Linoleoyl lysophosphatidylcholine is an efficient substrate for soybean lipoxygenase-1

Archives of Biochemistry and Biophysics 455 (2006) 119–126 www.elsevier.com/locate/yabbi

Linoleoyl lysophosphatidylcholine is an eYcient substrate for soybean lipoxygenase-1 Long Shuang Huang a, Mee Ree Kim b, Dai-Eun Sok a,¤ a

b

College of Pharmacy, Chungnam National University, Yuseong-ku, Taejon, Republic of Korea Department of Food and Nutrition, Chungnam National University, Yuseong-ku, Taejon, Republic of Korea Received 23 June 2006, and in revised form 15 September 2006 Available online 6 October 2006

Abstract Oxygenation of 1-linoleoyl lysophosphatidylcholine (linoleoyl-lysoPC) by soybean lipoxygenase-1 was monitored by measuring the increase of absorbance at 234 nm. In support of this, the hydroperoxy derivative of linoleoyl-lysoPC as a major product and its reduction product as a minor one were detected by LC/MS analyses. The greater part of the hydroperoxy derivative was found to contain hydroperoxide group at C-13 rather than C-9, consistent with the position speciWcity of soybean lipoxygenase-1 in oxygenation of linoleic acid. Such a preferential production of 13-hydroperoxy derivative of linoleoyl-lysoPC was also observed at pH 7.4, suggesting that the positional speciWcity of lipoxygenase-1 is not aVected greatly by pH. In addition, the pH-dependent oxygenation of linoleoyl-lysoPC, showing an optimal activity around pH 9, was similar to that of linoleic acid. In kinetic study, lipoxygenase 1-catalyzed oxygenation of linoleoyllysoPC followed Michaelis–Menten kinetics (Vm, 167.5 U/mg protein; Km, 12.9 M). In comparison, linoleoyl-lysoPC was no less eYcient than linoleic acid as a substrate of soybean lipoxygenase-1. Moreover, oxygenation of linoleoyl-lysoPC by LOX-1 was not aVected by detergent. Thus, linoleoyl-lysoPC could be utilized as a convenient substrate in the assay of soybean lipoxygeanse-1. © 2006 Elsevier Inc. All rights reserved. Keywords: Soybean lipoxgenase-1; Linoleoyl lysophosphatidylcholine; Oxygenation; Hydroperoxide; UV assay; HPLC

Lipoxygenase (linoleate:oxygen oxidoreductase, EC 1.13.11.12), a nonheme iron-containing enzyme, catalyzes the addition of molecular oxygen to fatty acids containing at least one (Z,Z)-pentadiene system to give corresponding hydroperoxides [1,2]. These enzymes are ubiquitously distributed in animals and plants, and have a key function in the formation of biologically active substances [2,3]. Generally, free unsaturated fatty acids, substrates for lipoxygenases, are liberated from membrane phospholipids via phospholipase-catalyzed reactions [2–4]. Although some lipoxygenase isoenzymes can oxidize certain phospholipids [5,6] or triglycerides [7], it is acknowledged that free polyunsaturated fatty acids are preferable substrates [1,2]. In mammalian systems, the direct eVect of lipoxygenases on

*

Corresponding author. Fax: +82 42 823 6566. E-mail address: [email protected] (D.-E. Sok).

0003-9861/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2006.09.015

phospholipids and biomembranes [8–11] suggests a role of lipoxygenases in some processes such as cellular maturation, which implies a change in the structure of the membrane [12,13]. Additionally, soybean lipoxygenase-1 (LOX-1)1 is known to induce oxidation of phospholipids in LDL, where phospholipids exist as solubilized state [14]. Since the lipoxygenation reaction requires the solubilization of fatty acids substrate, the detergent such as deoxycholate or Tween 20 have been routinely used for the determination of lipoxygenase activity [4,15–17]. In the presence of deoxycholate, polyunsaturated acyl moieties in phosphatidylcholine were converted by lipoxygenase to the respective hydroperoxide, although the oxidation rate was

1 Abbreviations used: linoleoyl-lysoPC, 1-linoleoyl lysophosphatidylcholine; LOX-1, lipoxygenase-1; HODE, hydroxyoctadecadienoic acid; DLPC, dilinoleoyl phosphatidylcholine; SP, straight-phase; RP, reverse phase.

