrifuged again, more than half of the enzyme activity was found in the supernatant. In oocyte homogenates, the 5- and 8-HETEs are formed by reduction of the primary lipoxygenase products, the fatty acids hydroperoxides Ž HPETEs. . After ammonium sulfate fractionation Ž 0–50%. of the cytosol, it was possible to recover the intact HPETEs after incubation with arachidonic acid. The 5-HPETE and 8-HPETE products were separated by SP-HPLC and identified by cochromatography with authentic HPETE standards and by their reduction with triphenylphosphine to the corresponding HETEs Žnot shown..
donic acid metabolism was examined using a dialyzed cytosol fraction of surf clam oocytes. The enzyme reaction was stimulated by calcium in a concentration-dependent manner and the maximal activity was observed with 0.3 mM calcium chloride ŽFig. 3. . Both the 5R- and 8R-lipoxygenase activities were stimulated. Magnesium was equally stimulatory, being maximally effective at around 1 mM. Stimulation of the enzyme activities by calcium chloride was not increased by the addition of ATP as occurs with the mammalian 5S-lipoxygenase w23x. When the time course of 5R- and 8R-HŽ P. ETE production from arachidonic acid was examined using surf clam oocyte cytosol at room temperature, the reaction of both 5- and 8-lipoxygenases with arachidonic acid was found to have almost ceased by 5 min Ždata not shown.. This short time course is observed with certain other lipoxygenases, for example the porcine leukocyte 12S-lipoxygenase, and is ascribed to self-catalyzed inactivation or ‘suicide inactivation’ w24x.
3.4. Additional properties of the lipoxygenase(s)
3.5. Identification of keto metabolites
Previously, we found that the 8R-lipoxygenase of starfish oocytes was calcium-dependent w6x, whereas the 8R-lipoxygenase of the gorgonian Plexaura homomalla is insensitive to calcium w12x. The effect of various calcium chloride concentrations on arachi-
5R-HETE and 8R-HETE are readily detected by their absorbance at 235 nm, and they are the major arachidonic acid metabolites in homogenates of surf clam oocytes. However, other compounds were detected by UV monitoring at 270 nm, and these products are formed in larger amounts in the incubations of the 10 000 = g pellet. The UV spectra of these compounds showed the smooth chromophore Ž lmax f 280 nm. characteristic of a conjugated dienone. They were identified as 5-keto and 8-keto derivatives by their co-chromatography with authentic 5-ketoand 8-ketoeicosatetraenoic acids on RP-HPLC and SP-HPLC, and by their conversion to 5-HETE and 8-HETE on treatment with sodium borohydride Ž data not shown.. Biosynthesis of the keto derivatives was examined in the 10 000 = g pellet using arachidonic acid, 5Rand 8R-HPETE, and 5R- and 8R-HPETE as substrates. Comparison of arachidonic acid and the 5R derivatives is illustrated in Fig. 4, and the corresponding experiment with the 8R derivatives in Fig. 5. In each case, arachidonic acid is converted to prominent keto derivatives Ž panels A. , and boiling eliminates
Fig. 3. Concentration-dependent stimulation of lipoxygenase product formation by CaCl 2 . The EDTA-free cytosol fraction of surf clam oocytes was assayed in various calcium concentrations. Lipoxygenase activity was assessed by incubation with w 14 Cxarachidonic acid followed by extraction and RP-HPLC analysis.
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the activity Ž panels B. . The HPETEs give rise to the corresponding keto product and the formation is largely, though not completely, eliminated by boiling Žpanels C and D respectively, Figs. 4 and 5. , indicating that there is a significant non-enzymatic component to this reaction. In contrast, no products at all are formed from the R-HETEs, either with active or boiled enzyme Ž panels E and F, Figs. 4 and 5. . 3.6. Resolution of 5R- and 8R-lipoxygenase actiÕities The ammonium sulfate fraction Ž 0–50%. of surf clam oocytes was dialyzed into 40 mM Tris buffer and subjected to DEAE anion-exchange chromatography. As shown in Fig. 6, after application of the ammonium sulfate fraction, half of the protein eluted
Fig. 5. Synthesis of 8-keto products by the 10 000= g pellet of surf clam oocytes. Comparison of the metabolic profiles of arachidonic acid, 8R-HPETE and 8R-HETE in a 10 000= g pellet of surf clam oocytes that showed mainly 8-lipoxygenase activity. Products were chromatographed as described in the legend to Fig. 4A,B, arachidonic acid incubated with 10 000= g pellet; ŽC. and ŽD., 8R-HPETE; ŽE. and ŽF., 8R-HETE. In the right-hand series of panels ŽB, D and F., substrates were incubated with boiled enzyme.
Fig. 4. Synthesis of 5-keto products by the 10 000= g pellet of surf clam oocytes. Comparison of the metabolic profiles of arachidonic acid, 5R-HPETE and 5R-HETE in the 10 000= g pellet of surf clam oocytes. Substrates were incubated with 10 000= g pellet in 50 mM Tris-HCl ŽpH 7.5. containing 2 mM CaCl 2 for 10 min. Products were extracted with diethyl ether and subsequently analyzed by SP-HPLC with hexanerisopropyl alcoholrglacial acetic acid Ž100:2:0.1, vrvrv. at 1 mlrmin. The right-hand series of panels ŽB, D, and F. are from substrates incubated with boiled enzyme. ŽA. and ŽB., arachidonic acid incubated with 10 000= g pellet; ŽC. and ŽD., 5R-HPETE; ŽE. and ŽF., 5R-HETE.
Fig. 6. Elution profile of protein and enzyme activity from anion-exchange chromatography on DEAE-Sephacel. A 50% ammonium sulfate fraction of surf clam oocytes was applied to the column, which was washed with 20 mM Tris-HCl buffer ŽpH 7.4. containing 1 mM EDTA and 1 mM dithiothreitol, and then, as indicated by the arrow, eluted with 0.4 M NaCl. Lipoxygenase activity in each fraction was assessed by incubation of an aliquot with w 14 Cxarachidonic acid, followed by diethyl ether extraction and RP-HPLC analysis with on-line detection of radioactivity.
