Received for publication June 23, 1987 and in revised form August 31, jasmonic acid pathway in plants (19). As a part of these studies,

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1 Plant Physil. (1987) 85, /87/85/ 173/6/$1./ Pathways f Fatty Acid Hydrperxide Metablism in Spinach Leaf Chlrplasts Received fr publicatin June 23, 1987 and in revised frm August 31, 1987 BRADY A. VCK* AND DON C. ZMMRMAN United States Department fagriculture, Agricultural Research Service, Metablism and Radiatin Research Labratry, State University Statin, Farg, Nrth Dakta 5815 ABSTRAC The metablism f 13-hydrperxylinlenic acid was examined in prtplasts and hmgenates prepared frm mature leaves f spinach (Spinacia leracea L.). Chlrplast membranes were the principal site fr metablism f the cmpund by at least tw highly hydrphbic enzyme systems, hydrperxide lyase and hydrperxide dehydrase, the new name fr an enzyme system frmerly knwn as hydrperxide ismerase and hydrperxide cyclase. Hydrperxide lyase was mst active abve ph 7 and culd be separated frm hydrperxide dehydrase by anin exchange chrmatgraphy. Hydrperxide dehydrase, measured by the frmatin f bth a-ketl prduct and 12-x-phytdienic acid, had its ptimum activity in the range f ph 5 t 7. Lyase was mre active than dehydrase activity when the enzymes were extracted by hmgenizatin. The reverse was true when the enzyme activities were measured in prtplasts, which are islated by gentle extractin methds. The variatin in enzyme activity ratis with extractin methds suggests that hydrperxide lyase is activated by plant injury and thus may functin in a wund respnse. n the absence f injury, the nrmal pathway f fatty acid hydrperxide metablism is prbably by hydrperxide dehydrase activity. The mlecular weights f bth the lyase and dehydrase were apprximately 22,, as estimated by gel filtratin. Fatty acid hydrperxides are prduced in plants frm plyunsaturated fatty acids and xygen in a reactin catalyzed by lipxygenase. Linleic and linlenic acids are the mst abundant plyunsaturated fatty acids in plants, and therefre are the mst likely substrates fr lipxygenase catalysis. Tw majr pathways fr the metablism f fatty acid hydrperxides have been characterized in plants (Fig. 1). n ne pathway, HL' catalyzes the cleavage f 13-hydrperxylinlenic acid int 12-x-ddecenic acid and hexenal. n the ther pathway, HD transfrms its substrate int a shrt-lived allene xide (8) that quickly underges hydrlysis t frm a- and y-ketls r rearranges t frm the cyclic cmpund 12-x-PDA. Prir t the characterizatin f the allene xide intermediate by Hamberg (8), the enzyme system respnsible fr the frmatin f ketls was referred t as hydrperxide ismerase (22), and the system that frmed 12-x- PDA was called hydrperxide cyclase (24). The latter prduct is a key intermediate in the bisynthesis f jasmnic acid, a cmpund that has grwth regulating prperties. Our recent investigatins have cncentrated n the rle f the 'Abbreviatins: HL, hydrperxide lyase; 12-x-PDA, 12-xcis,cis-1,15-phytdienic acid; topc-8:, (lr,2r)3-x-2-(2'-pentenyl)cyclpentanectanic acid; GAPDH, glyceraldehyde-3-phsphate dehydrgenase; HD, hydrperxide dehydrase. jasmnic acid pathway in plants (19). As a part f these studies, this paper will shw that a key enzyme f the jasmnic acid pathway, HD, is lcalized in the chlrplasts f spinach leaves. The study als shws that spinach chlrplasts pssess bth the HL and HD pathways f fatty acid hydrperxide metablism. These results supplement and clarify earlier investigatins f fatty acid hydrperxide metablism in plants. n the past, investigatrs f hydrperxide metablism have usually fcused n nly ne f the abve pathways in the study f a particular plant, and have nt dealt with the pssibility that bth pathways