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much lower for phosphatidylcholine substrate than free fatty acid substrate. For example, arachidonyl and linoleoyl moieties in phosphatidylcholine were converted to exclusively to the 15(S)-hydroperxyeicosatetraenoic acid and 13(S)-hydroperoxyoctadecadienoate analogs, respectively, suggesting that fatty acids esteriWed in phospholipids can be subjected to highly speciWc oxygenation by lipoxygenase [9,18]. Thus, biomembrane phospholipids in the solubilized state might be more likely susceptible to lipoxygenases-catalyzed oxidation. In this regard, the water-soluble lysophospholipid, containing polyunsaturated fatty acyl moiety, would be readily oxidized by lipoxygenases. Presumably in support of this, the peroxide form of linoleoyl lysophosphatidylcholine (linoleoyl-lysoPC) has been reported to exist in the seed extract [19,20] or heart tissue [21]. Nonetheless, thus far, lipoxygenase-catalyzed oxygenation of linoleoyl-lysoPC has not been examined extensively in respect of substrate substitution. Even although soybean lipoxygenase is employed to oxygenate linoleic acid in a coupled enzyme assay for the determination of PLA2 [22,23], it is not clariWed whether soybean LOX-1 can use linoleoyl-lysoPC, the other hydrolysis product of dilinoleoyl phosphatidylcholine, as another substrate. Here, it is demonstrated that linoleoyl-lysoPC is no less than eYcient as linoleic acid as substrate of soybean LOX1. Moreover, linoleoyl-lysoPC can be utilized as a substrate for the routine assay of lipoxygenase in the absence of detergents.

6.8), and the fractions of LOX-1 activity, showing a relatively homogeneous band in SDS–PAGE, were used as LOX-1 (SpeciWc activity, 114.3 U/mg protein). Protein amount was determined according to Lowry method [27].

Preparation of 1-linoleoyl lysophosphatidylcholine (linoleoyllysoPC) from dilinoleoyl phosphatidylcholine (DLPC) Linoleoyl-lysoPC was prepared from PLA2-catalyzed hydrolysis of DLPC as described previously with some modiWcation [6]. DLPC (2 mg), dissolved in chloroform, was dried under N2, and then rapidly dispersed in 10 ml of 50 mM borax buVer, pH 8.5 containing 100 mM CaCl2. The hydrolysis was started by adding PLA2(»120 U), and allowed to continue under N2 with constant stirring for 1 h at 25 °C. The reaction mixture was loaded directly onto C18 Sep-pack column (3 £ 1 cm), which was washed with three volumes of distilled water, and Wnally lysophospholipid products was eluted with methanol. Next, linoleoyl-lysoPC was puriWed by silica gel TLC in the solvent system (chloroform: methanol: concentrated ammonia water: H2O D 90: 54: 5.5: 5.5) as described previously [28]. The spot, containing linoleoyl-lysoPC, was scratched oV, extracted with methanol (10 ml) three times, and dried under N2.

pH-dependent oxygenation of linoleic acid or 1-linoleoyl lysoPC by LOX-1 LOX-1 (0.02 U/ml) was incubated with linoleoyl-lysoPC (100 M) in 500 l of buVers of various pHs (pH 6.0–11) at 25 °C as described before [15]; 200 mM phosphate (pH 6–8), 50 mM borax (pH 8.5–9.5), 300 mM sodium bicarbonate (pH 10–11). Separately, the oxygenation was started by including LOX-1 (0.02 U/ml) in the reaction mixture containing sodium linoleate (100 M) in the presence of Tween 20 (25 M).