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with 5R-lipoxygenase activity in the pass-through fraction. In contrast, 8R-lipoxygenase activity was retained on the column and was eluted subsequently with 0.4 M NaCl buffer. It should be stated at this point that this single experiment represents the only convincing chromatographic resolution of the two lipoxygenase activities that we were able to achieve, and there remains some doubt whether one or two lipoxygenase enzymes account for the 5R- and 8Rlipoxygenase activities of S. solidissima oocytes Žsee Section 4. . 3.7. Incubations in intact oocytes: formation of esterified products Incubation of w 14 Cxarachidonic acid Ž 50 m M. with intact S. solidissima oocytes was associated with a low conversion Žf 2–5%. to radiolabeled 5R-HETE and 8R-HETE. These products were detected mainly esterified in the oocyte lipids; they were recovered as the methyl esters after transmethylation of lipid extracts with sodium methoxide and identified by HPLC analysis. Additional minor radiolabeled peaks on the HPLC chromatogram were probably comprised of products formed initially through conversion of the hydroperoxides to ketoeicosatetraenoic acids. One of the more prominent additional products appeared on the back shoulder of the 5-HETE peak on RP-HPLC. This compound had no absorbance at 235 nm. It was purified by SP-HPLC Ž it chromatographed slightly ahead of 8-HETE methyl ester. and analyzed by GC-MS as the methyl ester TMS ether derivative Ž it co-eluted on GC with the same derivative of 8HETE.; the El mass spectrum showed structurally diagnostic ions at mrz 393 Ž M-15, 5% relative abundance., mrz 267 ŽC8-C20, 60% abundance. , mrz 243 ŽC1 -C8, 10% abundance. , with additional prominent ions in the scan range Ž mrz 100–500. observed at mrz values of 214 Ž45%. , 199 10%. , 187 Ž40%., 177 Žbase peak., 159, 135, 121, and 107. Based on these data the product was tentatively identified as the trienoic acid metabolite, 8-hydroxyeicosa-5,11,14-eicosatrienoic acid. There was no detectable formation of free or esterified HETEs or HEPEs following stimulation of intact oocytes with ionophore A23187 Ž 10 m M., or serotonin Ž10 m M., although as expected, these stim-
uli did induce re-initiation of meiosis in the cells as evidenced by germinal vesicle breakdown. 3.8. Induction of oocyte maturation: comparison of serotonin and fatty acid deriÕatiÕes The re-initiation of meiosis Ž oocyte maturation. in surf clam oocytes is induced by release of the natural agonist, serotonin w9x, and this activity is readily mimicked in washed preparations of oocytes in artificial sea water by addition of serotonin or a number of other agents including calcium ionophore A23187 w25x, elevated extracellular potassium ion concentration w15x, and by an unidentified low molecular weight substance obtained from surf clam body fluids w26x. In the surf clam Spisula sachalinensis from the Sea of Japan, arachidonic acid, 5-HETE and linoleic acid are active inducers of maturation, whereas eicosapentaenoic acid, 5-HEPE Ž 5-hydroxyeicosapentaenoic acid. and docosahexaenoic acid are inactive w10x. We used serotonin and A23187 as positive controls for maturation of S. solidissima oocytes. The ED50 to serotonin was typically between 10y7 and 10y8 M, and we noticed that addition of ethanol Ž a potential solvent for the fatty acids. was a potent inhibitor of the effect of serotonin. A final concentration of 1% ethanol Ž 10 m l EtOH to 1 ml sea water. induces a 100-fold shift to the right in the dose-response curve to serotonin Ž100-fold higher concentration required for maturation. . As a consequence of this observation, fatty acids and their derivatives were converted to the sodium salt with Na 2 CO 3 and added in aqueous solution, with appropriate vehicle controls. This effect of ethanol on S. solidissima oocyte maturation is in contrast to the complete lack of effect of 1% ethanol on 1-methyladenine-induced oocyte maturation in starfish w5x. In S. solidissima oocytes, ethanol did not affect the response to ionophore A23187. The following list of fatty acids and their derivatives were tested for induction of oocyte maturation or inhibition of the effects of serotonin using several batches of oocytes: arachidonic acid Ž C20.4v 6., eicosapentaenoic acid Ž C20.5 v 3., 5RS-HETE, 8RSHETE, and 8-ketoeicosatetraenoic acid. In addition, the following were tested on a single batch of oocytes, again using serotonin and A23187 as positive con-
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trols: linoleic acid Ž C18.2 v 6., a-linolenic acid ŽC18.3 v 3. , g-linolenic acid Ž C18.3 v 6., and dihomog-linolenic acid Ž C20.3 v 6.. Each fatty acid was found ineffective in induction of oocyte maturation at concentrations up to 100 m M, and also they were not inhibitory on oocyte maturation induced by serotonin Ž1 m M.. 3.9. Metabolism of arachidonic acid in testis of S. solidissima Although incidental to the main purpose of this study, it should be mentioned here that we also found prominent 5R- and 8R-lipoxygenase activities in testis homogenates from male S. solidissima. This parallels the situation in starfish ŽHughes and Brash, unpublished observations. , where the testis has very high 8R-lipoxygenase activity, again matching the activity in the ovary. The HETE products were identified by RP-HPLC, SP-HPLC, uv spectroscopy and chiral column HPLC analyses.
4. Discussion Arachidonic and eicosapentaenoic acids are metabolized by S. solidissima oocytes mainly to the 5R and 8R hydroxy derivatives. These products are formed by lipoxygenase activities followed by peroxidasecatalyzed reduction of the hydroperoxides to the corresponding HETEs and HEPEs, respectively. While 8R-lipoxygenase has been found before in marine invertebrates w5,12x, this is the first reported discovery of 5R-lipoxygenase activity. Catalysis by a single lipoxygenase enzyme is one possible explanation for the synthesis of both 5R- and 8R-hydroperoxides in S. solidissima oocytes. Formation of 5R and 8R products involves a simple ‘frame shift’ along the carbon chain of arachidonic acid Ž Fig. 7., and dual positional specificity of this type is precedented in certain other lipoxygenases. For example, the rabbit reticulocyte 15S-lipoxygenase forms 12S-HPETE and 15S-HPETE in a ratio of approximately 1:9 w27x, and the porcine leukocyte 12S-lipoxygenase forms the same two products in opposite proportions w28x. The phenomenon of dual positional specificity is shown also by lipoxygenases with singular specificity when they are presented with syn-
Fig. 7. Relationship of 5R-lipoxygenase and 8R-lipoxygenase activities. By analogy with other lipoxygenases, enzymatic reaction is expected to be initiated by abstraction of a hydrogen Žindicated with a curved arrow. from C-7 Ž5R-lipoxygenase. or C-10 Ž8R-lipoxygenase. of arachidonic acid.