have the ptential t functin simultaneusly. MATRALS AND MTHODS Chemicals. Linleic and linlenic acids were purchased frm NuChek Prep, nc.,2 lysian, MN. Sybean lipxygenase, Percll, and Tritn X-OOR (reduced frm f Tritn X-1) were frm Sigma Chemical C. Cellulysin and macerase were purchased frm Calbichem. Wacker silicne ils AR2 and AR2 were btained frm SWS Silicnes Crp., Adrian, M. BCA prtein assay reagent was a prduct f Pierce Chemical C. Nrmal phase HPTLC plates and LKC8F TLC plates were frm Whatman, nc. Plant Material and xtractin Prcedures. Spinach bunches (Spinacia leracea L., Califrnia-grwn) were purchased at a lcal supermarket. Spinach extracts fr clumn chrmatgraphy were prepared by first remving the midvein and then cutting the leaves int small pieces with a razr blade. A Brinkmann PT 1/35 Plytrn hmgenizer was used t hmgenize 1 g f leaves in 3 ml f.2 M K-phsphate (ph 7.5), cntaining.1 % Tritn X- OOR. After filtratin thrugh 7 gm nyln mesh, the extract was centrifuged twice at 12,g t remve cellular debris. Fr studies f enzyme ph ptimum, the extractin was cnducted at ph 7. and the rati was 1 ml f extractin buffer fr each g f leaves. Spinach chlrplasts frm 35 g f leaves were prepared in lw catin medium by the methd f Nakatani and Barber (12). The chlrplasts were resuspended in 4 ml f the lw catin medium and then further purified n linear Percll gradients by a prcedure similar t that described by Haas et al. (7). The gradients were prepared frm 14 ml f.33 M srbitl in Percll adjusted t ph 7.6 and 14 ml f lw catin medium, which cnsisted f.33 M srbitl adjusted t ph 7.6 with.5 mm Tris base. After centrifugatin fr 2 min at 33g, the gradient was separated int 2-ml fractins by pumping frm the bttm f the gradient, and the fractins were assayed fr varius enzymes. The intact chlrplasts present in the lwer band were then resuspended in 2 Names f prducts are included fr the benefit f the reader and d nt imply endrsement r preferential treatment by the United States Department f Agriculture. 173

2 174 VCK AND ZMMRMAN Plant Physil. Vl. 85, OOH 13-hdrerxllnbn 13-hydrperxyllnlenlc c"i acid d tra-2-hsxwf 12-x-fW acid cis-3-hexml lye.. drex reerreigenmet -COOH cyclizatin C O O 12-x-POA 1 N-CO _,, COOH jeemnic H~~O OOH + Z CO. 12-x--x-9-d d s b-_c nis. xide QH HCOOH COO H 12,13-kele COCOH 9,12-lull FG. 1. Pathways f 13-hydrperxylinlenic acid metablism. 2 ml f lw catin medium and centrifuged at 22g fr 2 After the supernatant was decanted, the chlrplasts were ruptured by resuspending them in 4 ml f 1 mm Tris (ph 7.5) cntaining mm DTT, and then recentrifuged n a Percll gradient identical t the ne described abve. Spinach leaf prtplasts were prepared by remving the midvein f the leaf and then slicing the leaf int mm strips. strips were flated fr 4 h in a slutin cntaining1% Cellulysin and.5% Macerase in.5 srbitl, 1 mm calcium chlride, and 5 mm Mes (ph 6.). The strips were then swirled and the brei filtered thrugh 7,m nyln mesh. After centrifugatin fr 5 min at5g 1 the sedimented prtplasts were purified by prcedure described by dwards et al (5). The rapid separatin f chlrplasts frm prtplasts was accmplished by silicne centrifugatin accrding t Rbinsn and Walker (13). The prtplasts, resuspended in 1 t,ul 15 f lw catin medium, were placed in a reservir n tp f a 4 ul tube. A 2 gm nyln mesh frmed the bttm supprt f the reservir. The lwer prtin f the tube cntained,ul 5 f.4 m sucrse as bttm phase,,l 1 f silicne il (19 parts AR2 t part AR2) as the middle phase, and 5 f.4 m srbitl as upper phase. The prtplasts were centrifuged fr 6 s in Beckman Micrfuge B. After passing thrugh the nyln mesh, the cmpnents f the brken prtplasts distributed int the lwer r upper phases f sucrse r srbitl. Gel Filtratin and n xchange