EVect of substrate concentration on LOX-1-catalyzed oxygenation of linoleic acid or linoleoyl-lysoPC

Materials and methods Materials Dilinoleoyl phosphatidylcholine (DLPC, 99%), soybean lysophosphatidylcholine and 1-plamitoyl-lysophosphatidylcholine were from Avanti Polar Lipid (Alabaster, AL, USA). Soybean lipoxygenase (lipoxidase Type I-B, EC 1.13.11.12, 187,400 Sigma U/mg protein), cholesterol esterase (EC 3.1.1.13, bovine pancreas), phospholipase A2 (EC 3.1.1.4, honey bee venom), 13(S)-hydroxyoctadecadienoic acid (HODE), 9(S)-HODE, and Tween 20 were purchased from Sigma–Aldrich Corp. (St. Louis, MO, USA). HPLC solvents were all of HPLC grade, and other chemicals were of analytical grade.

Assay of lipoxygenase 1 activity in oxygenation of linoleate Lipoxygenase-1 (LOX-1) activity was monitored at 25 °C by measuring the increase in absorbance at 234 nm due to the formation of hydroperoxide (234 D 25,000 M¡1 cm¡1) as described before [24,25]. The reaction was started by including LOX-1 (0.0065 U/ml) in 50 mM borax buVer, pH 9.0 (0.5 ml) containing sodium linoleate (100 M), which was prepared by suspending sodium linoleate (7 mg) in 2.5 ml of distilled water containing Tween 20 (7 mg). One unit is deWned as the amount of LOX-1 that can produce one micromole of conjugated diene per min.

PuriWcation of soybean lipoxygenase-1 LOX-1 was puriWed from soy bean lipoxidase preparation (Type I-B) according to the method of Finazzi Agro et al [26]; brieXy describing, soybean LOX-1 (100 mg) was dissolved in 0.1 M phosphate buVer (pH 6.8), and applied to DEAE Sephacel (1.5 £ 25 cm). The bound enzyme was eluted by concentration gradient (0.1–0.25 M) of phosphate buVer (pH

Soybean LOX-1 (0.02 U/ml) was incubated with linoleic acid or linoleoyl-lysoPC of various concentrations in 500 l of 50 mM borax buVer, pH 9.0 at 25 °C.

RP-HPLC separation of oxygenation products of linoleoyl-lysoPC Oxygenation of linoleoyl-lysoPC was started by including soybean LOX-1 (0.5 U/ml) in 150 l of 50 mM borax buVer, pH 9.0 containing linoleoyl-lysoPC (1 mM). After 10 min, the reaction products were subjected to RP-HPLC (Hitachi L-7100 pump, Japan) equipped with C18 column (300 £ 7.8 mm, Phenomenex, USA), which was eluted at a Xow rate of 1 ml/min with gradient solvent system of 0.1% formic acid/ acetonitrile (solvent B) in 0.1% formic acid/H2O (solvent A); 25% from 0 to 10 min; 25–45% from 10 to 25 min; 45% from 25 to 55 min; 45–100% from 55 to 70 min.

LC/ESI–MS analysis LC–MS was performed using a MSDI spectrometer (HP 1100 series LC/MSD, Hewlett Packard, USA) equipped with ZORBAX Eclipse XDB C18 column (5 m, 50 £ 4.6 mm, Agilent Technologies, USA), which was eluted (1 ml/min) with gradient solvent of 0.1% formic acid/acetonitrile (solvent B) in 0.1% formic acid/H2O (solvent A): 0–45% from 0 to 10 min and 45% from 10 to 55 min. The products from oxygenation of linoleoyllysoPC were monitored by ESI–MS system using positive-ion modes.

IdentiWcation of peroxy linoleic acid generated from cholesterol esterase-catalyzed hydrolysis of peroxy linoleoyl-lysoPC Linoleoyl-lysoPC or soybean lysoPC (1 mM) was incubated with LOX-1 (0.5 U/ml) under stirring in 10 ml of 50 mM borax buVer (pH 9.0).

