89

Biochimica et BiophysicaActa. 1083(1991)89-93 ~,31991 ElsevierSciencePublishersB.V.0005-2760/91/503.59 ADONIS 000527609100145Z

BBALIP53628

Catalytic properties of human platelet ! 2-1ipoxygenase as compared with the enzymes of other origins T a k a h i k o Hada, N~tsuo Ueda, Yoshitaka Tak8hashi and Shozo Yarnarnoto Departmentof Biochemistm', Tokushima Unit,ersitvSchoolof Medicine. Kuramata.cho. Takushima(Japan)

(Received11 September1990)

Keywords: 12-Lipoxygenase"Lipoxin;Arachidonicacid; (Humanplatdet) Arachidonate 12.1ipoxygenases of porcine and bovine leukocytes were different in substrate specificity and immunogenicity from the enzyme of bovine platelets (Arch. Biochem. Biophys. (1988) 266, 613). In order to extend the comparative studies on the two types o| 12-1ipoxygenase, we purified the enz~me from the cytosol of human platelets by immunoaffinit~' chromatography to a specific activity o| about 0.3 ttmol/mlu per mg protein at 37°C. Tkc l~u~di~d enzyme was active with dcosapolyenoic acids and docosahexaenoic acid. Linoleie and linolenic acids were poor substrates in contrast to the high reactivity of the leukocyte enzymes with these octadecapolyenoic acids. The liming that the human platelet enzyme catalyzed 15.oxygenation oi 5S.hydroxy.6,8,11,14-eicosatetraenoic acid, raised a question if lipoxins were produced by incubation of the enzyme with leukotriene A 4. However, the leukotriene A 4 was scarcely transformed to lipoxin isomers by 12-1ipoxygenases of human and bovine plate:e'.s. In sharp contrast, the porcine and bovine leukocyte enzymes converted leukotriene A4 to various lipoxin isomers h~ ihe reaction rates of 3% and 2% of the aruchidonate 12-oxygenation. Thus, 12qipoxygenases of human and bovine platelets were catalytically distinct from the porcine and bovine leukocyte enzymes in terms of their reaetivities not only with linoleic and linolenic acids, but also with leukotriene A 4 as lipoxin precursor.

Introduction

Arachidonate 12-1ipoxygenase incorporates one molecule of oxygen at C-12 of arachidonic acid and produces 12S-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12S-HPETE). 12-Lipoxygenase was found first in platelets !1,2]. and later in leukocytes [3,4]. Earlier it was reported that 12-1ipoxygenase of bovine platelets was active with arachidonic acid and other C:o acids but almost inactive with linoleic and l~nolenic acids (Cl~ acids) [2], On the other hand, 12-1ipoxygenase of porcine leukoeyles was active with the Cis acids as well as various C2o acids [4,51. Interestingly, linoleic acid as substrate was oxygenated by the bovine leukocyte 12-

lipoxygenase, but not by the platclet enzyme of the same animal species [6]. Furthermore, we demonstrated that the 12-1ipoxygenases of bovine leukocytes and platelets were distinct not only in the substrate specificity, but also in the reactivity with antibodies [7]. in this paper, we compared catalytic properties of 12-1ipoxygenase purified from human platelets with those of three 12-1ipoxygenases from porcine leukocytes, bovine leukocytes and bovine platelets. Since recent papers reported that human platelet suspensions converted leukotriene (LT)A a to lipoxin (LX) isomers presumably by the catalysis of 12-1ipoxygenase [8,91, the activities of these four 12-1ipoxygenases with LTA 4 were also examined. Materials and Methods

Abbreviations: LT, leukotriene;LX. tipoxin; 12$-H(P)ETE.12$-hydro(pero)xy-SZ,SZ,lOE,14Z-eicosatetraenoic acid; 5S,HETE, 5S-hydroxy-6E,8Z,llZ,14Z-eicosatetraenoic acid; 15S-H(P)ETE, lSS-hydro(pcro)xy-SZ,8Z,l IZ,I3E-eicosatetraenoLcacid; 5&ISS-diHPETE. 5S,I 5S.dihvdroperoxy.6E,SZ.11Z,!3t?-eicosatetraenoicacid~ Correspondence: S. Yamamoto, Department of Biochemistry. TokushimaUniversitySchoolof Medicine.Kuramoto-cho,Tokushima 770. Japan.

