a-Hydroxylation of Oleic Acid in Vicia sativa Microsomes - NCBI

Plant Physiol. (1993) 102: 1313-1318

a-Hydroxylation of Oleic Acid in Vicia sativa Microsomes' lnhibition by Substrate Analogs and lnactivation by Terminal Acetylenes

Franck Pinot**, Hubert Bosch, Carole Alayrac, Charles Mioskowski, Alain Vendais, Francis Durst, and Jean-Pierre Salaun lnstitut de Biologie Moleculaire des Plantes-Centre National de Ia Recherche Scientifique, Département d'Enzymologie Cellulaire et Moléculaire, 28 rue Goethe, F-67083 Strasbourg Cedex, France (F.P., F.D., J.-P.S); Centre National de Ia Recherche Scientifique-Faculté de Pharmacie, Laboratoire de Synthèse Bio-Organique, 74 route du Rhin, F-67048 Strasbourg Cedex, France (H.B., C.A., C.M.); and Service des Molécules Marquées, Département de Biologie Cellulaire et Moléculaire, C.E.A. 91 191 Gif-sur-Yvette, France (A.V.)

minor) microsomes, oleic acid is subjected to a cascade of reactions that involve at least three distinct enzymes: a peroxygenase, an epoxide hydrolase, and a Cyt P-450-dependent w-hydroxylase (Pinot et al., 1992). The latter enzymic system is able to w-hydroxylate oleic acid, (Z)-9,10-epoxystearic acid and 9,lO-dihydroxystearic acid. The interplay of these enzymes may account for the formation of the major Cls cutin monomers derived from oleic acid (Kolattukudy, 1981). Different studies performed with mammalian systems suggest that w-hydroxylation by Cyt P-450 enzymes is the first step in fatty acid catabolism (Gibson, 1989). The inactivation of these w-hydroxylases by terminal acetylenic compounds has been extensively studied (Kunze et al., 1983; Ortiz De Montellano and Reich, 1984). Acetylenic fatty acid analogs inactivate lauric acid w-hydroxylases from rat liver both in vitro and in vivo (Cajacob and Ortiz De Montellano, 1986). Cyt P-450LAw, purified from clofibrate-induced rat liver, oxidizes lauric acid to 11- and 12-hydroxydodecanoic acids in a 1:17 ratio (Cajacob et al., 1988). Inhibition of the enzyme with 10-undecynoic acid leads to the inactivation of only one-half of the enzymic activity, suggesting that the preparation contained two distinct lauric acid w-hydroxylases. Similarly, oxygenases, which w-hydroxylate leukotrienes in human polymorphonuclear leukocytes (Shak et al., 1985) and prostaglandins in rabbit lung (Muerhoff et al., 1989), are inactivated by terminal acetylenic fatty acids. Shak et al. (1985) showed that 12-hydroxy-16-heptadecynoicacid inactivates rabbit lung microsomal prostaglandin w-hydroxylase without affecting lauric acid w-hydroxylase. Few studies have addressed the autocatalytic inactivation of fatty acid w-hydroxylases in plant systems. Earlier studies from our laboratory have shown that lauric acid (Salaun et al., 1986) and a series of 6 7-10 Z and E monounsaturated analogs (Weissbart et al., 1992) are cu-hydroxylated in V. sativa microsomes. These w-hydroxylation reactions are in-

Oleic acid (18:l) i s hydroxylated exclusively on the terminal methyl by a microsomal cytochrome P-450-dependent system (wOAH) from clofibrate-induced Vicia sativa 1. (var minor) seedlings (F. Pinot, 1.-P. Salaun, H. Bosch, A. Lesot, C. Mioskowski, F. Durst [1992] Biochem Biophys Res Commun 184: 183-193). This reaction was inactivated by two terminal acetylenes: (Z)-9-octadecen-l7ynoic acid (17-ODCYA) and the corresponding epoxide, (Z)-9,10epoxyoctadecan-17-ynoic acid (17-EODCYA). lnactivation was mechanism-based, with an apparent binding constant of 21 and 32 ~LM and half-lives of 16 and 19 min for 17-ODCYA and 17EODCYA, respectively. We have investigated the participation of one or more w-hydroxylase isoforms i n the oxidation of fatty acids in this plant system. Lauric acid (12:O) is w-hydroxylated by the cytochrome P-450 w-hydroxylase w-LAH (1.-P. Salaun, A. Simon, F. Durst [1986] Lipids 21: 776-779). Half-lives of w-OAH and w-LAH in the presence of 40 p~ 17-ODCYA were 23 and 41 min, respectively. lnhibition of oleic acid w-hydroxylation was competitive with linoleic acid (18:2), but noncompetitive with lauric acid (12:O). In contrast, oleic acid did not inhibit w-hydroxylation of lauric acid. Furthermore, 1-pentadecyltriazole inhibited w-hydroxylation of oleic acid but not of lauric acid. These results suggest that distinct monooxygenases catalyze w-hydroxylation of medium- and longchain fatty acids i n V. sativa microsomes.