L.S. Huang et al. / Archives of Biochemistry and Biophysics 455 (2006) 119–126 After 30 min incubation, cholesterol esterase (5 U/ml) was included into the above mixture to hydrolyze 1-acylated lysophosphatidylcholine [29]. Separately, linoleoyl-lysoPC or soybean lysoPC was incubated with LOX1 in 50 mM phosphate buVer (pH 7.4) for 30 min. Then, the above mixture was acidiWed to inactivate remaining LOX-1 activity, followed by the pH adjustment to pH 9.0 with borate buVer for the cholesterol esterase hydrolysis. After another 60 min incubation, the oxygenation products were loaded into C18 Sep-pack column (3 £ 1 cm), which was washed with distilled water. The methanol eluate was concentrated, and then an aliquot (100 l) was subjected to RP-HPLC analysis using C18 column (5 m, 50 £ 4.6 mm), which was eluted with solvent gradient of 0.1% formic acid/ acetonitrile (solvent B) in 0.1% formic acid/ H2O (solvent A): 10–25% from 0 to 10 min; 25% from 10 to 20 min; 25–45% from 20 to 45 min; 45–100% from 45 to 70 min. The fractions of the peak (retention time, 51 min) were collected, and reduced with sodium borohydride as described before [30]. The reduction products, after ethyl acetate extraction at pH 3.0, were subjected to SP-HPLC equipped with silica gel column (4 m, 150 £ 3.9 mm, Waters, USA), which was eluted (1 ml/min) with n-hexane/isopropyl alcohol/acetic acid (100:1:0.1). Finally, the identiWcation of the product was performed by coinjection with each standard compound, 13(S)-hydroxyoctadecadienoic acid (HODE) or 9 (S)-HODE.

Results When soybean LOX-1 was incubated with linoleoyllysoPC as substrate in 50 mM borax buVer (pH 9), the UV spectral change corresponding to the enzymatic hydroperoxidation of linoleoyl-lysoPC (100 M) was observed with a maximal absorbance at 234 nm, consistent with the formation of conjugated dienes (Fig. 1). The nature of the spectral change during the enzyme assay showed that the formation of the oxygenation product was proportional to time up to 3 min (Fig. 1, inset). Above 3 min, the oxygenation rate seems to be retarded, suggestive of a gradual deactivation of LOX-1 activity. The lack of absorbance in the 270 to 280 nm region indicates that there is no signiWcant formation of oxodienes during the short incubation time. This allowed us to propose the notion that soybean LOX-1

Fig. 1. Change of UV spectrum during oxygenation of linoleoyl-lysoPC by soybean LOX-1. LOX-1 (0.0065 U/ml) was incubated with linoleoyllysoPC (100 M) in 0.5 ml of 50 mM borax buVer, pH 9.0 at 25°C. The change of UV spectra were scanned with a speed of 2000 nm/min every min. Inset, the change of absorbance at 234 nm was monitored continuously for 15 min in the presence of LOX-1 (1) or heat-treated LOX-1 (2).

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oxidized linoleoyl-lysoPC to readily generate corresponding hydroperoxide form, suggesting that linoleoyl-lysoPC can be used as a substrate for soybean LOX-1. Separately, although soybean lysoPC (100 M), containing 42% linoleoyl-lysoPC, was also found to be oxygenated by LOX-1, it showed a shorter period of linearity in the oxygenation rate under the conditions used. To identify the product from LOX-1-catalyzed oxygenation of linoleoyl-lysoPC, linoleoyl-lysoPC was incubated with LOX-1 in 50 mM borax buVer, pH 9.0, and the lipoxygenation products were partially puriWed using C18 extraction column as described before [6]. When the partially puriWed products were injected into RP-HPLC column, which was eluted with 45% acetonitrile containing 0.1% formic acid, a major peak (retention time, 49 min), showing an absorbance at 234 nm, was obtained (Fig. 2), in addition to linoleoyl-lysoPC, which appeared as a major peak with a retention time of 72 min (arrow symbol) in a separate experiment monitoring the absorbance at 205 nm. To provide an evidence for the formation of hydroperoxy derivative from linoleoyl-lysoPC, the oxygenation products were subjected to LC/ESI–MS analyses. As demonstrated in Fig. 3A, a major peak (retention time, ca. 47.0 min), the mass spectrum of which corresponds to hydroperoxy derivative of linoleoyl-lysoPC appeared (Fig. 3B); molecular ion at m/z 552.3 (MH+), m/z 574.3 ([M + Na]+), and m/z 590.3 ([M + K]+). Additionally, the peak (elution time, 45.5 min), which appeared as a minor peak, was found to contain hydroxyl derivative of linoleoyl-lysoPC; molecular ion at m/z 536.3 (MH+), m/z 558.3 ([M + Na]+) and m/z 574.3 ([M + K]+). Another minor peak (elution time, 46 min) was