thetic polyunsaturated fatty acids that have ‘frameshifted’ double bonds w29x. In the case of the lipoxygenase activity in S. solidissima oocytes, we obtained some chromatographic evidence that two distinct enzymes are present Ž Fig. 6. . Each data point of lipoxygenase activity in Fig. 6 is based on an incubation and RP-HPLC analysis that can readily distinguish the products, 5-HŽP.ETE and 8-HŽP.ETE., and therefore there is no doubt that resolution of 5R- and 8R-lipoxygenase activities was achieved in this experiment. However, we were not able to reproduce this result. A factor that may contribute to this inconsistency relates to the finding of major changes in the proportions of 5RHETE and 8R-HETE formed under different conditions by S. solidissima oocyte extracts. In an early season sample, collected before the ovary was ripe with oocytes responsive to serotonin, the only significant product was 5-HETE. When ripe ovaries became available, the oocytes formed mixtures of 5-HETE and 8-HETE Ž typified by the chromatogram in Fig. 1.. On several occasions, oocyte homogenates that originally made a certain mixture of 5-HETE and 8-HETE gave a completely different profile after storage at y708C for a few days or weeks. We should note here that there is a recent precedent in the literature describing the apparent changing of lipoxygenase positional specificity: Shen and colleagues reported that a human 15-lipoxygenase transgenically expressed in rabbit macrophages formed variable proportions of 12- and 15-HETE when the cells were harvested and incubated with arachidonic acid ex vivo w30x. Under the conditions of our experi-
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ments it appeared that the 5R-lipoxygenase could disappear on storage at y708C and be replaced by 8R-lipoxygenase activity. Similarly, the activities were found to change in an unpredictable way when samples were fractionated with ammonium sulfate or passed through an ion exchange column. Based on these observations, it remains an open issue whether there exist separate 5R- and 8R-lipoxygenase enzymes in S. solidissima oocytes. Formation of the keto fatty acid derivatives in S. solidissima oocytes occurs exclusively from the corresponding hydroperoxide, not from the HETE. The reaction appears to be mainly enzymatic in character, although some conversion is observed in the boiled 10 000 = g pellet fraction. Whether this reaction is of significance in vivo is unknown. The enzymatic formation of keto products directly from the corresponding hydroperoxides parallels closely the reported conversion of 12-HPETE and 15-HPETE directly to their 12-keto and 15-keto derivatives in microsomes from neonatal rat epidermis, and also their further conversion to eicosatrienoic acids w31,32x, as we observed for 8R-HETE in intact oocytes. Neither arachidonic acid, 5R-HETE or 8R-HETE, nor related derivatives, have activity on induction of oocyte maturation in S. solidissima. This appears to parallel a situation that applies to approximately half of all species of starfish – the oocytes have the capacity to synthesize a product that is an active inducer of maturation in other related species. In the case of the surf clam, the work of Varaksin et al. indicates that arachidonic acid and 5-HETE are active inducers of maturation in S. sachalinensis w10x. Although the chirality of the 5-HETE used by Varaksin et al. is not specified in their paper, it was probably racemic 5-HETE produced by the convenient chemical transformation from arachidonic acid w13x. It seems very likely that activity will be found to be restricted to the R-HETE enantiomers, just as 8R-HETE is the only active enantiomer in starfish oocytes w5x. We would anticipate also that the activity of arachidonic acid in S. sachalinensis is related to its metabolism to 5R-HETE andror 8R-HETE. It also remains to be determined what, if any, is the active metabolite of linoleic acid in S. sachalinensis. The lack of maturation-inducing activity of eicosapentaenoic acid and its 5-hydroxy derivative 5-HEPE in S. sachalinensis represents a most striking difference from the 20.4v 6
analogs. This contrasts with the situation in starfish in which both the 20.4v 6 and 20.5 v 3 fatty acids and their 8R-hydroxy derivatives are active in the ‘responding’ species of starfish w5x. The role of the lipoxygenase enzymes in the ‘non-responders’, which in the surf clam we have shown to include S. solidissima, remains an open question.
Acknowledgements This work was supported by NIH grants GM15431, GM-49502 and HD-05970. A fellowship for T.H. was provided by Teijin Ltd.
References w1x H.W. Gardner, Biochim. Biophys. Acta 1084 Ž1991. 221– 239. w2x S. Yamamoto, Biochim. Biophys. Acta 1128 Ž1992. 117– 131. w3x D.J. Hawkins, A.R. Brash, J. Biol. Chem. 262 Ž1987. 7629–7634. w4x G. Perry, D. Epel, Dev. Biol. 107 Ž1985. 47–57. w5x L. Meijer, A.R. Brash, R.W. Bryant, K. Ng, J. Maclouf, H. Sprecher, J. Biol. Chem. 261 Ž1986. 17040–17047. w6x A.R. Brash, M.A. Hughes, D.J. Hawkins, W.E. Boeglin, W.C. Song, L. Meijer, J. Biol. Chem. 266 Ž1991. 22926– 22931. w7x L. Meijer, J. Maclouf, R.W. Bryant, Prostaglandins, Leukotrienes Med. 23 Ž1986. 179–184. w8x H. Tosuji, T. Kanazawa, Zool. Sci. 4 Ž1987. 1046. w9x S. Hirai, T. Kishimoto, A.L. Kadam, H. Kanatani, S.S. Koide, J. Exp. Zoo. 245 Ž1988. 318–321. w10x A.A. Varaksin, G.S. Varaksina, O.V. Reunova, N.A. Latyshev, Comp. Biochem. Physiol. 101C Ž1992. 627–630. w11x K.E. Peers, D.T. Coxon, Chem. Phys. Lipids 32 Ž1983. 49–56. w12x A.R. Brash, S.W. Baertschi, C.D. Ingram, T.M. Harris, J. Biol. Chem. 262 Ž1987. 15829–15839. w13x E.J. Corey, S. Hashimoto, Tetrahedron Lett. 22 Ž1981. 299–302. w14x T.A. Dix, L.J. Marnett, J. Biol. Chem. 260 Ž1985. 5351– 5357. w15x R.D. Allen, Biol. Bull 105 Ž1953. 213–239. w16x G.H. Bligh, W.J. Dyer, Can. J. Biochem. Physiol. 37 Ž1959. 911–917. w17x A.R. Brash, D.J. Hawkins, Method Enzymol. 187 Ž1990. 187–195. w18x J. Folch, M. Lees, G.H. Sloane-Stanley, J. Biol. Chem. 226 Ž1957. 497–509. w19x W.R. Morrison, L.M. Smith, J. Lipid Res. 5 Ž1964. 600–604.
T. Hada et al.r Biochimica et Biophysica Acta 1346 (1997) 109–119 w20x M. Paradis, R.G. Ackman, Lipids 12 Ž1976. 170–176. w21x C.D. Ingram, A.R. Brash, Lipids 23 Ž1988. 340–344. w22x J.M. Boeynaems, A.R. Brash, J.A. Oates, W.C. Hubbard, Anal. Biochem. 104 Ž1980. 259–267. w23x K. Ochi, T. Yoshimoto, S. Yamamoto, K. Taniguchi, T. Miyamoto, J. Biol. Chem. 258 Ž1983. 5754–5758. w24x Y. Takahashi, N. Ueda, S. Yamamoto, Arch. Biochem. Biophys. 266 Ž1988. 613–621. w25x A.W. Schuetz, J. Exp. Zool. 191 Ž1975. 443–446. w26x T. Toyaya, Y. Nagahama, H. Kanatani, S.S. Koide, Experientia 43 Ž1987. 885–886. w27x R.W. Bryant, J.M. Bailey, T. Schewe, S.M. Rapoport, J. Biol. Chem. 257 Ž1982. 6050–6055.
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w28x T. Yoshimoto, Y. Miyamoto, K. Ochi, S. Yamamoto, Biochim. Biophys. Acta 713 Ž1982. 638–646. w29x H. Kuhn, H. Sprecher, A.R. Brash, J. Biol. Chem. 265 ¨ Ž1991. 16300–16304. w30x J. Shen, H. Kuhn, ¨ A. Petho-Schramm, L. Chang, FASEB J. 9 Ž1995. 1623–1631. w31x J. Van Wauwe, M.-C. Coene, G. Van Nyen, W. Cools, J. Goossens, L. Le Jeune, W. Lauwers, P.A.J. Janssen, Eicosanoids 4 Ž1991. 155–163. w32x J. Van Wauwe, M.-C. Coene, G. Van Nyen, W. Cools, L. Le Jeune, W. Lauwers, Eicosanoids 5 Ž1992. 141–146.