Materials

Arachidonic acid and other unsaturated fatty acids were obtained (as described in Ref. 5). Nordihydroguaiaretic acid and soybean lipoxygenase (Type I) were purchased from Sigma (St. Louis, MO). 5S,12S-Dihydroxy.6E,8Z, lOE,14Z-eicosatetraenoic acid was a gift from Drs. J. Rokach and B. Fitzsimmons of Merck

Frosst Canada (Dorval). prostaglandin B, as an internal standard for HPLC from Ono Pharmaceutical Company (Osaka). and LTA 4 methyl ester from Dr. S. Terao of Takeda Research Laboratories (Osaka). Preparaiit,~ of 5S-HETE [10], 12S-HETE [5], 15S-H(P)ETE [111. and 5S,15S-diHPETE I11] was described previously. Saponification of LTA 4 methyl ester was performed by treatment with methanol/50~ sodium hydroxide (9:1. v/v) at 4°C for 1 h [121. Various LX isomers as standard compouvds were biosynthesized by the rc,~ctions of 5S.15S-diHPETE with 5- and 12-1ipoxygenases ot porcine [eukocytes [13.14].

Enzyme preparation Human platelets were prepared from a buffy coat as described previously [15]. The isolated platelets were suspended in 10-times the volume (v/w) of ice-cold 20 mM Tris-HCl buffer (pH 7.4) and sonicated at 20 kHz twice for 15 s by the use of a Branson sonifier model 185D. The sonicate was centrifuged at 10000 × g for 10 min and then at 105000×g for 60 rain at 4°C. The high-speed supernatant solution was stored at -80°C as the cytosol fraction. 12-Lipoxygenase was partially purified from the cytosol fraction of human platelets by immunoaffinity chromatography using a monoclonat antibody, HPLO-3, which was raised against human platelet 12-1ipoxygenase [7]. The enzyme purification procedure was essentially the same as that of bovine platelet 12-1ipoxygenase [7]. The purified enzyme gave a specific enzyme activity of about 0.3 p.mol/min per mg protein at 37°C, and converted arachidonic acid to 12S-HPETE, which was identified as 12S-HETE after borohydride-reduction by ultraviolet and mass spectra and reverse- and chiral-phase HPLCs as described previously [7]. 12-Lipoxygenases of porcine leukocytes [51, bovine leukocytes [7], and bovine plalelets 171 were purified by imnmnoaffinity chromatography as described previously. Spectrophotometric assay The purified 12-1ipoxygenase was allowed to react with various polyunsaturated fatty acids (50 #M) at 37°C in 1.0 ml of 50 mM potassium phosphate buffer (pH 7.0). The increase in absorption at 240 nm or at 270 nm was monitored continuously by the use of a Hitachi spectrophotometer model 330. Molecular absorption coefficients used were 22000/M per cm at 240 nm for a conjugated dicnc of monohydro×y acid [16] and 50000/M per cm at 270 nm for a conjugated triene of dihydroxy acid [17]. ItPLC analysis The purified 12-1ipoxygenases from various sources were incubated with 50 #M 15S-HPETE for 5 min (the leukocyte enzymes) or for 30 min (the platelet enzymes)