Fig. 2. RP-HPLC of products from oxygenation of linoleoyl-lysoPC with LOX-1. Oxygenation products from 10 min incubation of linoleoyllysoPC (1 mM) with soybean LOX-1 (0.5 U/ml) in 150 l of 50 mM borax buVer (pH 9.0) were injected into C18 column (300 £ 7.8 mm), which was eluted (1 ml/min) with 0.1% formic acid/ acetonitrile (solvent B) in 0.1% formic acid/H2O (solvent A); 25% from 0 to 10 min; 25–45% from 10 to 25 min; 45% from 25 to 55 min; 45–100% from 55 to 70 min. Oxidized linoleoyl-lysoPC and linoleoyl-lysoPC were monitored at 234 and 205 nm, respectively.

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found to contain oxo derivative of linoleoyl-lysoPC as a decomposition product of hydroperoxy linoleoyl-lysoPC (data not shown); molecular ion at m/z 534.3 (MH+), m/z 556.3 ([M + Na]+, and m/z 572.3 ([M + K]+). From this, it is clearly shown that hydroperoxy linoleoyl-lysoPC is obtained as a major oxygenation product during enzymatic oxygenation of the linoleoyl chain of linoleoyl-lysoPC. To establish the position of oxygenation of linoleoyl chain in LOX-1-catalyzed oxygenation of linoleoyl-lysoPC, the hydroperoxy derivative of linoleoyl-lysoPC was hydrolyzed by cholesterol esterase, a hydrolytic enzyme which is known to hydrolyze 1-monoacyl lysophosphatidylcholine [29,31], to generate hydroperoxy linoleic acid. For this purpose, the oxygenation products, which were produced from the exposure of linoleoyl-lysoPC to LOX-1 in 50 mM borax buVer (pH 9.0), were further incubated in the presence or absence of cholesterol esterase for 60 min. Then, the oxygenation products were analyzed by RP-HPLC. As exhibited in Fig. 4A, the oxygenated derivative of linoleoyllysoPC appeared as a major peak (retention time, 34 min) in the absence of cholesterol esterase. Meanwhile, the inclusion of cholesterol esterase gave rise to another major peak with retention time of ca. 51 min (Fig. 4B), comigrating with hydroperoxy linoleic acid, which was produced from the exposure of linoleic acid to LOX-1 (Fig. 4C). Then, the fraction containing hydroperoxy linoleic acid was collected, reduced with sodium borohydride, and then the reduction products were subjected to SP-HPLC analysis. As

displayed in the chromatograms (Fig. 5A), a major peak, showing an absorbance at 234 nm, appeared with an elution time of around 6.5 min, and a minor peak, with elution time of around 13.2 min. Separately, when each peak was coinjected with each standard linoleic acid hydroxide, 13HODE or 9-HODE, into SP-HPLC column (Fig. 5A), it was found that the major peak migrated with 13-HODE (solid line arrow) while the minor peak did with 9-HODE (dotted line arrow). Based on the area of the peak, the quantitative ratio of 13-HODE to 9-HODE is estimated to be approximately 9:1. From this, it is suggested that LOX1-catalyzed oxygenation of linoleoyl-lysoPC selectively at position C-13 of linoleoyl group. In addition, a similar ratio of major peak to minor one was also obtained when LOX-1 was incubated with linoleoyl-lysoPC at pH 7.4 (Fig. 5B), showing that the positional speciWcity of LOX-1 in oxygenation of linoleoyl-lysoPC was not altered signiWcantly by the change of pH. Separately, a similar result was reproduced when LOX-1 was incubated with soybean lysoPC at both pHs (Figs. 5C and D). Although 9-HODE derivative appeared to be generated in the incubation, a similar amount of 9-hydroperxy derivative was also produced in the incubation of soybean lysoPC with heat-treated lipoxygenase, suggesting that the formation of 9-HODE might be ascribed to non-enzymatic oxygenation caused by the lengthy procedure employing hydrolysis and concentration. After establishing the conversion of linoleoyl-lysoPC to hydroperoxy derivative of linoleoyl-lysoPC, the optimal