Discovery of 5R-lipoxygenase activity in oocytes of the surf clam, Spisula solidissima Takahiko Hada 1, Larry L. Swift, Alan R. Brash
)
Departments of Pharmacology and Pathology, Vanderbilt UniÕersity Medical Center, NashÕille, TN 37232-6602, USA Received 12 November 1996; accepted 26 November 1996
Abstract Arachidonic acid and 5-hydroxyeicosatetraenoic acid Ž5-HETE. are reported to induce reinitiation of meiosis in oocytes of the surf clam Spisula sachalinensis from the Sea of Japan ŽVaraksin et al., Comp. Biochem. Physiol. 101C, 627–630 Ž1992.. As the Atlantic surf clam Spisula solidissima is a commonly used model for the study of meiosis reinitiation, we examined these cells for the possible occurrence of lipoxygenases and for the bioactivity of the products. Incubation of w 14 Cxarachidonic acid with homogenates of S. solidissima oocytes led to the formation of two major metabolites: 5R-HETE, a novel lipoxygenase product, and 8R-HETE. The products were identified by HPLC, uv spectroscopy, and GC-MS. The corresponding hydroperoxy fatty acids, the primary lipoxygenase products, were isolated from incubations of ammonium sulfate fractionated oocyte cytosol. Arachidonic and eicosapentaenoic acids were identified as constituents of S. solidissima oocyte lipids and the free acids were equally good lipoxygenase substrates. We examined the activity of C18 and C20 polyunsaturated fatty acids and their lipoxygenase products on meiosis reinitiation in Spisula solidissima oocytes, using serotonin and ionophore A23187 as positive controls. The fatty acids and their derivatives were inactive. We conclude that in the surf clam, Žas in starfish., there are responding and non-responding species in regard to the maturation-inducing activity of the oocyte lipoxygenase products, and that the lipoxygenase has another, as yet uncharacterized, function in oocyte physiology. Keywords: Lipoxygenase; Arachidonic acid; Surf clam; Oocyte; Maturation
1. Introduction
Abbreviations: HŽP.ETE, hydroŽpero.xyeicosatetraenoic acid; HŽP.EPE, hydroŽpero.xyeicosapentaenoic acid; HPLC, high pressure liquid chromatography; RP-HPLC, reversed-phase HPLC; SP-HPLC, straight-phase HPLC; ED50 , 50% effective dose; GCMS, gas chromatography-mass spectrometry; El, electron impact. ) Corresponding author. Fax: q1 Ž615. 3224707. E-mail: [email protected] 1 Present address: Pharmaceutical Discovery Research Laboratories, Teijin Institute for Biomedical Research, 4-3-2 Agahi-gaoka, Hino, Tokyo 191, Japan.
Lipoxygenases are a class of dioxygenase that are widespread in plants, and in higher animals are best known for their occurrence in different types of blood cells and epithelial tissues w1,2x. Their conversion of polyunsaturated fatty acids to hydroperoxy derivatives is implicated in the production of different classes of signalling molecules, and in the synthesis of specific hydroxylated fatty acids w1,2x. One of the highest levels of expression of lipoxygenase activity in animal cells occurs in the eggs or
0005-2760r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 5 - 2 7 6 0 Ž 9 6 . 0 0 1 7 9 - 8
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oocytes of several species of marine invertebrate. Eggs of the sea urchin Strongylocentrotus purpuratus contain high levels of 11R- and 12R-lipoxygenase activity w3x. The eggs of several other sea urchin species do not have comparable activity w4x, and the function of the S. purpuratus egg lipoxygenaseŽs. has not been determined. Oocytes of starfish have a prominent 8R-lipoxygenase activity w5,6x. In many species, arachidonic acid and the 8R-lipoxygenase product 8R-hydroxy-eicosatetraenoic acid Ž 8R-HETE. are fully active agonists that mimic the activity of the hormone 1-methyadenine in the induction of starfish oocyte maturation Ž re-initiation of meiosis. w5x. In other starfish species, arachidonic acid and 8R-HETE have no maturation-inducing activity, although the oocytes still contain an 8R-lipoxygenase w6x. In these species there is some evidence that arachidonic acid or an arachidonic acid metabolite may have an inhibitory influence on 1-methyladenine-induced oocyte maturation w7,8x. The surf clam oocyte is another cell used for study of the reinitiation of meiosis. In the surf clam, serotonin has been identified as a natural hormone that induces oocyte maturation w9x. Recently it was reported by Varaksin and colleagues that in oocytes of Spisula sachalinensis from the Sea of Japan, the effects of serotonin are mimicked by certain polyunsaturated fatty acids including arachidonic acid, and by 5-HETE, the 5-hydroxy metabolite of arachidonic acid w10x. These results appear analogous to the activity of arachidonic acid and its 8R-hydroxy metabolite on oocyte maturation in starfish. To examine this system further, we investigated the metabolic fate and activity of arachidonic acid in oocytes of the most closely related animal that was available to us, the Atlantic surf clam Spisula solidissima.
2. Materials and methods 2.1. Materials and reagents Unlabeled arachidonic acid was purchased from Nu Chek Prep Inc. ŽElysian, MN., and eicosapentaenoic acid from Cayman Chemical Ž Ann Arbor, MI. . w1- 14 CxArachidonic and w1- 14 Cxeicosapentaenoic acids were obtained from Dupont-New England Nuclear.
Racemic HŽP.ETE and HŽP.EPE standards were prepared by vitamin E-controlled autoxidation w11x. The 8R-HETE and 8R-HEPE were prepared by incubation of arachidonic and eicosapentaenoic acids respectively with acetone powder of the coral Plexaura homomalla w12x. Authentic 5R- and 5S-HETEs were prepared by resolution of racemic 5-HETE as the methyl ester, dehydroabietylamine derivative w13x. Ketoeicosatetraenoic acid standards were isolated from the mixture of products formed on treatment of the corresponding HPETE with hematin w14x. Surf clams were supplied by Ocean Resources ŽIsle Allhaut, ME. . 2.2. Isolation of cells and enzyme preparation Ovaries were removed and placed in ice-cold Shapiro’s calcium-free artificial sea water w452 mM NaCl Ž 26.43 grl. , 10 mM KCl Ž0.75 grl., 29.8 mM MgCl 2 P 7H 2 O Ž4.24 grl., 10 mM Tris base Ž1.21 grl.x adjusted to pH 7.9 with HCl. Oocytes were freed from the ovary by tearing the tissue with fine forceps, followed quickly by filtration through three layers of cheesecloth to remove tissue fragments w15x. The oocytes were allowed to settle to the bottom and the supernatant fluid was discarded. This washing process was repeated at least three times. The washed oocytes were disrupted using a Potter-Elvehjem homogenizer in 5 volumes of ice-cold 20 mM Tris-HCl ŽpH 7.4. containing 1 mM EDTA and 1 mM dithiothreitol. Fractionation of the enzyme activity was carried out by ammonium sulfate precipitation and DEAE column chromatography. The homogenate was initially centrifuged at 10 000= g for 10 min and then 105 000 = g for 60 min at 48C. The cytosol was brought to 50% saturation with solid ammonium sulfate, stirred for 1 h and then centrifuged at 10 000 = g for 20 min. The pellet was resuspended in 20 mM Tris ŽpH 7.4. containing 1 mM EDTA and 1 mM dithiothreitol. DEAE column chromatography was carried out at room temperature. The ammonium sulfate fraction Ž 2 ml. was applied to a DEAE Sephacel column Ž 11 = 50 mm. equilibrated with 20 mM Tris-HCl Ž pH 7.4. containing 1 mM EDTA and 1 mM dithiothreitol. After the column was washed with 12 ml of the equilibration buffer, 0.4 M NaCl in
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Incubations with homogenate or enzyme preparation were carried out in 50 mM Tris-HCl buffer Ž pH 7.4., with addition of calcium Ž 2 mM. . Arachidonic or eicosapentaenoic acids Ž 25 m M., including the corresponding 14C fatty acid Ž 0.1–0.2 m Ci. , were added in ethanol solution. Incubation were conducted at room temperature for 1–30 min. After the incubation, the products were acidified to pH 3–4 with 1 N HCl and extracted with diethyl ether, or the incubation was extracted directly using the Bligh and Dyer procedure w16x. Incubations of intact oocytes used a 10% Ž by volume. suspension of cells in artificial sea water, with gentle rocking of the cell suspension during the incubation. Subsequently the cells and sea water were extracted together using the Bligh and Dyer procedure w16x.