both in 1.0 ml of 50 mM potassium phosphate buffer (pH 7.0) at 24°C. Incubation with 5S-HETE was also ca,:ried out under the same conditions. In the reaction with 60 #M LTA 4 as substrate, the incubation was performed for 5 min (the leukocyte enzymes) or for 20 rain (the platelet enzymes) both at 24°C in 1.0 ml of 50 mM potassium phosphate buffer (pH 7.0) containing 0.1% bovine serum albumin. After the enzyme reactions, the products were treated with sodium borohydride, acidified to pH 3 with 2 M HCI, and extracted with ethyl ether. The solvent was evaporated, and the dried materials were applied to reversc-phase HPLC. Dihydroxy acids from 15S-HPETE or 5S-HETE were analyzed by a TSK-gel column (type ODS-120T, 4.6 x 250 mm. 5 #m. Tosoh) equipped with a 6000A pump and an injector model U6K (Waters). Absorption at 270 nm was monitored by a multiwavelength detector model 490 (Waters) and C-R4A chromatopac (Shimadzu). The solvent system was a mixture of methanol/water/acetic acid (70:30:0.01, v/v) at a flow rate of 1.0 ml/min. LX isomers produced from LTA 4 were analyzed by a Nova-Pak C18 column (3.9 x 150 ram, Waters) monitoring absorption at 300 nm for a conjugated tetraene. A mixture of acetonitrile/methanol/water/acetic acid (425:425:1150:1, v/v) was used at a flow rate of 1.0 ml/min.

Results and Discussion 12-Lipoxygenase was partially purified from the cytosol fraction of human platelets by immunoaffinity chromatography. The purified 12-1ipoxygenase was allowed to react with various unsaturated fatty acids, and the increase in absorption at 240 nm was monitored continuously to detect the production of hydroperoxy acids. As shown in Table i, the human platelet enzyme was active with several eicosapolyenoic acids and docosahexaenoic acid. K m values for arachidonic acid, 5,8,11,14,17-eicosapentaenoic acid, and 8,11,14-eicosatrienoic acid were approx. 8, 3 and 35 #M, respectively. in contrast, octadecapolyenoic acids were scarcely oxygenated. As cited in Table 1, such a substrate specificity of the human platelet 12-tipoxygenase was similar to that of bovine platelet enzyme [7], and different from those of the porcine [5] and bovine [71 leukocyte enzymes, which were active for various octadecapolyenoic acids as well as eicosapolyenoic acids and docosahexaenoic acid. We reported previously that the bovine platelet 12lipoxygenase and the bovine leukocyte 12-1ipoxygenase exhibited different reaction time courses [7]. Therefore, tb: reaction time course of the human platelet 12tipo~.ygen;~se w~s recorded ~nd compared with !hose of bovine platelets and porcine and bovine leukocytes (Fig. 1). The reactions of both the porcine and bovine leukocyte enzymes with arachidonic acid ceased within 5 rain,

, lla .......

TABLE I Sub.+lrare .+peclfR'tlies of fi+ltr I-

~imJ.x[r~eml.~e~

0.00..,

The purified 12-1ipoxygenase from human plalclets Was allowed to react with various unsaturated fatty acids at 511 ~M. The inaial rea~:i.ioti rates were de:':rmir:cd spectrophmometricalb m,,,~it,,ring absorption at 270 nm for 5S-HETE and 15S-HPETE as substrates and absorption at 240 nm for other substrates. The data for the other three enzymes were cited from previous papers as indicated in the table. The initial rate with arachidonic acid presented as 100 is 1.54 nnm!/min, 5 ~g ptotc,,i ,at 37°C L,r hum.".n 21:~:.%t ,.+~7v,'.,¢. !.'5, nmol/min, 6.2 gg protein at 37°C for bovine platelct cnz)me. 6.5 nmol/min, 6.2 ttg protein at 37+C for bovine Icukoc)te enzyme, and 26 nmol/min, 5.2 t~g protein at "-0°C for porcine leukocyte enzyme. n.d., not detectable. Substrate

R6ative initial vdocity 1'7 )

Human Bovine Bovine Porcine platelet platelet leuktx:yte leuhx-,te enzyme * enzyme enzyme enz',me Araehidonic acid Linoleic acid a-Linolenic acid ¥-Linolenic acid I 1.14-Eicosadienoic acid ] 1,14.17-Eicosatrienoic acid 8.1 l,l&Eicosatricnoic acid 5,8,11,14,17-Eicosapcntaenoic acid 4,7,10,13,16,19- Dt~:osahexae3oic acid 5S.HETE 15S-HPETE LTAa

100 rt.d. n.d. 4

IO0 n.d+ n.d. n,d.