Fig. 3. LC/ESI–MS Analysis of oxygenation products of linoleoyl-lysoPC. (A) Oxygenation products from 10 min incubation of linoleoyl-lysoPC (1 mM) with soybean LOX-1 (0.5 U/ml) in 150 l of 50 mM borax buVer (pH 9.0) were injected into ZORBAX Eclipse XDB C18 column (5 m, 50 £ 4.6 mm), which was eluted (1 ml/min) with gradient solvent of 0.1% formic acid/acetonitrile (solvent B) in 0.1% formic acid/H2O (solvent A): 0–45% from 0 to 10 min and 45% from 10 min to 55 min. The oxygenation products were detected at 234 nm. (B) A representative mass spectrum of peroxy derivative of linoleoyl-lysoPC. The mass spectrum of the major peak (retention time, 47.0 min) in Fig. 3A was obtained by ESI–MS system using positive-ion mode.

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Fig. 4. RP-HPLC of products from cholesterol esterase-catalyzed hydrolysis of oxidized linoleoyl-lysoPC. (A) Linoleoyl-lysoPC (1 mM) was incubated with LOX-1 (0.5 U/ml) in 10 ml of 50 mM borax buVer, pH 9.0 for 30 min. (B) The reaction mixture from (A) was further incubated in the presence of cholesterol esterase (5 U/ml) for 60 min. (C) Separately, sodium linoleate (3.3 mM) was incubated with LOX-1 (0.5 U/ml) for 10 min. The aliquot (50 l) from each incubation, after concentration on C18 Sep-pack column, was injected into C18 HPLC column (50 £ 4.6 mm), which was eluted by solvent gradient of 0.1% formic acid/ acetonitrile (solvent B) in 0.1% formic acid/H2O (solvent A): 10–25 % from 0 to 10 min; held at 25% from 10 to 20 min; 25–45 % from 20 to 45 min; 45–100% from 45 to 70 min.

Fig. 5. Determination of oxygenation position of oxidized linoleoyl-lysoPC or soybean lysoPC by SP-HPLC. Linoleoyl-lysoPC or soybean lysoPC was incubated with LOX-1 in 50 mM buVer (pH 7.4 or 9.0) for 30 min, and then the mixture was further incubated in the presence of cholesterol esterase for 60 min as described in Materials and methods. The products, corresponding to peroxy linoleic acid, were reduced with sodium borohydride, and the reduction products, after ethyl acetate extraction at pH 3.0, were separated by SP-HPLC equipped with silica gel column (150 £ 3.9 mm), which was eluted (1 ml/min) with n-hexane/isopropyl alcohol/acetic acid (100:1:0.1). (A) The incubation of linoleoyl-lysoPC with LOX-1 at pH 9.0. (B) The incubation of linoleoyl-lysoPC with LOX-1 at pH 7.4. (C) The incubation of soybean lysoPC with LOX-1 at pH 9.0. (D) The incubation of soybean lysoPC with LOX-1 at pH 7.4. Arrow indicates the coinjection with standard 13(S)-HODE (solid line) or 9(S)-HODE (dotted line).

condition for the oxygenation of linoleoyl-lysoPC by LOX1 was investigated in further studies. First, when the eVect of pH on the oxygenation of linoleoyl-lysoPC by LOX-1 was examined (Fig. 6), the oxygenation of linoleoyl-lysoPC varied with pH, with the optimal pH range being about 9, slightly diVerent from the optimal pH value reported for oxygenation of linoleic acid by LOX-1 [5,21,33]. Separately,

the eVect of detergent on oxygenation of linoleoyl-lysoPC by LOX-1 was examined. However, the oxygenation of linoleoyl-lysoPC was not inXuenced by Tween 20, contrary to the Wnding [17] that the oxygenation of linoleic acid by LOX-1 was enhanced by Tween 20. Even 1-palmitoyl lysoPC (1–3 mM), a native detergent, failed to aVect LOX1-catalyzed oxygenation of linoleoyl-lysoPC.