Oleic acid and its oxygenated derivatives form an important part of the fatty acids found in cuticular membranes of plants (Kolattukudy, 1981). Previous investigations from our laboratory have demonstrated that, in Vicia sativa L. (var ~~

F.P. and H.B. are currently supported by fellowships from Ministère de la Recherche et de la Technologie and from Centre National de la Recherche Scientifique-Région Alsace. C.A. was supported by RhÔne-Poulenc. This study has been conducted under the BIOAVENIR program: Groupe de recherches "Banières Cuticulaires" financed by Rhone-Poulenc with the contribution of the Ministère de la Recherche et de 1'Espace and the Ministère de 1'Industrie et du Commerce Extérieur. Present address: Department of Entomology, University of California, Davis, CA 95616-8584. * Corresuondine. author; fax 1-916-752-1537.

.,

Abbreviations: 17-EODCYA, (Z)-9,10-epoxyoctadecan-l7-ynoic acid; 17-ODCYA, (Z)-9-octadecen-l7-ynoicacid; K,, binding constant; w-LAH, omega hydroxylase of lauric acid; w-OAH, omega hvdroxvlase of oleic acid. i

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hibited by 11-dodecynoic acid, the terminal acetylenic analog of lauric acid (Salaun et al., 1988; Weissbart et al., 1992). Based on the results obtained with mammalian and plant systems, we have now synthesized (Z)-9-octadecen-l7-ynoic acid and the corresponding (Z)-9,10-epoxide in an effort to inactivate the o-hydroxylases involved in the oxidation of the Cls fatty acid family. These compounds are potential tools for mechanistic studies of these enzymes and for investigating the impact of selective oxygenase inhibitors on the composition of the cuticle. We report here, for the first time, the inactivation of an oleic acid w-hydroxylase by acetylenic analogs of CI8 fatty acids. To investigate the involvement of a single or of multiple forms of Cyt P-450 in w-hydroxylation of lauric and oleic acids, competition and mechanism-based inactivation experiments were performed to compare the inhibition and inactivation of both activities. MATERIALS AND METHODS Chemicals

Radiolabeled [l-'4C]-oleic acid (2.09 GBq/mmol) and [ l ''C]-lauric acid (1.74 GBq/mmol) were from CEA (Gif sur Yvette, France). TLC plates (Silica gel G60 F254) were from Merck (Darmstadt, Germany). NADPH was purchased from Sigma Chimie (La Verpillière, France). 1-Pentadecyltriazole was a generous gift of Drs. Anding and Greiner from RhÔnePoulenc Agrochimie (Lyon, France). Synthesis of 17-ODCYA

8-Bromo-1-octanolwas refluxed with 1.2 eq of triphenylphosphine, in anhydrous acetonitrile, overnight to give (7hydroxyocty1)-triphenylphosphonium bromide (yield: 95%). The alkylidenephosphorane of (7-hydroxyoctyl)-triphenylphosphonium bromide, generated by means of 2 eq of lithium diisopropylamide in tetrahydrofuran at -4OoC, was reacted with 1 eq of 7-bromoheptanal to give 1-bromo-7-pentadecene-15-01 (yield: 66%).The alcohol was protected (Bollit and Mioskowski, 1988) as its tetrahydropyrannyl ether (90%), and the bromide converted to iodide by reaction with Na1 in acetone (97%). The resulting 1-iodo-l5-(tetrahydropyrannyloxy)-7-pentadecenewas reacted with 1.5 eq of the carbanion of trimethylsilylacetylene (generated from trimethylsilylacetylene and n-butyllithium at O°C in tetrahydrofuran) to give 1-trimethylsilyl- 17-(tetrahydropyranny1oxy)-9-heptadecene1-yne with 74% yield. The reagent PPh3/CBr4 then allows the conversion from the tetrahydropyrranyl ether into the corresponding l-trimethylsily1-17-bromo-9-heptadecene1yne (55%). The resulting bromide is substituted by cyanide (as KCN) in DMSO at 80°C to l-trimethylsilyl-17-cyano-9heptadecene-1-yne (86%), which is finally hydrolyzed by 50% aqueous KOH/ethanol to 9-octadecene-17-ynoic acid with 86% yield and as a mixture Z/E of 79/21. A11 intermediates were characterized by 'H and 13C NMR spectroscopy, IR spectroscopy, and elemental analysis. Synthesis of 17-EODCYA