80:20:0.01 Ž by volume. at a flow rate of 1 mlrmin. UV spectra and the profiles at 205 nm, 220 nm, 235 nm and 270 nm were recorded using a HewlettPackard 1040A diode array detector, and radioactivity was monitored on-line using a Radiomatic Instruments Flo-One detector. The main radiolabeled peaks were further purified by SP-HPLC using a Beckman Ultrasphere 5 m silica column Ž 25 = 0.46 cm. and a solvent of hexanerisopropyl alcoholrglacial acetic acid 100:2:0.1 Ž by volume. . Steric analysis of the hydroxy products was carried out on the methyl ester derivatives by chiral phase HPLC using a Chiralcel OB column Ž 250 = 4.6 mm, Daicel, Japan. and a solvent mixture of hexane:isopropyl alcohol, 100:5 Ž vrv. for 5-HETE methyl ester and 100:2 Ž vrv. for 8-HETE methyl ester w17x. The order of elution of R and S enantiomers was established using chiral standards prepared as described in Section 2.1. In chiral analysis of the 5-hydroxyeicosapentaenoates, we assumed that the order of elution of R and S on the chiral column is identical to the order observed for the HETEs.
2.4. Transmethylation
2.6. Gas chromatography-mass spectrometry
Transesterification was carried out by dissolving an aliquot of the lipid extract Ž equivalent to 0.5 ml of the original cell incubation. in dry methanol Ž 100 m l. plus 10 m l dichloromethane to help solubilize the extract; the sample was treated with 100 m l of 0.5 M sodium methoxide in methanol Ž a 10-fold dilution of the 5M Aldrich product.. After 10 min at room temperature, 600 m l of 0.1 N HCl was added together with 100 m l of 1 M KH 2 PO4 to help stabilize the pH, and the solution was extracted using the Bligh and Dyer method w16x. The organic phase was washed with Bligh and Dyer upper phase Ž prepared with water. and then taken to dryness with a stream of nitrogen and stored in 200 m l methanol under argon at y708C.
Products were derivatized to the methyl ester using ethereal diazomethane:methanol Žf 4:1 vrv., and trimethylsilyl derivatives were prepared by treatment with BSTFArpyridine Ž 2:1 vrv. for 30 min at room temperature. Mass spectra were recorded in the positive ion electron impact mode using a Finnegan Incos 50 instrument operated at 70 eV and coupled to a Hewlett-Packard 5890 gas chromatograph equipped with a SPB-1 fused silica capillary column Ž 5 m= 0.25 mm inner diameter. . Samples dissolved in hexane were injected on column at an oven temperature of 1908; after 1 min the temperature was programmed to 3008C at 108C or 208Crmin.
2.5. HPLC analysis
Lipids were extracted from oocytes using the method of Folch et al. w18x. Fatty acids in the total lipid extract were methylated using boron trifluoride-methanol w19x and analyzed by gas chromatography using a Hewlett-Packard 5890A gas chromatograph equipped with a 6 ft = 2 mm i.d.
equilibration buffer was used to elute enzyme activity. 2.3. Incubation conditions
Typically, the extracted materials were analyzed initially by reversed-phase HPLC using a Beckman Ultrasphere 5 m m ODS column Ž 25 = 0.46 cm. with a solvent of methanolrwaterrglacial acetic acid
2.7. Analysis of fatty acid content
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glass column packed with 10% SP 2330 on 100r120 Chromosorb W Ž Supelco, Bellefonte, PA. . Fatty acid methyl esters were identifed by comparison of retention times to those of known standards.
3. Results 3.1. Fatty acid composition of S. solidissima oocytes Both arachidonic acid and eicosapentaenoic acid are constituents of surf clam oocyte lipids Ž Table 1. . Eicosapentaenoate predominates by a ratio of ; 15:1 over arachidonate. For comparison, the reported content of arachidonic and eicosapentaenoic acids in a North Atlantic species of starfish Ž Asterias Õulgaris. is about 4:1 in favor of the eicosapentaenoate w20x. Our studies on lipoxygenase activity focussed mainly on arachidonic acid, as this fatty acid is active in inducing re-initiation of meiosis in Spisula sachalinensis, whereas eicosapentaenoic acid is without ef-
fect w10x. The work described below indicates that the two polyunsaturated fatty acids are equally good substrates for the lipoxygenase activity in S. solidissima oocytes. Several GC peaks in our fatty acid analyses Žapprox. 13% by area. did not correspond to available standards. Some of these may comprise fatty acids with branched chains or non-methylene-interrupted double bonds are reported to occur in other marine invertebrates w20x. 3.2. Major metabolites in oocyte homogenates Incubation of w 14 Cxarachidonic acid with homogenates of S. solidissima oocytes led to the formation mainly of HETE products. As illustrated in the reversed-phase HPLC analysis in Fig. 1, the typical radio-chromatogram shows two major peaks, designated 1 Ž17.5 min. and 2 Ž 20.5 min. . The retention times of peaks 1 and 2 correspond to authentic 8-HETE and 5-HETE, respectively, and identification was further substantiated by their co-chromatography
Table 1 Fatty acid composition of S. solidissima oocytes Fatty acid
Retention time Žmin.