Chrmatgraphy. HL and HD were partially purified by gel filtratin and anin exchange chrmatgraphy. A spinach extract (6 ml) was applied t a Sephacryl S-3 clumn (2.5 x 29 cm) and eluted with.2 m phsphate (ph 7.5) cntaining.1% (w/v) TritnX-1OO R. flw rate was 1.9 ml/min and 2.5-ml fractins were cllected. The ml wt f the fatty acid hydrperxide-metablizing enzymes were estimated by their elutin vlumes n this clumn. The ml wt standards were ferritin (44,),,B-amylase (2,), alchl dehydrgenase (15,), bvine serum albumin (67,), and valbumin (43,). Fractins that actively metablized fatty acid hydrperxides were cmbined and applied t a DA Sepharse CL-6B clumn (2.5 x 21 cm). Prteins were eluted with 3 ml f a gradient ranging frm zer t.2 M NaCl in 5 mm K-phsphate (ph 7.), cntaining.1% (w/v) Tritn X-1OOR. The flw rate was 3.2 ml/min and 4.2-ml fractins were cllected. nzyme Assays. 13-Hydrperxylinlenic acid substrate was prepared by reacting 1 ml f 8 mm linlenic acid slutin prepared accrding t Surrey (14) with 32 ml f a slutin cntaining 2 mg f sybean lipxygenase in.6 mm brate (ph 9). After 2 min at rm temperature, the hydrperxide substrate was ready fr use. The cncentratin f the substrate slutin was abut 23 Mm (estimated frm a mlar absrptivity f 25 L ml-' cm-'). HL and HD activities were measured simultaneusly in a GC assay with the use f an internal standard. The reactin mixture cntained abut 35 um 13-hydrperxylinlenic acid substrate (prepared as described abve),.5 t 2. ml f enzyme slutin, and 5 mm K-phsphate (ph 7.) in a final vlume f 1 ml. After 1 min the reactin was stpped by the additin f1 ml f 1 N HC cntaining 1 nml f topc-8: (2) as an internal standard. The lipid prducts were cllected by passing the reactin mixture thrugh a precnditined C,8 slid phase extractin clumn and eluting with diethyl ether. T remve the small amunt f water present in the ether, the slutin was briefly centrifuged and the ether layer carefully decanted t a clean test tube. The prducts were esterified with diazmethane and the ether evaprated under a stream f N2. T remve Tritn X- OOR intrduced with the enzyme, the sample was disslved in ml f hexane, applied t a silica extractin clumn, and eluted with 5 ml f diethyl ether. Under these cnditins the xygenated fatty acid metablites were eluted, whereas the Tritn X-1OOR remained n the clumn. The slvent was evaprated and the prducts were redisslved in 3,l f hexane. The amunt f prducts frmed was determined by the injectin f an aliqut f the sample int a Hewlett-Packard 588 gas chrmatgraph equipped with a 25 m x.31 mm (i.d.) methyl silicne clumn (Hewlett-Packard). The gas chrmatgraph was perated in the splitless mde and the temperature was prgrammed frm 9 t 27 C at1 C/min. Hydrperxide lyase activity was measured by the use f the internal standard t calculate the amunt f 12-x-trans- 1 -ddecenic acid frmed. This prduct pssesses ana,,3-unsaturated aldehyde grup that reacts in varying degrees with diazmethane t frm a cyclprpane ring at the duble bnd psitin (2). Therefre, this esterificatin by-prduct was included in the calculatin f the lyase activity. Hydrperxide dehydrase activity was similarly determined by measuring the amunt f a-ketl r 12-x-PDA prducts that resulted frm the hydrlysis r rearrangement, respectively, f the allene xide. NADP+-dependent GAPDH was measured accrding t the methd reprted by Heber et al. (1). The relative prtein cncentratin f fractins frm the gel filtratin and anin exchange clumns was estimated by their absrptin at 28 nm. Fr the specific activity determinatins at varying ph, prtein was assayed with Pierce BCA prtein assay reagent, with BSA as the standard. Chl was estimated by its absrptin at 652 nm (1). Characterizatin f Metablites. Metablites were prepared by the reactin f 13-hydrperxylinlenic acid with a spinach enzyme preparatin that had been partially purified n a Sephacryl S-3 clumn. The reactin mixture cntainedgm hydrperxylinlenic acid, 12 ml f partially pure enzyme, and 5mm K-phsphate (ph 5.3; the lw ph was used t reduce the