The experimental procedure is very close to phase transfer epoxidation by hydrogen peroxide described by Venturello

Plant Physiol. Vol. 102, 1993

et al. (1983). Tlhe epoxidation was performed on a 0.5-mmol 17-ODCYA sc,ale, the phase transfer catalyst was Adiogen 464, and the reaction time was 2 h. Yield was up to 79'%, Microsomal Preparations

Vicia sativa 1,. (var minor) seedlings were purchased from S.A. Blondeau (Bersie, France). Four-day-old etiolated seedlings were agetl for 48 h in a 1 mM clofibrate solution before isolation of the microsomal fractions as already described (Salaun et al., 1986). Microsomal proteins were quantified by a microassay procedure from Bio-Rad using BSA as a standard. Enzyme Activities and Their lnhibitions

Enzyme activities were measured as described (Pinot et al., 1992; Weissbart et al., 1992) by following by TLC the rate of metabolite formation from radiolabeled substrates cluring incubation of microsomes from clofibrate-induced V. sativa seedlings. The metabolite generated by microsomes from oleic acid has been previously assigned to w-hydroxyoleic acid (Pinot et al., 1992). Lauric acid was converted to 12-hydroxylaurate exclusively (Salaun et al., 1986). The standard assay contained 0.19 to 0.43 mg of microsomal protein, 20 mM phosphate buffer (pH 7.4), and radiolabeled substrate in a final volume of 0.2 mL. w-Hydroxylase activities were measured in the presence of 1 mM NADPH plus a regenerating system (consisting of a final concentration of 6.7 mM Glc-6P and 0.4 IU of Glc-6-P dehydrogenase) and 2.5 rnM Pmercaptoethanol. Concentrations of substrates and inhibitors and times of incubation are mentioned in the legend of each figure. Solvents containing substrates or inhibitors were evaporated under a stream of argon before incubations. Reactions were initiated by adding NADPH at 27OC and stopped with 0.2 mL of acetonitri1e:acetic acid (99.8:0.2, v/v). Reaction products were extracted twice into 1 mL of diethyl ether and resolved by TLC. lnactivation with Mechanism-Based lnhibitors

Following a procedure similar to that already described (Salaun et al., 1988), microsomes were preincubated at 27OC with 1 m~ NADPH plus a regenerating system (see above) in the presence of different concentrations of (Z)-9-octadecen-17-ynoic or (Z)-9,10-epoxyoctadecan-l7-ynoicacids. After different periods of time, a small volume (30 ILL) of preincubated microsomes was added to incubation medium at 27OC that contained [l-'4C]oleic acid or [l-'4C]lauric acid, 1 m~ NADPH, and phosphate buffer in a final volume of 0.2 mL. Incubations were allowed to continue a further 5 min at 27OC and then stopped as described above. Chromatographic Analysis

Metabolites generated from incubations were resolved by silica gel TLC. Plates were developed with a mixture of diethyl ether:light petroleum (bp 40-60°):formic acid (50:50:1, v/v/v). The areas corresponding to 18-hydroxyoleic ( R F = 0.2) and 12-hydroxylauric (RF -- 0.2) acids were scraped directly into vials and quantified by liquid scintillation (Pinot et al., 1992).

w-Hydroxylation of Oleic Acid in Vicia sativa Microsomes

RESULTS

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lnactivation of w-OAH by 17-ODCYA and 17-EODCYA