III1.1 I1 17 32

l(~) 69 56 I1,~}

n.d.

n.d+

15

44

n.d.

n.d.

28

36

27

7

78

55

107

q4

73

67

7 n.d. 6 n.d. *

q2 15 IS 2"

56 50 5~ 3"

7

7

5

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Reference

* Determined in the present work+

E

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0.4 / 7

I

/ ~

/'.

i i/is (

I

0.I

/ E 0

. . . . . .

o

6

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,+ (A} I



3o

Time (min) Fig. I. Reaction iime-cnurses of four 12.1ipoxygena.ses+The purified 12-1ipoxygenases from porcine leuh~cytes 16.3 ag) tar, bovine leukt',cytes (87pC) (B), bovine platdets (25 ,g) (C), human platelets (7,2 v.gl (D), and human platdet (healed+ 7.2 ttg) (El were allowed to react with 50 +aM arachidonic acid in 1+0 ml of reaction mixture at 37"(.'. Absorption at 240 nm was monitored continuously.

E ¢;

l=

i,g

o.ooi

~'; ..... lil-I

,~n

~ t

(B)

0,0~

/I ?" ,4 RetentionTime(mini Fig. 2. Reaction~ of tSS-HPETF with four 12-1ipox.',genases. The purified 12+lipox~,genasesfrom porcine teukc,cytcs 1,,8.4lag) IAl, bo,'ine leukocylCS (17..* PC) (BI. bosine platelets 150 ~tgl (C}, and human plateleb, (14.4 ~gl tD} were incubated with 50 ~.M ISS-HPETE at 24°t" for 5 mm CA and B) or for 311 rain (C and Dr. Re','crxe-phas¢ HPLC ~ a,,, performed as described in Materitds and Methods. Prosiagl,mdin B2. (PGB,) (0.6 ~g} was added as an internal standard. Peak numbers are described in the text.

whereas those of the human and bovine platetet enzymes proceeded almost linearly for more than 30 min. Thu,~. in terms of the reaction time-course the human platelet 12-tipoxygenase was close to the bovine platelet 12-1ipoxygenase. but different from the porcine and bovine leukocyte 12-1ipoxygenases. These enzymological results were consistent with our

previous immunological observ,.tions distinguishing two types of 12-1ipoxygenase. Namely, a monoclonal antibody against the human platelet 12-1ipoxygenase reacted with the bovine platelet enzyme, but not with the bovine leukocyte enzyme [71. On the other hand, a monoclonaL antibody against porcine leukocyte 12lipoxygenase reacted with the bovine leukocyte enzyme [7]. hut not with the human and bovine platelct enzymes liSl. Reactivity of 12-1ipoxygenase with 15S-HPETE was previously reported for the enzymes of porcine leukocytcs [51, bovine leukocytes [71 and bovine platelets [7]. In comparison with the three enzymes previously studied, the human platelet 12-1ipoxygenase was allowed to react with 15S-HPETE. The products were reduced with borohydride, and analyzed by reversephase HPLC monitoring absorption at 270 nm to detect a conjugated triene (Fig. 2Dr+ The same major peaks were obtained with the porcine leukocyte enzyme (Fig. 2A), the bovine leukocyte e,,.zyme (Fig. 2B)+ and the bovine platelet enzyme (Fig. 2C). These products were identified previously as follows [5]: la and lb. 14R,15Sand I,:IS,! 5S-dihydroxy-5Z.SZ, 10 E. 12 E-eicosatetra-