Percent composition 3 individuals
14.0 16.0 16.1 ? ? 18.0 18.1 v 9 18.2 v 6 ? 18.3 v 3 20.1 ? ? 20.4v 6 ? 20.5 v 3 22.4v 6 22.5 v 3 22.6 v 3 Total identified Unidentified
Mean value
a1
a2
a3
4.4 25.0 6.4 0.5 0.3 6.9 11.1 0.4 0.6 0.8 5.7 8.0 2.1 1.1 1.3 14.9 1.6 0.7 8.2
4.3 23.4 7.0 0.5 0.3 5.6 9.6 0.8 0.6 0.9 5.9 7.8 2.5 1.3 1.2 16.3 1.9 0.8 9.3
4.1 22.0 8.0 0.5 0.3 5.4 11.2 0.5 0.7 0.8 5.6 6.9 2.1 1.2 1.1 18.7 1.8 0.8 8.3
4.3 23.5 7.1 0.5 0.3 6.0 10.6 0.6 0.6 0.8 5.7 7.6 2.2 1.2 1.2 16.6 1.8 0.8 8.6 87.6% 12.4%
1.72 2.78 3.24 3.53 4.02 4.47 5.05 5.98 6.24 7.26 7.60 8.14 8.88 10.50 11.50 12.30 14.54 16.56 17.35
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with the respective standards on SP-HPLC. The UV spectrum of each product was typical of a conjugated diene chromophore. Subtle differences in the lmax of the two chromophores Žobserved at 237 nm and 235 nm respectively for peaks 1 and 2. also matched precisely the spectra of the standards w21x. The product identities were confirmed as 8-HETE Ž peak 1. and 5-HETE Ž peak 2. by gas chromatography-mass spectrometry of the methyl ester trimethylsilyl ether derivatives Ž data not shown. w22x. The absolute configuration of the products was determined by chiralphase HPLC of the methyl ester derivatives w17x.
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Fig. 2. Steric analysis of surf clam oocyte products by chiral column HPLC. The HETEs were converted to the methyl ester derivatives, repurified by SP-HPLC, and the enantiomers resolved using a Chiralcel OB column Ž25=0.46 cm., a flow rate of 1 mlrmin, and with UV detection at 235 nm. A solvent of hexanerisopropanol 100:5 Žvrv. was used for 5-HETE methyl ester analysis, and hexanerisopropanol 100:2 Žvrv. for 8-HETE methyl ester.
These steric analyses revealed that both products were greater than 97% of the ‘R’ configuration Ž Fig. 2.. In all incubations of oocyte homogenates, the HEPE metabolites of eicosapentaenoic acid were formed from endogenously released substrate and were evident on HPLC analysis with UV detection at 235 nm. The ratio of 5- to 8-hydroxy was always very similar for the 20.5 metabolites and the HETEs formed from added 20.4 substrate. Study of the metabolism of w 14 Cxeicosapentaenoic acid confirmed that the metabolic fate is essentially the same as for arachidonic acid. The 20.5 v 3 substrate was converted to its 5R-HEPE and 8R-HEPE metabolites ŽFig. 1B..
Fig. 1. RP-HPLC analysis of products from surf clam oocytes incubated with w1- 14 Cxarachidonic acid or w1- 14 Cxeicosapentaenoic acid. Panel A: w1- 14 CxArachidonic acid Ž25 m M. was incubated with a homogenate of surf clam oocytes for 30 min at room temperature. Products were extracted using the Bligh and Dyer procedure w16x. HPLC was carried out on a Beckman 5 m Ultrasphere ODS column at 1 mlrmin with a solvent of methanolrwaterrglacial acetic acid Ž80:20:0.01, by volume.; the solvent was changed to 100% methanol to elute the arachidonic acid. Panel B: An oocyte homogenate was incubated as above with radiolabeled eicosapentaenoic acid and the products were analyzed by RP-HPLC using the solvent methanolrwaterrglacial acetic acid Ž85:15:0.01, by volume..
Table 2 Subcellular distribution of the lipoxygenase activity in surf clam oocytes Fraction
Total activity Ž%.
Specific activity Žnmolr30 minr mg protein.
Homogenate 10,000=g pellet 10,000=g supernatant 100,000=g pellet 100,000=g supernatant
100 70 67 18 59
1.9 4.3 1.8 3.0 2.6
Each fraction was allowed to react with w1- 14 Cxarachidonic acid Ž25 m M. at room temperature for 30 min. The products were extracted and analyzed by RP-HPLC.
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3.3. Fractionation of enzymatic actiÕity The specific enzyme activity of the oocyte homogenate was 1.9 nmolr30 minrmg protein at room temperature. As shown in Table 2, enzymatic activity was detected in all subcellular fractions although it was most abundant in the 10 000 = g pellet. When the 10 000 = g pellet was resuspended and centrifuged again, more than half of the enzyme activity was found in the supernatant. In oocyte homogenates, the 5- and 8-HETEs are formed by reduction of the primary lipoxygenase products, the fatty acids hydroperoxides Ž HPETEs. . After ammonium sulfate fractionation Ž 0–50%. of the cytosol, it was possible to recover the intact HPETEs after incubation with arachidonic acid. The 5-HPETE and 8-HPETE products were separated by SP-HPLC and identified by cochromatography with authentic HPETE standards and by their reduction with triphenylphosphine to the corresponding HETEs Žnot shown..
donic acid metabolism was examined using a dialyzed cytosol fraction of surf clam oocytes. The enzyme reaction was stimulated by calcium in a concentration-dependent manner and the maximal activity was observed with 0.3 mM calcium chloride ŽFig. 3. . Both the 5R- and 8R-lipoxygenase activities were stimulated. Magnesium was equally stimulatory, being maximally effective at around 1 mM. Stimulation of the enzyme activities by calcium chloride was not increased by the addition of ATP as occurs with the mammalian 5S-lipoxygenase w23x. When the time course of 5R- and 8R-HŽ P. ETE production from arachidonic acid was examined using surf clam oocyte cytosol at room temperature, the reaction of both 5- and 8-lipoxygenases with arachidonic acid was found to have almost ceased by 5 min Ždata not shown.. This short time course is observed with certain other lipoxygenases, for example the porcine leukocyte 12S-lipoxygenase, and is ascribed to self-catalyzed inactivation or ‘suicide inactivation’ w24x.
3.4. Additional properties of the lipoxygenase(s)
3.5. Identification of keto metabolites
Previously, we found that the 8R-lipoxygenase of starfish oocytes was calcium-dependent w6x, whereas the 8R-lipoxygenase of the gorgonian Plexaura homomalla is insensitive to calcium w12x. The effect of various calcium chloride concentrations on arachi-
5R-HETE and 8R-HETE are readily detected by their absorbance at 235 nm, and they are the major arachidonic acid metabolites in homogenates of surf clam oocytes. However, other compounds were detected by UV monitoring at 270 nm, and these products are formed in larger amounts in the incubations of the 10 000 = g pellet. The UV spectra of these compounds showed the smooth chromophore Ž lmax f 280 nm. characteristic of a conjugated dienone. They were identified as 5-keto and 8-keto derivatives by their co-chromatography with authentic 5-ketoand 8-ketoeicosatetraenoic acids on RP-HPLC and SP-HPLC, and by their conversion to 5-HETE and 8-HETE on treatment with sodium borohydride Ž data not shown.. Biosynthesis of the keto derivatives was examined in the 10 000 = g pellet using arachidonic acid, 5Rand 8R-HPETE, and 5R- and 8R-HPETE as substrates. Comparison of arachidonic acid and the 5R derivatives is illustrated in Fig. 4, and the corresponding experiment with the 8R derivatives in Fig. 5. In each case, arachidonic acid is converted to prominent keto derivatives Ž panels A. , and boiling eliminates
Fig. 3. Concentration-dependent stimulation of lipoxygenase product formation by CaCl 2 . The EDTA-free cytosol fraction of surf clam oocytes was assayed in various calcium concentrations. Lipoxygenase activity was assessed by incubation with w 14 Cxarachidonic acid followed by extraction and RP-HPLC analysis.