Microsomes from V. sativa seedlings were preincubated for various lengths of time with 1 mM NADPH and 17-ODCYA at concentrations ranging from 20 to 80 PM, and the residual w-OAH activity was measured (Fig. 1). No inhibition was observed in incubations with 17-ODCYA alone. However, there was a slight but sizable loss of activity in the presence of NADPH alone. This was also observed in similar experiments with mammalian (Cajacob and Ortiz De Montellano, 1986) and other plant sytems (Salaun et al., 1988). Preincubation with 17-ODCYA produced a time- and concentrationdependent inhibition of oleic acid hydroxylation. The inactivation rates followed roughly pseudo first-order kinetics, suggesting autocatalytic destruction of the enzyme. After correction for the activity loss observed with NADPH alone, a Ki of 21 ~ L and M a kinactivation of 7.2 X 10-4 s-l were calculated. The half-life of the enzyme at saturating inhibitor concentration was 16 min. Recently, we demonstrated that cis-9,lO-epoxystearicacid was w-hydroxylated by a Cyt P-450 system in microsomes from V. sativa (Pinot et al., 1992). We have now synthesized 17-EODCYA, the terminal acetylene analog of this substrate, to determine whether w-OAH might be implicated in this reaction. I'reincubation of microsomes with 17-EODCYA produced a concentration- and time-dependent inactivation of w-OAH similar to that produced by 17-ODCYA (data not shown). After correction for the effect of preincubation with NADPH alone, a Ki of 32 PM and a kinactivatianof 6 X 10-4 s-l were determined. The half-life of the enzyme at saturating

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[OLEIC ACID] (mmol)-l Figure 2. lnactivation of w-OAH activity by linoleic acid. Lineweaver-Burk plot of w-OAH activity as a function of oleic acid in the absence (O) and in the presence of 50 p~ (X), 100 PM (O), or 250 /IM (A)linoleic acid. Microsomal incubation time was 30 min. The plot is based on duplicate experimental measurements.

17-EODCYA concentration (19 min) was comparable to that determined for 17-0DCYA, suggesting that w-OAH catalyzes the w-hydroxylation of cis-9,10-epoxystearicacid. Competition with Linoleic Acid

In addltion to oleic acid derivatives, cutin contains minor amounts of oxidized derivatives of linoleic acid, the 18:2 analog of oleic acid. As shown in Figure 2, linoleic acid is a competitive inhibitor of o-OAH. A Ki of 200 PM was calculated, suggesting that the apparent affinity of w-OAH for linoleic acid is considerably lower than the affinity for oleic acid (K, = 110 p ~ ) . Oleic and Lauric Acid Are Hydroxylated by Distinct Cyt P-450 Enzymes

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PREINCUBATION TIME (min) Figure 1. lnactivation of w-OAH by 17-ODCYA. Semilogarithmic plot of w-OAH activity as a function of time of microsome preincubation with NADPH and 20 PM (O), 40 PM (m), or 80 PM (X) 17ODCYA. The activity after preincubation with NADPH alone is also shown (O). The time required to lose half of the activity (t1/2) is given for each concentration of 17-ODCYA and for NADPH incu. w-OAH bated alone. Oleic acid concentration was 128 ~ L MControl activity was 109 pmol min-' mg-' protein.

We had previously reported that lauric acid is w-hydroxylated by a Cyt I'-450 enzyme (a-LAH) in V. sativa microsomes (Salaun et al., 1986). w-LAH was shown to be specific for short (CIO)and medium (C12-cl4) fatty acids (Simon, 1987). Microsomes were preincubated for different lengths of time with NADPH and 40 ~ L M17-0DCYA, and the residual w-hydroxylase activities for oleic and lauric acids were measured (Fig. 3). Both activities were inactivated, but at different rates: the half-life for w-LAH was 41 min compared with 23 min for w-OAH. Without preincubation, 17-ODCYA had no competitive effect on w-LAH activity. The different inactivation rates may indicate that w-hydroxylation of oleic and lauric acids are catalyzed by distinct Cyt P-450 isoforms. To further investigate this hypothesis, we performed competition experiments. Figure 4 shows that lauric acid is a noncompetitive inhibitor of w-OAH activity. In contrast, w-LAH activity measured at 30 PM lauric acid was not affected by oleic acid at concentrations ranging from 125 to 375 PM (Fig. 5). Another type of Cyt I'-450 inhibitor is provided by nitrogen-containing molecules, notably azoles, which inhibit Cyt P-450 by bonding to the heme iron (Ortiz De Montellano and Reich, 1986). We have studied the effect of l-pentade-


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Effeci of oleic acid (C18:l) on w-LAH activity. Substrate concentration vvas 30 p~ (about the K, of w-LAH for lauric acid). Oleic acid concentrations are indicated in the plot. The plot is based on triplicate experimental measurements.