3 activity f HL). After 1 h the mixture was adjusted t ph 3 and the slutin was passed thrugh a C18 slid phase extractin clumn. The metablites were eluted with diethyl ether and esterified with diazmethane. The prducts were reacted vernight with.1 ml f 2% (w/v) methxyamine-hc in pyridine t prepare the methxime derivatives. Water (5 ml) was added, the mixture was adjusted t ph 3, and then passed thrugh anther C,8 slid phase extractin clumn. After elutin with diethyl ether, the slutin was dried with anhydrus sdium sulfate and cncentrated t 2,l. The sample was analyzed with a Hewlett-Packard 5992 gas chrmatgraph-mass spectrmeter that had a cl, n-clumn injectr and a 25 m x.31 mm i.d. crss-linked methyl silicne clumn (Hewlett-Packard). The temperature was prgrammed frm 12 t 25 C at 1 C/min. HYDROPROXD MTABOLSM N SPNACH RSULTS ntracellular Lcatin f nzymes f Fatty Acid Hydrperxide Metablism. Purified spinach prtplasts were subjected t centrifugatin thrugh a zne f silicne il psitined between a layer f.4 M sucrse belw it and a layer f.4 M srbitl abve it. With this technique, the prtplasts were ruptured as they passed thrugh a 2,um nyln mesh, and the cellular cntents then mved int the upper srbitl phase. Accrding t Rbinsn and Walker (13), nly the chlrplasts mve thrugh the zne f silicne il and int the lwer sucrse phase. Table shws the results f a typical experiment in which apprximately 75, prtplasts were subjected t silicne il centrifugatin. The majrity f the activity (67%) f the chlrplast marker enzyme, NADP+-GAPDH was fund in the lwer sucrse phase. Likewise, HL and HD activities were predminantly in the chlrplast fractin. The smewhat higher percent f recrded HL (81%) can prbably be attributed t increased experimental variability frm interfering chrmatgraphic peaks that becme mre significant at lw levels f enzyme activity. We nted that in sme silicne il centrifugatin experiments there was minr cntaminatin f the lwer phase with catalase and Cyt c xidase, raising the pssibility that perxismes r mitchndria may als pssess enzymes that metablize fatty acid hydrperxides. Hwever, additinal experiments in which spinach leaf rganelles were separated n a discntinuus Percll gradient indicated that there was n metablism f fatty acid hydrperxides in perxismes r mitchndria (data nt shwn). The results suggest that the spinach enzymes that metablize fatty acid hydrperxides reside principally in the chlrplasts. An additinal experiment was cnducted t determine whether the enzymes f fatty acid hydrperxide metablism were present in the strma r in the membranes f the chlr- Table. Distributin fnzyme Activities between Chlrplast and Cytplasm Fractins fspinach LeafPrtplasts Spinach leaf prtplasts were subjected t a silicne il centrifugatin prcedure (13) in which chlrplasts were separated frm the cytplasm and cytplasmic rganelles. Details f the prcedure are described in "Materials and Methds." Ttal nzyme Activity in Prtplast Fractins nzyme ~~~~~~Lwer, sucrse nzyme Upper, srbitl L r se phase (cytplasm) phase nml/min % nml/min % GAPDH 32.4 (33) 66.5 (67) HL 1.6 (19) 7. (81) HD (12-x-PDA) 5.4 (35) 1.1 (65) HD (a-ketl).6 (26) 1.7 (74) plast. Spinach chlrplasts that were prepared by differential centrifugatin were further purified n a Percll gradient. Figure 2 shws that HL and HD enzymes were present bth in the lwer band f intact chlrplasts and in the upper band cnsisting primarily f brken chlrplast membranes. This result