enoic acids; Ila and lib. 8S.15S- and 8R,15S-dihydroxy-5Z,gE,11Z,13E-eicosatetraenoic ~cids; and IIla and tllb, 8R,15S- and 8S,15S-dihydroxy5Z,gE,I lE,13E.eicosatetraenoic acids. Compounds llla and lilb were known as major hydrolytic products of 14,15-LTA 4, whereas compounds la and Ila were produced by the borohydride-reduction of 14R,15S- and 8S.15S-dihydroperoxy acids [I9]. It should be noted that the IIa pe:lk was higher than the lib peak with the leukocyte enzymes, while the lib peak was higher than ~h," i!a peak with the platelet enzymes. This is a subject fl)r further investigations. These findings indicated that like the other 12-]ipoxygenases the human platelet enzyme possessed the 14,15-LTA 4 synthase activity and the 8- and 14-oxygenase activities. However, the reaclion rate of the human platelet enzyme with 15S-HPETE as examined spectrophotometrically was only 2% of that of the arachidonate 12-oxygenation, and this value was lower than the reactivities of 15S-HPETE with the 12-1ipoxygenases of porcine and bovine leukocytes (Table I). The human platelet 12-1ipoxygenase was also active for 5S-HETE. As analyzed by reverse-phase HPLC, 5S, ]2S-dihydroxy.6E.8Z.lOE,14Z-eicosatetraenoic acid and 5S,15S,dihydroxy-6E,8Z,11Z,13E-eicosatetraenoic acid were produced in a ratio of about 8:2. The reaction rate was only 3% of that of arachidonate 12oxygenation. In contrast, the porcine and bovine leukocyte 12-1ipoxygenases oxygenated 5S-HETE at higher reaction rates (Table I). According to recent reports, a human platelet suspension converted LTA4 to several LX isomers. It was presumed that the platelet 12-1ipoxygenase catalyzed the 15-oxygenation of LTA 4 and produced a 15-hydroperoxy-5,6-epoxide [8,9]. Indeed the purified 12-1ipoxygena.se of human platelets showed the 15-oxygenase activity with 5S-HETE as mentioned above. We attempted to examine whether or not the purified 12-1ipoxygenases of various origins could produce LX isemers from LTA,,, The products were analyzed by reversephase HPLC monitoring absorption at 300 nm for a conjugated tetraene. As shown in Figs. 3A and 3B, the porcine and bovir~e leukocyte enzymes converted LTA 4 to compounds eluting as peaks 1 + 2, 3 + 4, 5, and 6. Peaks 1 + 2 and 3 + 4 showed absorption spectra with a maximum at 302 nm and shoulders around 287 and 316 nm, which were characteristic of a conjugated tetraene. The retention times of these products were compared with those of authentic LXs. Peaks 5 and 6 were coeluted with LXA 4 and 6S-LXA 4, respectively, Peak I + 2 was clearly separated intc~ two peaks by further HPLC on a TSK-gel column ~solvent system, acetonitrile/ water/acetic acid, 35 : 65 : 0.05, v/v), and the two peaks cochromatographed with authentic 8-trans-LXB4 and 14S-8-trans-LXB4. Similarly, peak 3 + 4 was a mixture of l l-trans-LXA 4 and 6S-11-trans-LXA 4. These LX

I

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Retention Time (min) Fig, 3. Reactions of LTA4 with four 12-lipoxygcnascs. The purified ]2-lipoxygenase from porcine Ieukocytes (21 Pg) (A), bovine leukocytes (220 pg) (B). bovine platelets (34 vg) (C), human platelets (18 #g) (D). and human platelcts (heated, 18 Fg) (E) were incubated with 60 ,aM LTA4 at 24°C for 5 rain (A and B) or for 20 rain (C. D and