Figure 5.

Figure 3. Time-dependent inactivation of w-OAH and w-LAH activities by 17-ODCYA. w-OAH (O) and w-LAH ( X ) activities are plotted in a semilogarithmic fashion against time of microsome preincubation in the presence of NADPH plus 40 ~ L M17-ODCYA. Control activities were 76 and 407 pmol min-' mg-' protein for wOAH and w-LAH, respectively.The concentration of oleic and lauric

acid was 100 p

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cyltriazole, an alkane azole analog, on the w-hydroxylation of lauric and oleic acids (Fig. 6). Formation of 12-hydroxylauric acid was decreased by less than 10%, but that of 18hydroxyoleic acid was reduced by 34% in the presence of 60 ILM of the triazole. DISCUSSION

The physiological roles of fatty acid o-hydroxylases, in animals as well as in plants, remain obscure. In mammals, it has been demonstrated that 20-hydroxyeicosatetraenoic acid constricts the rat aortic ring (Schwarzman et al., 1989) and inhibits ion transport in the rabbit kidney loop of Henle (Escalante et al., 1991). The existence of isoenzymes that catalyze only this particular reaction, the specific induction of one of these isoenzymes by clofibrate, and the enhanced excretion of dicarboxylicacids under conditions of high fatty acid flux suggest that fatty acid w-hydroxylation could be

involved in a catabolic process in mammals (Tamburini et al., 1984). Interestingly, clofibrate also induces fatty acid oxidation by plants (Salaun et al., 1986; Pinot et al., 1992). Furthermore, Palma et al. (1991) have shown that this hypolipidemic drug leads, as in mammalian systems, to proliferation of peroxisomes and mitochondria in leaves of Pisum sativum. These analogies suggest that w-hydroxylation of fatty acids in plants could be the first step in their catabolism through 0-oxidation. However, severa1 lines of evidence also suggest that w-hydroxylation of long-chain fatty acids may be implicated in biosynthetic pathways. Studies of the biosynthesis of plant cutins and suberins have shown that Cls fatty acids are incorporated into the cuticle after w-hydroxylation by Cyt P-450 isoenzymes (Kolattukudy, 1980). We have recently demonstrated that the w-hydroxylation of oleic acid, 9,lOepoxystearic acid, and 9,l O-dihydroxystearic acid is performed by a Cyt P-450 from V. sativa microsomes (Pinot et al., 1992). We hypothesize that this reaction controls the elongation of the cutin polymer and is therefore a key step in the synthesis of the plant cuticle. To test this hypothesis, we are now developing inhibitors of the Cls w-hydroxylase. The two acetylenes 17-ODCYA and 17-EODCYA proved

Figure 4. lnhibition of w-OAH activity by lauric acid. Lineweaver-Burk plot of w-OAH activity as a function of oleic acid concentration in the absence (x) or in the presence of 50 p~ (O) or

100 ~ L M(A)lauric acid. Microsomal incubation time was 30 min. The plot is based on duplicate experimental measurements.

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w-Hydroxylation of Oleic Acid in Vicia sativa Microsomes

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[l -Pentadecyltriazole] Figure 6. Effect of 1-pentadecyltriazole o n w-OAH and W-LAH activities. Relative w-LAH (black bars) and w-OAH (white bars) activities are plotted against 1-pentadecyltriazole concentration. Microsomal incubation time was 10 min. Substrate concentration was 100 ~ L MResults . are expressed as percent of maximal activities measured without 1-pentadecyltriazole, which were 99 and 323 pmol min-' mg-' protein for w-OAH and w-LAH, respectively. Each value is the mean of duplicate experiments.