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the activity Ž panels B. . The HPETEs give rise to the corresponding keto product and the formation is largely, though not completely, eliminated by boiling Žpanels C and D respectively, Figs. 4 and 5. , indicating that there is a significant non-enzymatic component to this reaction. In contrast, no products at all are formed from the R-HETEs, either with active or boiled enzyme Ž panels E and F, Figs. 4 and 5. . 3.6. Resolution of 5R- and 8R-lipoxygenase actiÕities The ammonium sulfate fraction Ž 0–50%. of surf clam oocytes was dialyzed into 40 mM Tris buffer and subjected to DEAE anion-exchange chromatography. As shown in Fig. 6, after application of the ammonium sulfate fraction, half of the protein eluted
Fig. 5. Synthesis of 8-keto products by the 10 000= g pellet of surf clam oocytes. Comparison of the metabolic profiles of arachidonic acid, 8R-HPETE and 8R-HETE in a 10 000= g pellet of surf clam oocytes that showed mainly 8-lipoxygenase activity. Products were chromatographed as described in the legend to Fig. 4A,B, arachidonic acid incubated with 10 000= g pellet; ŽC. and ŽD., 8R-HPETE; ŽE. and ŽF., 8R-HETE. In the right-hand series of panels ŽB, D and F., substrates were incubated with boiled enzyme.
Fig. 4. Synthesis of 5-keto products by the 10 000= g pellet of surf clam oocytes. Comparison of the metabolic profiles of arachidonic acid, 5R-HPETE and 5R-HETE in the 10 000= g pellet of surf clam oocytes. Substrates were incubated with 10 000= g pellet in 50 mM Tris-HCl ŽpH 7.5. containing 2 mM CaCl 2 for 10 min. Products were extracted with diethyl ether and subsequently analyzed by SP-HPLC with hexanerisopropyl alcoholrglacial acetic acid Ž100:2:0.1, vrvrv. at 1 mlrmin. The right-hand series of panels ŽB, D, and F. are from substrates incubated with boiled enzyme. ŽA. and ŽB., arachidonic acid incubated with 10 000= g pellet; ŽC. and ŽD., 5R-HPETE; ŽE. and ŽF., 5R-HETE.
Fig. 6. Elution profile of protein and enzyme activity from anion-exchange chromatography on DEAE-Sephacel. A 50% ammonium sulfate fraction of surf clam oocytes was applied to the column, which was washed with 20 mM Tris-HCl buffer ŽpH 7.4. containing 1 mM EDTA and 1 mM dithiothreitol, and then, as indicated by the arrow, eluted with 0.4 M NaCl. Lipoxygenase activity in each fraction was assessed by incubation of an aliquot with w 14 Cxarachidonic acid, followed by diethyl ether extraction and RP-HPLC analysis with on-line detection of radioactivity.
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with 5R-lipoxygenase activity in the pass-through fraction. In contrast, 8R-lipoxygenase activity was retained on the column and was eluted subsequently with 0.4 M NaCl buffer. It should be stated at this point that this single experiment represents the only convincing chromatographic resolution of the two lipoxygenase activities that we were able to achieve, and there remains some doubt whether one or two lipoxygenase enzymes account for the 5R- and 8Rlipoxygenase activities of S. solidissima oocytes Žsee Section 4. . 3.7. Incubations in intact oocytes: formation of esterified products Incubation of w 14 Cxarachidonic acid Ž 50 m M. with intact S. solidissima oocytes was associated with a low conversion Žf 2–5%. to radiolabeled 5R-HETE and 8R-HETE. These products were detected mainly esterified in the oocyte lipids; they were recovered as the methyl esters after transmethylation of lipid extracts with sodium methoxide and identified by HPLC analysis. Additional minor radiolabeled peaks on the HPLC chromatogram were probably comprised of products formed initially through conversion of the hydroperoxides to ketoeicosatetraenoic acids. One of the more prominent additional products appeared on the back shoulder of the 5-HETE peak on RP-HPLC. This compound had no absorbance at 235 nm. It was purified by SP-HPLC Ž it chromatographed slightly ahead of 8-HETE methyl ester. and analyzed by GC-MS as the methyl ester TMS ether derivative Ž it co-eluted on GC with the same derivative of 8HETE.; the El mass spectrum showed structurally diagnostic ions at mrz 393 Ž M-15, 5% relative abundance., mrz 267 ŽC8-C20, 60% abundance. , mrz 243 ŽC1 -C8, 10% abundance. , with additional prominent ions in the scan range Ž mrz 100–500. observed at mrz values of 214 Ž45%. , 199 10%. , 187 Ž40%., 177 Žbase peak., 159, 135, 121, and 107. Based on these data the product was tentatively identified as the trienoic acid metabolite, 8-hydroxyeicosa-5,11,14-eicosatrienoic acid. There was no detectable formation of free or esterified HETEs or HEPEs following stimulation of intact oocytes with ionophore A23187 Ž 10 m M., or serotonin Ž10 m M., although as expected, these stim-
uli did induce re-initiation of meiosis in the cells as evidenced by germinal vesicle breakdown. 3.8. Induction of oocyte maturation: comparison of serotonin and fatty acid deriÕatiÕes The re-initiation of meiosis Ž oocyte maturation. in surf clam oocytes is induced by release of the natural agonist, serotonin w9x, and this activity is readily mimicked in washed preparations of oocytes in artificial sea water by addition of serotonin or a number of other agents including calcium ionophore A23187 w25x, elevated extracellular potassium ion concentration w15x, and by an unidentified low molecular weight substance obtained from surf clam body fluids w26x. In the surf clam Spisula sachalinensis from the Sea of Japan, arachidonic acid, 5-HETE and linoleic acid are active inducers of maturation, whereas eicosapentaenoic acid, 5-HEPE Ž 5-hydroxyeicosapentaenoic acid. and docosahexaenoic acid are inactive w10x. We used serotonin and A23187 as positive controls for maturation of S. solidissima oocytes. The ED50 to serotonin was typically between 10y7 and 10y8 M, and we noticed that addition of ethanol Ž a potential solvent for the fatty acids. was a potent inhibitor of the effect of serotonin. A final concentration of 1% ethanol Ž 10 m l EtOH to 1 ml sea water. induces a 100-fold shift to the right in the dose-response curve to serotonin Ž100-fold higher concentration required for maturation. . As a consequence of this observation, fatty acids and their derivatives were converted to the sodium salt with Na 2 CO 3 and added in aqueous solution, with appropriate vehicle controls. This effect of ethanol on S. solidissima oocyte maturation is in contrast to the complete lack of effect of 1% ethanol on 1-methyladenine-induced oocyte maturation in starfish w5x. In S. solidissima oocytes, ethanol did not affect the response to ionophore A23187. The following list of fatty acids and their derivatives were tested for induction of oocyte maturation or inhibition of the effects of serotonin using several batches of oocytes: arachidonic acid Ž C20.4v 6., eicosapentaenoic acid Ž C20.5 v 3., 5RS-HETE, 8RSHETE, and 8-ketoeicosatetraenoic acid. In addition, the following were tested on a single batch of oocytes, again using serotonin and A23187 as positive con-
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trols: linoleic acid Ž C18.2 v 6., a-linolenic acid ŽC18.3 v 3. , g-linolenic acid Ž C18.3 v 6., and dihomog-linolenic acid Ž C20.3 v 6.. Each fatty acid was found ineffective in induction of oocyte maturation at concentrations up to 100 m M, and also they were not inhibitory on oocyte maturation induced by serotonin Ž1 m M.. 3.9. Metabolism of arachidonic acid in testis of S. solidissima Although incidental to the main purpose of this study, it should be mentioned here that we also found prominent 5R- and 8R-lipoxygenase activities in testis homogenates from male S. solidissima. This parallels the situation in starfish ŽHughes and Brash, unpublished observations. , where the testis has very high 8R-lipoxygenase activity, again matching the activity in the ovary. The HETE products were identified by RP-HPLC, SP-HPLC, uv spectroscopy and chiral column HPLC analyses.