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L.S. Huang et al. / Archives of Biochemistry and Biophysics 455 (2006) 119–126 Table 1 Kinetic parameters for oxygenation of linoleoyl-lysoPC or linoleic acid Km (M) Linoleic acid Soybean lysoPC¤ Linoleoyl-lysoPC

12 § 0.70 16 § 0.66 12.9 § 1.64

Vm (U/mg) 114.3 § 6.0 170.4 § 10.9 167.5 § 10.4

Vm/Km (U/mg/M) 9.53 § 0.53 10.65 § 1.01 12.98 § 2.54

Data were expressed as means § SD of results from at least three independent experiments. ¤ Concentration of soybean lysoPC was estimated based on its average molecular weight.

Discussion

Fig. 6. EVect of pH on the rate of LOX-1-catalyzed oxygenation of linoleoyl-lysoPC. LOX-1 (0.02 U/ml) was incubated with 100 M of linoleoyllysoPC (䊏), 100 M of sodium linoleate (䉬) or 250 M of soybean LPC (䉱) in buVers of various pHs; 200 mM phosphate (pH 6–8), 50 mM borax (pH 8.5–9.5) and 300 mM sodium bicarbonate (pH 10–11).

Next, the eVect of linoleoyl-lysoPC concentration on LOX-1-catalyzed oxygenation of linoleoyl-lysoPC was examined. Fig. 7 showed that the enzyme activity followed classical Michaelis–Menten kinetics when linoleoyl-lysoPC concentration was varied. Lineweaver Burke plot for the kinetic data obtained resulted in a linear relationship (Fig. 7, inset), from which the Km and Vm values were estimated to be 12.9 M and 167.5 U/mg protein, respectively. When the kinetic values for the oxygenation of linoleoyllysoPC was compared to those for oxygenation of linoleic acid by LOX-1 (Table 1), the eYciency as substrate was somewhat greater for linoleoyl-lysoPC than for linoleic acid, indicating that linoleoyl-lysoPC might be no less eYcient as a substrate for LOX-1 than linoleic acid.

Fig. 7. EVect of substrate concentration on oxygenation of linoleoyllysoPC by LOX-1. LOX-1 (0.02 U/ml) was incubated with linoleoyllysoPC of various concentrations (3–100 M) in 50 mM borax (pH 9.0). Inset, Lineweaver Burke plot for oxygenation of linoleoyl-lysoPC by soybean LOX-1. Data were expressed as means § SD of results in at least three independent experiments.

It is generally acknowledged that free polyunsaturated fatty acids are the preferred substrates for lipoxygenase oxygenation. Nonetheless, dilinoleoyl phosphatidylcholine had been reported to be oxygenated by soybean LOX-1 [6], although to a lower extent, compared to linoleic acid; for example, the rate for the lipoxygenation of dilinoleoyl phosphatidylcholine by soybean LOX-1 was 23% of that obtained using linoleic acid as a substrate in borate buVer (pH 10) containing 10 mM deoxycholate [23]. Additionally, reticulocyte lipoxygenase had been reported to utilize phosphatidylcholines as substrates [32]. Here, we state that under experimental conditions used, linoleoyl-lysoPC corresponds to an eYcient substrate for soybean LOX-1, based on UV spectrum and LC/MS analyses. Although the lack of absorbance in the 270 to 280 nm region in UV spectrum indicates that there is no formation of oxodienes [33], the products such as hydroxyl or keto derivatives were observed to be generated as decomposition products during LC/MS analyses. The nature of the spectral change during the enzyme assay showed that the formation of the reaction product was relatively proportional to time. However, the oxygenation rate tended to decrease during the lengthy incubation (>3 min) or in the presence of excess enzyme activity, suggestive of a possible inhibition of LOX-1 by peroxylated products. Such phenomena were had been reported for LOX-1-catalyzed oxygenation of polyunsaturated fatty acids [34,35]. Previously, detergents such as deoxycholate or Tween 20 have been used in the assay of soybean LOX-1 [6,23,25], where linoleic acid is used as substrate. The beneWcial eVect of detergents on soybean LOX1-catalyzed oxidation of linoleic acid could be the result of an enhancement in the susceptibility of linoleic acid toward the enzymatic attack. Alternatively, it is possible that the positive eVect of detergents could be due to their direct eVect on the enzyme molecule. However, the latter possibility is ruled out from no eVect of detergent on LOX1-catalyzed oxidation of linoleoyl-lysoPC. Then, the lower activity displayed by soybean LOX-1 toward dilinoleoyl phosphatidylcholine might be the result of micellar structure adopted by the substrate [16,17], which could aVect the availability of substrate monomer. No eVect of Tween 20 on LOX-1-catalyzed oxidation of linoleoyl-lysoPC, together with previous observations [4,15], strongly favor a model in which LOX-1 has a substantially higher aYnity