to be potent inactivators of w-OAH. As noted by others (Cajacob and Ortiz De Montellano, 1986), a non-negligible loss of w-hydroxylase activity is recorded in the presence of NADPH alone. This could be due to the production during abortive catalytic cycles of reactive oxygen, which, in the absence of substrate, may react with the enzyme and inactivate it. After correction for the NADPH effect, inactivation rate constants of 7.2 X 10-4 s-' and 6 X 10e4 s-' were determined for 17-ODCYA and l'/-EODCYA, respectively. Surprisingly, 17-ODCYA also inactivated, although at a slower rate, the medium-chain fatty acid hydroxylase w-LAH. It is well established that affinity of the inhibitor is not the prime factor in mechanism-based enzyme inactivation, but rather it is the ratio of alkylation over normal product formation that is important (Walsh, 1982). Because the different Cyt P-450 isoforms share basically the same reaction mechanism, even modest affinity of nontarget Cyt P-450 forms for an inactivator may produce their inactivation. Although oleic acid itself is not an inhibitor of w-LAH (Fig. 5), one could assume that the rigid and linear acetylene group of 17ODCYA modifies the conformation of the substrate and enables recognition by the enzyme active site. One alternative explanation to our data would be that a fraction of lauric acid hydroxylation is catalyzed by w-OAH and is therefore sensitive to 17-ODCYA. In this context, the noncompetitive inhibition of w-OAH by laurate would be due to the binding of laurate at a different site of the heme pocket than oleate. To test further for the involvement of distinct Cyt P-450 forms in the w-hydroxylation of mediumand long-chain fatty acids, we have used an azole alkane analog 1-pentadecyltriazole. Azole compounds inhibit Cyts P-450 by forming a noncovalent bond with the iron of the prosthetic group (Ortiz De Montellano and Reich, 1986). The inhibitory character of the azole is here enhanced by the presence of an aliphatic chain that interacts with the hydrophobic domain of the active site. The inhibition by o-OAH

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was 4 times higher than that by w-LAH. This confirms earlier studies from our laboratory that showed that w-LAH is inhibited only by alkyltriazoles with aliphatic chains shorter than 13 carbon atoms (our unpublished data). Our data are best reconciled with the existence of at least two distinct Cyt P-450-dependent w-hydroxylase systems in V. sativa microsomes: an a-LAH catalyzing the oxidation of lauric acid and different laurate analogs (Salaun et al., 1986; Simon, 1987; Weissbart et al., 1992), and an w-OAH that hydroxylates Cls fatty acids. This latter enzyme may also catalyze a part of lauric acid oxidation, as evidenced by the effects of 17-ODCYA and 1-pentadecyltriazoleand the noncompetitive inhibition of w-OAH by laurate. We have recently shown that linoleic acid is oxidized by Cyt P-450 in V. sativa microsomes (Pinot, 1992). The competitive inhibition of w-OAH by linoleate (Fig. 2) suggests that these two CI8 fatty acids are substrates of the same Cyt P-450 enzyme. The irreversibility of mechanism-based inactivators such as 17-ODCYA and 17-EODCYA makes them attractive as probes for the study of the biochemical and physiological roles of w-OAH. It remains to be shown that these compounds are active in vivo. In mammals, terminal acetylenic fatty acid analogs are rapidly inactivated in vivo by /3-oxidation (Cajacob et al., 1988). To increase the efficacy of our inhibitors, we plan to replace the carboxylic acid group by a sulfate, which is not subjected to P-oxidation (Cajacob et al., 1988). Preliminary studies in our laboratory have shown that a carboxylic group can be replaced by a sulfate without affecting recognition of the molecule by plant w-hydroxylases. We have also synthesized radiolabeled [1-14C]17-ODCYA and [ 1-I4C]17-EODCYA. These compounds provide new tools to study further the mechanism of enzyme inactivation. It has been suggested (Cajacob et al., 1988) that acetylenes inactivate w-hydroxylases by alkylating the Cyt P-450 apoprotein. These radiolabed inactivators will now be used to test this hypothesis and may serve as markers during hydroxylase purification. Received February 9, 1993; accepted May 7, 1993. Copyright Clearance Center: 0032-0889/93/102/1313/06. LITERATURE ClTED

Bollit V, Mioskowski C (1988)Triphenylphosphinehydrobromide: a mild and efficient catalyst for tetrahydropyranylationof tertiary alcohols. Tetrahedron Lett 2 9 4583-4586 Cajacob CA, Chan WK, Shephard E, Ortiz De Montellano PR (1988) The catalytic site of rat hepatic lauric acid w-hydroxylase. J Biol Chem 2 6 3 18640-18649 Cajacob CA, Ortiz De Montellano PR (1986)Mechanism based in vivo inactivation of lauric acid hydroxylases. Biochemistry 25: 4705-4711

Escalante B, Erlij D, Falck JR, McGiff JC (1991) Effect of cytochrome P450 arachidonate metabolites on ion transport in rabbit kidney loop of Henle. Science 251: 799-802 Gibson GG (1989) Comparative aspects of the mammalian cytochrome P450 IV family. Xenobiotica 1 9 1123-1148 Kolattukudy PE (1980) Biopolyester membranes of plants: cutin and suberin. Science 208: 990-1000