suggested that the enzymes are assciated with the membranes f the chlrplasts. n cntrast, the chlrplast marker enzyme NADP+-GAPDH was nly active in the intact chlrplasts, as expected fr a strmal enzyme. The assciatin f the enzymes f fatty acid hydrperxide metablism with the chlrplast membrane was cnfirmed in an additinal experiment in which the intact chlrplasts were ruptured by smtic shck and then repurified n an identical Percll gradient (Fig. 3). This time the activity f the three enzymes was lcated in the lighter band f brken membranes. xcept fr a small amunt f a-ketl frmatin, n enzymes f hydrperxide metablism were evident in the upper regin f the gradient which cntained sluble enzymes f the strma, such as NADP+-GAPDH. We rutinely nticed that when chlrplasts were prepared by hmgenizatin and purificatin n Percll gradients, high amunts f lyase prducts were synthesized relative t 12-x- PDA (Fig. 2). Cnversely, 1 2-x-PDA synthesis was cnsistently higher than the frmatin f lyase prducts in chlrplasts islated frm prtplasts (Table ). Partial Purificatin f nzymes and Mlecular Weight Determinatin. lutin f a spinach extract frm a Sephacryl S-3 clumn failed t separate the HL and HD enzyme activities (Fig. a: C w'c W P L. x - B z 1.35 i, L m A < *6 L3 L,.4 w a. -, *2 p n cn 2w U LU 175 w - c FRACTON FG. 2. Percll gradient f spinach chlrplasts islated by differential centrifugatin in lw catin buffer. The lwer band cnsists f intact chlrplasts, and the upper band is cmpsed f brken chlrplast membranes. (A), Chl; (A), GAPDH, chlrplast marker enzyme; (O), HL; (), HD (a-ketl frmatin); (U), HD ( 2-x-PDA frmatin); (), refractive index.

4 176 : a 9 43 ' _ Xs. a C.*.- 'a FRACTON VCK AND ZMMRMAN x La z 1.35 *1.345 i *1.34 w (flc :< - a c -.C: an w N C ' FG. 3. Percll gradient f chlrplast membranes btained by the breakage f intact chlrplasts (lwer band, Fig. 1) thrugh smtic shck. (A), Chl; (A), GAPDH, chlrplast marker enzyme; (), HL; (), HD (a-ketl frmatin); (U), HD (12-x-PDA frmatin); (), refractive index. z 2 a 2 c N cn.2 X( c x Ux -a FRACTON LaJ nc L U N,- :c w a cn t hj 2._ cn a N - FG. 4. Separatin f enzymes f fatty acid hydrperxide metablism n a Sephacryl S-3 clumn (2.5 x 29 cm). (), HL; (), HD (aketl frmatin); (U), HD(1 2-x-PDA frmatin); (), prtein. Plant Physil. Vl. 85, ). The ml wt f the tw enzymes was estimated at 22, by cmparisn f their elutin vlumes with thse f knwn ml wt standards. When Tritn X-1OOR was remved frm the elutin buffer, the activities eluted with the vid vlume. This suggests that the enzymes are highly hydrphbic and, in the absence f a detergent, aggregate t frm high ml wt cmplexes. Such a degree f hydrphbicity wuld be expected frm enzymes assciated with the chlrplast membranes. The fractins frm the Sephacryl S-3 clumn that metablized fatty acid hydrperxides were cmbined and applied t a DA Sepharse CL-6B anin exchange clumn. When Tritn X-OOR was nt present with the enzyme and the elutin buffer, the enzymes adsrbed strngly t the DA Sepharse CL-6B clumn and culd nt be eluted even in the presence f 1 M NaCl. Hwever, when Tritn X-OOR was added t the buffer, the enzymes shwed very weak adsrptin t the anin exchanger (Fig. 5). The strng adsrptin f hydrphbic prteins t Sephadex and Sepharse clumns in the absence f detergent has been bserved by thers and may be due t a strng interactin with the hydrphbic ether prtin f the resin (11). Chrmatgraphy n the DA Sepharse CL-6B clumn shwed that, althugh nt cmpletely separated, there is a clear distinctin between HL and HD (Fig. 5). This was nt true with every sample f spinach tested. With many samples f spinach that were examined, the enzymes f fatty acid hydrperxide metablism culd nt be separated by the DA Sepharse CL- 6B clumn. Whether