L.S. Huang et al. / Archives of Biochemistry and Biophysics 455 (2006) 119–126

for monomeric substrate than for free fatty acid incorporated into micelles. This might be further supported by the present Wnding that linoleoyl-lysoPC is utilized as substrate at concentrations below its c.m.c. values; the Km value (12.9 M) of linoleoyl-lysoPC is much lower than its c.m.c. value (>50 M), which is estimated from c.m.c. values of lysoPC with various acyl groups [35]. Although dilinoleoylphosphatidylcholine was reported to be oxygenated by LOX-1 [6], it is less eYcient than linoleoyl-lysoPC as substrate of soybean LOX-1. Previously, it had been reported that the activity of phospholipase A2 (PLA2) could be determined by the coupled assay [22,23], where soybean LOX-1 was used for the oxidation of linoleic acid released from dilinoleoyl phosphatidylcholine. However, it is not clear whether the increase of absorbance at 234 nm is due to the oxygenation of linoleic acid or linoleoyl-lysoPC, since both products are substrates for soybean LOX-1 in our study. In this regard, the coupled enzymes assay of PLA2 activity is to be reexamined using phosphatidylcholine containing saturated fatty acyl group at C-1, so that the oxygenation of linoleic acid released can be selectively measured. It is well known [6,24] that when acting on free linoleic acid, soybean LOX-1 produces two chiral compounds, 13(S)-hydroperoxyoctadecadienoic acid [13(S)-HPODE] and 9(S)-hydroperoxyoctadecadienoic acid [9(S)-HPODE]. The former of the peroxides is generated over a broad pH range, while 13(S)HPODE prefers to be formed at pH levels above 8.5 [6]. According to the previous proposal [36], the non-ionized carboxylic acid form of linoleic acid, more predominant at lower pHs, may arrange itself at the active site in two opposite orientations giving rise to either 9(S)-HPODE or 13(S)HPODE. However, the lipoxygenation of dilinoleoyl phosphatidylcholine, forming the 13-hydroperoxide form exclusively, was not aVected by the lowering of pH, suggesting that the esteriWcation of linoleoyl groups at glycerol backbone provides only one possible orientation of linoleoyl groups for LOX-1 oxygenation [6]. The same might explain why the preferential production of 13-hydropeorxy derivative after the oxidation of linoleoyl-lysoPC by soybean LOX-1 was commonly observed at pH 7.4 and pH 9.0. Thus, it seems that the esteriWcation of linoleoyl group at glycerol backbone in linoleoyl-lysoPC may result in the selective oxygenation of linoleoyl group at 13-position. This might be further supported by earlier Wnding [37] that barley embryo LOX, which oxygenated linoleic acid to form 9(S)-HPODE as a major product, was found to oxygenate esteriWed lipids to generate 13(S)-HPODE derivative as a main oxygenation product. Additionally, the esteriWcation of linoleoyl group at glycerol backbone caused the pH proWle of the linoleoyl-lysoPC lipoxygenation to shift somewhat to higher pH regions, which remains to be clariWed. Linoleoyl-lysoPC as substrate can be prepared simply from PLA2-catalyzed hydrolysis of dilinoleoyl phosphatidylcholine. Instead, soybean lysoPC, containing 42% linoleoyllysoPC, can be utilized as a substrate for soybean LOX-1.

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