E). Reverse-phase HPLC was performed as described in Materials and Methods. ProstaglandinI~ (PGI~) (0.4 ,ag) was added as an internalstandard. Peaknumbersarc describedin the text. isomers produced by the leukocyte enzymes were the same as the products formed by non-enzymatic hydrolysis of synthetic 15S-hydroxy-5,6-LTA4 [20]. Presumably, LTA 4 as such (not as its hydrolytic 5,6-dihydroxy products), was oxygenated at C-15 position by the 12lipoxygenases to produce 15S-hydroperoxy-5,6-epoxide, which was then hydrolyzed to the LX isomers corresponding to peaks 1-6 in Fig. 3 (Scheme 1). This pathway was presumed for human platele:, by other

+Oz

LTA4 ~1[

12-Lipoxygenllse I

COOH O(O)H

15.hydr o(pero)xy.5,6-LTA4 +HsO

~

OH

OH

6H 8-trans.LXB4

OH 11.trans-LXA4

OH

OH

~ OH OH 14S-8-tr8ns-LXB4 bS-11-Irans-LXA4

OH

OH

LXA4 OH

OH 6S-LXA4

Scheme I. Biosynthetic pathway of LX isomers from LTA4 by 12-

lipoxygenase.

investigators [8,9]. An alternative pathway, namely, the epoxide hydrolysis followed by 15-oxygenatiop of the degradation products, did not explain the formation of LXB4 isomers. We incubated LTA 4 with soybean 15lipoxygenase and found essentially the samz profile of LX isomers on HPLC. LX production from LTA4 by soybean enzyme was also mentioned in a previous paper

This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare of Japan, and grants from the Japanese Foundation of Metabolism and Diseases, and the Tokyo Biochemical Research Foundation,

[91. Production of various LX isomers from LTA 4 by the porcine and bovine leukocyte 12-1ipoxygen:3ses proceeded at 3% and 2% the rate of arachidonate 12oxygenation, respectively (Table l). The reaction by the porcine leukocyte enzyme was completely inhibited by 100 .aM nordihydroguaiaretic acid, and the heat-denaturmed enzyme produced no significant LX peaks. Therefore, the LX production is enzyme-catalyzed. In contrast, the human and bovine platelet enzymes produced several minor peaks with retention times similar to those of LX isomers (Figs. 3C and 3D). However, they did not show ultraviolet absorption characteristic of a conjugated tetraene, and these compounds were also produced by the heat-denatured enzyme to the same extent (Fig. 3E), Definite LX peaks were not observed even when the amount of the human platelet enzyme was increased 5-fold. This amount of the enzyme (derived from about 10 + cells) catalyzed arachidonate 12-oxygenation almost at the same rate of the porcine leukocyte enzyme shown in Fig. 3A, Thus, the platelet enzymes had much lower, if any, activities of the LX production from LTA 4 as compared wtth the leukocyte enzymes. The LX production from LTA 4 by intact human platelets was shown by Eden{us et al. [8] and Serhan et al. [91 using about 10 ~ cells. This discrepancy awaits further investigations, Previously we described two types of 12-1ipoxygenase, which were distinguishable each other in terms of the reactivity with octadecapolyenoic acids and the immunogenicity [7]. The present study added another catalytic property, namely, the reactivity with LTA 4. which distinguished the platelet-type and leukocyte-type of 12-1ipoxygenase.

Acknowledgements We are grateful to Ryoichi Kunai for his technical assistance in recording the mass spectrum of 12-HETE.

References 1 Hamherg, M , and S~muels,~m, B. {19741 Prec. Nail, Acad. Sci. USA 71, 34O0 ~404.

2 Nuglcren. D.H.

Catalytic properties of human platelet 12-lipoxygenase as compared with the enzymes of other origins.

Arachidonate 12-lipoxygenases of porcine and bovine leukocytes were different in substrate specificity and immunogenicity from the enzyme of bovine pl...
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