these incnsistencies were due t varietal, envirnmental, r prcedural differences is nt knwn. ffect f ph n nzymes f Fatty Acid Hydrperxide Metablism. There was a marked difference between the ph activity prfile f HL and HD (Fig. 6). The activity f HL decreased sharply belw ph 7, but was still high even at ph 9, the highest ph tested. n cntrast, the frmatin f 12-x-PDA (Fig. 6B) and a-ketl (Fig. 6C) as a result f HD activity had similar ph activity prfiles in which bth prcesses were mst active in the range f ph 5 t 7. Characterizatin f Metablites. When 13-hydrperxylinlenic acid was reacted with a spinach enzyme preparatin that had been partially purified n a Sephacryl S-3 clumn, three majr prducts were identified in the reactin mixture. The HL prduct (R, = 6.85 min), was identified by its methxime, methyl ester derivative as 12-x-trans-1 -ddecenic acid by the characteristic ins at m/z 255 [MJt and 224 [M-OCH3]t, as earlier reprted by Zimmerman and Cudrn (23). The secnd prduct (R, = 12.1 min) was identified as 12-x-PDA, the rearrangement prduct f the allene xide. Mass fragments f m/z 335 [M]t, 34 [M-OCH3]t, and 266 [M-C5H9 + H]t were characteristic f the methxime, methyl ester f 12-x-PDA (18). The third prduct (R, = min) was characterized as the a-ketl hydrlysis prduct f the allene xide. Distinguishing mass fragments f the methxime, methyl ester derivative f 13-hydrxy-12-x-cis,cis-9,15-ctadecadienic acid were m/z 322 [M-OCH3Jt, 36 [322-H2O]t, 284 [M-C5H9]t, 252 [284-CH3H]t, and 192 [M-C6H, -(OCH3)2]t. DSCUSSON Hamberg (8) has recently characterized an intermediate in the enzymic cnversin f 13-hydrperxylinleic acid t a-ketl. The cmpund, 12,13-(S)-xid-9(Z), 1 1-ctadecadienic acid, has a half-life f abut 33 s at C. This prduct is frmed by the enzyme-atalyzed lss f water frm the hydrperxide. A similar allene xide intermediate wuld be expected in the reactin with 13-hydrperxylinlenic acid (Fig. 1). Nnenzymic hydrlysis f the allene xide intermediate wuld lead t the frmatin f a- and y-ketls. n previus literature this prcess was attributed t a hydrperxide ismerase enzyme. Alternately, the allene xide can underg nnenzymic rearrangement

5 HYDROPROXD MTABOLSM N SPNACH 177 z a. i (N ffi c 3 lu D X 2 W 1 llj < ~J U wl L. x - C LL tlx _ L. cn <: - L C] C V a O L ui X FG. 5. Separatin f enzymes f fatty acid hydrperxide metablism n a DA Sepharse CL-6B clumn (2.5 x 21 cm). (OJ), HL; (), HD (a-ketl frmatin); (-), HD (12-x- PDA frmatin); (x), prtein. FRACTON with ring clsure t frm 12-x-PDA, a prcess previusly credited t a hydrperxide cyclase enzyme. The rearrangement reactin f the allene xide mst likely ccurs thrugh the frmatin f an xidpentadienyl catin with antarafacial ring clsure. The same mechanism has been suggested fr the synthesis f similar cyclic cmpunds in the cral Clavularia viridis (4). Thus, Hamberg has prpsed that the names 'hydrperxide ismerase' and 'hydrperxide cyclase' be discntinued and replaced by the term 'hydrperxide dehydrase.' Accrding t the prpsed scheme, bth ketls and 12-x-PDA are prducts f the hydrperxide dehydrase reactin. Hwever, nly the first step, the frmatin f the allene xide, is enzymic. Because the hydrperxide dehydrase enzyme can give rise t tw prducts in sme kind f nnrandm manner, we wuld like t suggest that a parenthetical expressin be added t the name t indicate the specific rute f allene xide transfrmatin under discussin. Fr example, the cyclic rearrangement path wuld be referred t as hydrperxide dehydrase (cycl), r HD (cycl); the hydrlysis path wuld be hydrperxide dehydrase (ketl), r HD (ketl). The reactin mechanism prpsed by Hamberg is cnsistent with bservatins by us in this reprt and previus reprts cncerning the prperties f hydrperxide ismerase and hydrperxide