4. Discussion Arachidonic and eicosapentaenoic acids are metabolized by S. solidissima oocytes mainly to the 5R and 8R hydroxy derivatives. These products are formed by lipoxygenase activities followed by peroxidasecatalyzed reduction of the hydroperoxides to the corresponding HETEs and HEPEs, respectively. While 8R-lipoxygenase has been found before in marine invertebrates w5,12x, this is the first reported discovery of 5R-lipoxygenase activity. Catalysis by a single lipoxygenase enzyme is one possible explanation for the synthesis of both 5R- and 8R-hydroperoxides in S. solidissima oocytes. Formation of 5R and 8R products involves a simple ‘frame shift’ along the carbon chain of arachidonic acid Ž Fig. 7., and dual positional specificity of this type is precedented in certain other lipoxygenases. For example, the rabbit reticulocyte 15S-lipoxygenase forms 12S-HPETE and 15S-HPETE in a ratio of approximately 1:9 w27x, and the porcine leukocyte 12S-lipoxygenase forms the same two products in opposite proportions w28x. The phenomenon of dual positional specificity is shown also by lipoxygenases with singular specificity when they are presented with syn-
Fig. 7. Relationship of 5R-lipoxygenase and 8R-lipoxygenase activities. By analogy with other lipoxygenases, enzymatic reaction is expected to be initiated by abstraction of a hydrogen Žindicated with a curved arrow. from C-7 Ž5R-lipoxygenase. or C-10 Ž8R-lipoxygenase. of arachidonic acid.
thetic polyunsaturated fatty acids that have ‘frameshifted’ double bonds w29x. In the case of the lipoxygenase activity in S. solidissima oocytes, we obtained some chromatographic evidence that two distinct enzymes are present Ž Fig. 6. . Each data point of lipoxygenase activity in Fig. 6 is based on an incubation and RP-HPLC analysis that can readily distinguish the products, 5-HŽP.ETE and 8-HŽP.ETE., and therefore there is no doubt that resolution of 5R- and 8R-lipoxygenase activities was achieved in this experiment. However, we were not able to reproduce this result. A factor that may contribute to this inconsistency relates to the finding of major changes in the proportions of 5RHETE and 8R-HETE formed under different conditions by S. solidissima oocyte extracts. In an early season sample, collected before the ovary was ripe with oocytes responsive to serotonin, the only significant product was 5-HETE. When ripe ovaries became available, the oocytes formed mixtures of 5-HETE and 8-HETE Ž typified by the chromatogram in Fig. 1.. On several occasions, oocyte homogenates that originally made a certain mixture of 5-HETE and 8-HETE gave a completely different profile after storage at y708C for a few days or weeks. We should note here that there is a recent precedent in the literature describing the apparent changing of lipoxygenase positional specificity: Shen and colleagues reported that a human 15-lipoxygenase transgenically expressed in rabbit macrophages formed variable proportions of 12- and 15-HETE when the cells were harvested and incubated with arachidonic acid ex vivo w30x. Under the conditions of our experi-
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ments it appeared that the 5R-lipoxygenase could disappear on storage at y708C and be replaced by 8R-lipoxygenase activity. Similarly, the activities were found to change in an unpredictable way when samples were fractionated with ammonium sulfate or passed through an ion exchange column. Based on these observations, it remains an open issue whether there exist separate 5R- and 8R-lipoxygenase enzymes in S. solidissima oocytes. Formation of the keto fatty acid derivatives in S. solidissima oocytes occurs exclusively from the corresponding hydroperoxide, not from the HETE. The reaction appears to be mainly enzymatic in character, although some conversion is observed in the boiled 10 000 = g pellet fraction. Whether this reaction is of significance in vivo is unknown. The enzymatic formation of keto products directly from the corresponding hydroperoxides parallels closely the reported conversion of 12-HPETE and 15-HPETE directly to their 12-keto and 15-keto derivatives in microsomes from neonatal rat epidermis, and also their further conversion to eicosatrienoic acids w31,32x, as we observed for 8R-HETE in intact oocytes. Neither arachidonic acid, 5R-HETE or 8R-HETE, nor related derivatives, have activity on induction of oocyte maturation in S. solidissima. This appears to parallel a situation that applies to approximately half of all species of starfish – the oocytes have the capacity to synthesize a product that is an active inducer of maturation in other related species. In the case of the surf clam, the work of Varaksin et al. indicates that arachidonic acid and 5-HETE are active inducers of maturation in S. sachalinensis w10x. Although the chirality of the 5-HETE used by Varaksin et al. is not specified in their paper, it was probably racemic 5-HETE produced by the convenient chemical transformation from arachidonic acid w13x. It seems very likely that activity will be found to be restricted to the R-HETE enantiomers, just as 8R-HETE is the only active enantiomer in starfish oocytes w5x. We would anticipate also that the activity of arachidonic acid in S. sachalinensis is related to its metabolism to 5R-HETE andror 8R-HETE. It also remains to be determined what, if any, is the active metabolite of linoleic acid in S. sachalinensis. The lack of maturation-inducing activity of eicosapentaenoic acid and its 5-hydroxy derivative 5-HEPE in S. sachalinensis represents a most striking difference from the 20.4v 6
analogs. This contrasts with the situation in starfish in which both the 20.4v 6 and 20.5 v 3 fatty acids and their 8R-hydroxy derivatives are active in the ‘responding’ species of starfish w5x. The role of the lipoxygenase enzymes in the ‘non-responders’, which in the surf clam we have shown to include S. solidissima, remains an open question.
Acknowledgements This work was supported by NIH grants GM15431, GM-49502 and HD-05970. A fellowship for T.H. was provided by Teijin Ltd.
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