Kolattukudy PE (1981) Structure, biosynthesis and biodegradation of cutin and suberin. Annu Rev Plant Physiol32: 539-567 Kunze KL, Mangold BLH, Wheeler C, Beilan HS, Ortiz De Montellano PR (1983) The cytochrome P450 active site. Regiospecificity of prosthetic heme alkylation by olefins on acetylenes. J Biol Chem 258: 4202-4207

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Muerhoff AS, Williams DE, Reich NO, Cajacob CA, Ortiz De Montellano PR, Masters BSS (1989) Prostaglandin and fatty acid w- and (w-1)-oxidationin rabbit lung. Acetylenic fatty acid mechanism-based inactivators as specific inhibitors. J Biol Chem 264 749-756 Ortiz De Montellano PR, Reich NO (1984) Specific inactivation of hepatic fatty acid hydroxylases by acetylenic fatty acids. J Biol Chem 259: 4136-4141 Ortiz De Montellano PR, Reich NO (1986)Inhibition of cytochrome P450 enzymes. In PR Ortiz De Montellano, ed, Cytochrome P450: Structure, Mechanism and Biochemistry. Plenum Press, New York, pp 273-314 Palma JM, Garrido M, Rodriguez-Garcia MI, De1 Rio LA (1991) Peroxisome proliferation and oxidative stress mediated by activated oxygen species in plant peroxisomes.Arch Biochem Biophys 287: 68-74 Pinot F (1992) Etude des systemes doxydation de l’acide oleique dans les microsomes de Vicia sativa. Mise en evidence d u n e nouvelle monooxygenase i cytochrome P450 hydroxylant l’acide oleique et ses dérivb oxydés. PhD thesis. Université Louis Pasteur, Strasbourg, France Pinot F, Salaun JP, Bosch H, Lesot A, Mioskowski C, Durst F (1992) w-Hydroxylation of Z9-octadecenoic, Z9,lO-epoxystearic and 9,l O-dihydroxystearic acids by microsomal cytochrome P450 systems from Vicia sativa. Biochem Biophys Res Commun 184 183-193 Salaun JP, Simon A, Durst F (1986) Specific induction of lauric acid w-hydroxylase by clofibrate, diethylhexyl-phtalate and 2,4-dichlorophenoxyaceticacid in higher plants. Lipids 21: 776-779 Salaun JP, Simon A, Durst F, Reich NO, Ortiz De Montellano PR


(1988) Differential inactivation of a plant lauric acid w- and inchain-hydroxylase by terminally unsaturated fatty acids. Arch Biochem Biophys 260 540-545 Schwarzman ML, Falck JR, Yadagiri P, Escalante B (1989) Metabolism of 20-h,ydroxyeicosatetraenoic acid by cyclooxygenase: formation and iclentification of novel endothelium dependent vasoconstrictor metabolites. J Biol Chem 264 11658-11662 Shak K, Reich NO, Goldstein IM, Ortiz De Montellano PR (1985) Leukotriene 84 w-hydroxylase in human polymorphonuclear leukocytes. Suicida1inactivation by acetylenicfatty acids. J Biol Chem 260 13023-13028 Simon A (1987) Hydroxylation dacides gras i chaine courte ou moyenne par des monooxygénases a cytochrome P450 chez les végétaux supirieurs. PhD thesis. Université Louis Pasteur, Strasbourg, France Tamburini PP, Masson HA, Bains SK, Makowski RJ, Morris B, Gibson GG (1984) Multiple form of hepatic cytochrome P-450. Purification, characterization and comparison of a novel clcifibrate induced isoenzyme with other major forms of cytochrome P450. Eur J Biochem 139 235-246 Venturello C, Alneri E, Ricci M (1983) A new, effective catalytic system for epoxidation of olefins by hydrogen peroxide under phase-transfer conditions. J Org Chem 4 8 3831-3833 Walsh C (1982) Suicide substrates: mechanism-based inactivators. Tetrahedron 38: 871-909 Weissbart D,Salaun JP, Durst F, Pflieger P, Mioskowski C (1992) Regioselectivity of a plant lauric acid omega hydroxylase. Omega hydroxylation of cis and trans unsaturated lauric acid analogues and epoxygenation of the terminal olefin by plant cytochrome P450. Biochim Biophys Acta 1124 135-142