cyclase. These tw activities always c-elute n gel filtratin and anin exchange clumns (16, 17, 2). The tw activities have never been separated, and this prperty prmpted speculatin that bth activities were assciated with the same prtein. The similarity in ph activity prfiles (Fig. 6, B and C) als supprts the suppsitin that the tw enzyme activities are attributable t the same prtein. Past investigatins int fatty acid hydrperxide metablism have typically fcused n either the HL system r HD system. This may have led t the incrrect impressin that the tw systems are mutually exclusive in plants. The results presented here demnstrate that bth systems are ptentially functinal within a given plant tissue. n spinach leaves the principal intracellular site fr the tw systems is the chlrplast. The identificatin f chlrplasts as a site f metablism f fatty acid hydrperxides is nt new. Chlrplasts frm cucumber peel (21), tea leaf (9), and endive (6) have previusly been shwn t have high HL activity. Hwever, the demnstratin f HD activity in chlrplasts has nt been previusly reprted. t is nw clear that in spinach leaves the HL enzyme and the HD enzyme cexist in the chlrplast, thus creating tw divergent pathways fr fatty acid hydrperxide metablism. nzyme assays with purified, brken chlrplasts indicated that bth enzyme systems are predminantly assciated with the membranes. The results d nt specifically exclude the chlrplast envelpe as a site fr hydrperxide metablism, but we believe, as thers have cncluded (6, 9), that the thylakid membranes are the principal site fr metablism f fatty acid hydrperxides in phtsynthetic tissue. As wuld be expected fr prteins bund t membranes, bth HL and HD are highly hydrphbic enzymes. This was evident frm the tendency f the enzymes t aggregate in the absence f detergent. t was als apparent frm the strng binding bserved when the enzymes were applied t a DA Sepharse CL-6B clumn. This binding was clearly due t hydrphbic interactins because the enzymes were nt eluted by high inic strength eluents, but were quickly eluted by the additin f Tritn X- loor detergent. A curius feature f this research was ur inability t demnstrate lipxygenase activity in spinach leafextracts. Lipxygenase culd nt be detected by either spectrphtmetric assays r plargraphic (2 cnsumptin) assays at varying phs. Whether lipxygenase was truly absent r inhibitrs were present is nt knwn. t is unlikely that spinach leaves have n lipxygenase activity because that wuld restrict HL and HD t substrates that arise frm autxidatin r phtchemical xidatin. n cntrast t ur wrk, Takagi et al. (15) have reprted the purificatin f lipxygenase frm the chlrplasts f spinach leaves. The enzyme was lcated n the internal surface f the chlrplast, and nt n the uter surface. t is pssible that lipxygenase inhibitrs such as Chl (3) r cartenids (15) may have been respnsible fr the lack f lipxygenase activity in ur spinach extracts. The presence f tw pathways fr fatty acid hydrperxide metablism suggests that certain physilgical cntrls exist fr directing the hydrperxide t the HL r the HD pathway. One pssible mechanism culd be thrugh the alteratin f ph in the micrenvirnment fthe HL r HD enzymes. At lw ph ranges,

6 178 VCK AND ZMMRMAN Plant Physil. Vl. 85, 1987 mechanical stress the nrmal pathway f fatty acid hydrperxide metablism is prbably the HD pathway. Acknwledgment-We acknwledge the skillful technical assistance f Diana Cardwell. ph FG. 6. ffect f ph n the activity f(a) HL, (B) HD 12-x-PDA frmatin), and (C) HD (a-ketl frmatin). (),.1.1 K-phsphate; (A),.1 Tris. Na-acetate; (U), like thse expected inside the thylakid lumen during the light reactin f phtsynthesis, HL wuld be relatively inactive (Fig. 6) and HD wuld predminate. n regins f high ph, HL wuld be mre active. Anther factr relevant t the divergence f the HL versus HD pathways was bserved in the experiments with the spinach prtplasts. HL activity was lw in relatin t 12- x-pda synthesis when the enzymes riginated frm prtplasts (Table). But when the enzymes were prepared by hmgenizatin f spinach leaves, lyase prducts were frmed mre rapidly than 12-x-PDA (Figs. 2-6). This suggests that HL activity is stimulated by harsh extractin techniques. n viv this culd mean that HL is activated when the plant tissue is wunded r subjected t mechanical stress. Under cnditins f lw LTRATUR CTD 1. ARNON D 1949 Cpper enzymes in islated chlrplasts. Plyphenlxidase in Beta vulgaris. Plant Physil 24: BLACK TH 1983 The preparatin and reactins f diazmethane. Aldrichimica Acta 16: COHN B-S, S GROSSMAN, A PNSKY, BP KLN 1984 Chlrphyll inhibitin f lipxygenase in grwing pea plants. J Agric Fd Chem 32: CORY J, M D'ALARCAO, SPT MATSUDA, PT LANSBURY JR, Y YAMADA 1987 ntermediacy f 8-(R)-HPT in the cnversin f arachidnic acid t preclavulne A by Clavularia viridis. mplicatins fr the bisynthesis f marine prstanids. J Am Chem Sc 19: DWARDS G, SP ROBNSON, NJC TYLR, DA WALKR 1978 Phtsynthesis by islated prtplasts, prtplast extracts, and chlrplasts f wheat. Plant Physil 62: GOTZ-SCHMDT -M, M WNZL, P SCHRR 1986 C6-Vlatiles in hmgenates frm green leaves: lcalizatin f hydrperxide lyase activity. Lebensm-Wiss u-technl 19: HAAS R, HP SBRTZ, K WRAG, HNZ 198 Lcalizatin f sulflipid labeling within cells and chlrplasts. Planta 148: HAMBRG M 1987 Mechanism f crn hydrperxide ismerase: detectin f 12,13(S)-xid-9(Z), 1 -ctadecadienic acid. Bichim Biphys Acta 92: HATANAKA A, T KAJWARA, J SKYA, S NOUY 1982 Slubilizatin and prperties f the enzyme-cleaving 13-L-hydrperxylinlenic acid in tea leaves. Phytchemistry 21: HBR U, NG PON, M HBR 1963 Lcalizatin f carbxydismutase and trisephsphate dehydrgenases in chlrplasts. Plant Physil 63: JOHNsN RA, DL GARBRS, SJ PLKS 1976 Sme kinetic and chrmatgraphic prperties f detergent-dispersed adenylate cyclase. J Supraml Struct 4: NAKATAN HY, J BARBR 1977 An imprved methd fr islating chlrplasts retaining their uter membranes. Bichim Biphys Acta 461: ROBNSON SP, DA WALKR 1979 Rapid separatin f the chlrplast and cytplasm fractins frm intact leaf prtplasts. Arch Bichem Biphys 196: SURRY K 1964 Spectrphtmetric methd fr determinatin f lipxidase activity. Plant Physil 39: TAKAG S, M MATSUGAM, T MORTOK 1977 ffect f cartenid n leaf lipxygenase activity.. Purificatin f spinach chlrplast lipxygenase and the inhibitin by lutein t its activity. Okayama Daigaku Ngakubu Gakujutsu Hkku 49: VCK BA, DC ZMMRMAN 1981 Lipxygenase, hydrperxide ismerase, and hydrperxide cyclase in yung cttn seedlings. Plant Physil 67: VCK BA, DC ZMMRMAN 1981 Partial purificatin f hydrperxide ismerase and hydrperxide cyclase frm flaxseed. Plant Physil 67: S VCK BA, DC ZMMRMAN 1984 Levels f xygenated fatty acids in yung crn and sunflwer seedlings. Plant Physil 69: VCK BA, DC ZMMRMAN 1984 Bisynthesis fjasmnic acid by several plant species. Plant Physil 75: VCK BA, DC ZMMRMAN 1986 Characterizatin f 12-x-phytdienic acid reductase in crn. The jasmnic acid pathway. Plant Physil 8: WARDAL DA, A LAMBRT, T GALLARD 1978 Lcalizatin f fatty acid hydrperxide cleavage activity in membranes f cucumber fruit. Phytchemistry 17: ZMMRMAN DC 1966 A new prduct f linleic acid xidatin by a flaxseed enzyme. Bichem Biphys Res Cmmun 23: ZMMRMAN DC, CA COUDRON 1979 dentificatin f traumatin, a wund hrmne, as 12-x-trans-1-ddecenic acid. Plant Physil 63: ZMMRMAN DC, P FNG 1978 Characterizatin f a prstaglandin-like metablite f linlenic acid prduced by a flaxseed extract. Lipids 13:

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