THE JOURNAL OF BIOLOGKXL CHEMISTRY Vol. 265, No. 3, Issue of January 25, pp. l-771-1776, 1990 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S. A.

Identification of a Novel Arachidonate 12-Lipoxygenase in Bovine Tracheal Epithelial Cells Distinct from Leukocyte and Platelet Forms of the Enzyme*

(Received for publication, August 11, 1989)

J. Randall HansbroughS$, Yoshitaka Takahashill, Natsuo Ueday 11, Shozo Yamamotollll , and Michael J. Holtzman$** From the *Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110 and the llDepartment of Biochemistry, Tokushima University School of Medicine, Tokushima 770, Japan

We examined the characteristics of an arachidonate 12-lipoxygenase in bovine tracheal epithelial cells in relation to the enzyme expressed in leukocytes and platelets. Homogenous preparations of intact or dis- rupted tracheal epithelial cells metabolized arachi- donic acid predominantly to (12S)-hydroxyeicosa- tetraenoic acid, and subcellular fractionation by dif- ferential centrifugation demonstrated that the 12-L poxygenase activity was localized predominantly to the 100,000 X g supernatant (cytosol fraction). Analy- sis of cytosolic enzymatic activity for pH dependence (maximum activity at pH 7.4-&O), divalent cation ef- fects (no dependence on cations), and kinetic charac- teristics (lag phase elimination by addition of hydro- peroxide) exhibited similarity to leukocyte and platelet 12-lipoxygenases. Immunoprecipitation experiments demonstrated that the epithelial 12-lipoxygenase re- acted with a monoclonal antibody (lox-2) directed against leukocyte 12-lipoxygenase but not with an an- tibody (HPLO-3) against the platelet enzyme. Immu- noaffinity chromatography of the epithelial 100,000 x g supernatant fraction using lox-2 linked to Affi-Prep 10 yielded a single predominant protein band (Mr = 72,000) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis identical in apparent mass to the bo- vine leukocyte lipoxygenase. Western blotting using a polyclonal antibody to leukocyte 12-lipoxygenase showed peroxidase staining of the same 72-kDa pro- tein band. Activity assays of the purified enzymes dem- onstrated that substrate specificity for the epithelial 12-lipoxygenase was similar to that of the leukocyte enzyme, but the epithelial enzyme more efficiently con- verted 18-carbon fatty acids to the corresponding monohydroxylated conjugated dienes. We conclude that bovine tracheal epithelial cells express a 12-lipox- ygenase that has immunological reactivity similar to leukocyte and distinct from platelet 12-lipoxygenase and possesses substrate specificity distinct from both enzymes. We further suggest that lipoxygenase heter-

* This research was supported in part by Grant HL 40078 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article _ - must therefore be-hereby marded”aduertkement” inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Parker B. Francis Fellow. 11 Supported by grants-in-aid for scientific research from the Min-

istry of Education, Science, and Culture and the Ministry of Health and Welfare of Japan.

** The Schering Career Investigator of the American Lung Asso- ciation. To whom correspondence and reprint requests should be addressed: Washington University School of Medicine, 660 S. Euclid Ave., Box 8052, St. Louis, MO 63110.

ogeneity may provide a basis for different functional roles for the enzyme in different cell types.

The arachidonate 12-lipoxygenase was the first of the li- poxygenases to be discovered in mammalian tissues. The enzyme was described initially in platelets (1, 2) and then later in porcine and bovine polymorphonuclear leukocytes (3- 5) and murine eosinophils (6). It catalyzes the reaction of arachidonic acid with molecular oxygen to form (12S)-hydro- peroxyeicosatetraenoic acid (12-HPETE),’ which, under most conditions, is reduced to the corresponding hydroxy fatty acid (12-hydroxyeicosatetraenoic acid, 12-HETE) and released from the cell. The precise function of 12-lipoxygenase activity remains uncertain, but a number of the enzymatic products have been reported to have potent effects as specific mediators in a variety of tissues (reviewed in Ref. 7).

The proposed heterogeneity of 12-lipoxygenase function may be explained by the hypothesis that the enzyme serves different functional roles in different cell types. That possi- bility is supported by evidence of biochemical heterogeneity among 12-lipoxygenases isolated from leukocytes uersus platelets. Initial studies of bovine and porcine lipoxygenases demonstrate that the leukocyte form of the enzyme exhibits a much broader range of substrate acceptability in comparison with the platelet enzyme (2, 5, 8). More recently, the 12- lipoxygenases from bovine leukocytes and platelets have also been distinguished from one another by immunoprecipitation with distinct monoclonal antibodies (9). Furthermore, the immunoaffinity-purified enzyme from platelets shows little activity for octadecapolyenoic acid substrates relative to the leukocyte enzyme.

To define the capabilities of lipid mediator generation in the pulmonary airway epithelium, we have investigated the capacity of isolated bovine tracheal epithelial cells to metab- olize arachidonic acid. We chose to study bovine cells in view of the well characterized bovine platelet and leukocyte lipox- ygenases and thereby aimed to avoid possible species differ- ences (10). We discovered that the predominant arachidonic acid metabolite from bovine tracheal epithelial cells was (lBS)-HETE (ll), implicating the action of an arachidonate 12-lipoxygenase. The present experiments were designed to

1 The abbreviations used are: HPETE, hydroperoxyeicosatetrae- noic acid; HETE, hydroxyeicosatetraenoic acid; HPODE, hydro- peroxyoctadecadienoic acid; HODE, hydroxyoctadecadienoic acid; PG, prostaglandin; Hepes, 4-(2-hydroxyethyl)-l-piperazineethane- sulfonic acid, EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; HPLC, high pressure liquid chromatography; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis.

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1772 12-Lipoxygenase in Tracheal Epithelial Cells

characterize the enzyme definitively using immunoprecipita- tion and immunoaffinity chromatography with anti-12-lipox- ygenase monoclonal antibodies and to determine whether the epithelial enzyme differed in its immunological or kinetic characteristics from the leukocyte and platelet forms of the enzyme. The results represent the first reported purification and characterization of an epithelial cell lipoxygenase.

EXPERIMENTAL PROCEDURES

Sources of Materials-[ 1-Y]Arachidonic acid (54.9 mCi/mmol), [“Hs]arachidonic acid (95.1 C’/ i mmol), and [l-‘4C]linoleic acid (50.5 mCi/mmol) were from Du Pont-New England Nuclear; unlabeled arachidonic, linoleic, y-linolenic, cu-linolenic, 11,14-eicosadienoic, 11,14,17-eicosatrienoic, 8,11,14-eicosatrienoic, and 4,7,10,13,16,19- docosahexaenoic acid were from NuChek Prep Inc. (Elysian, MN); 5,8,11,14,17-eicosapentaenoic acid, Type I collagenase, Type XIV protease (Pronase E), Triton X-305, and Tween 20 were from Sigma; protein A-bearing Staphylococcus aureus and anti-mouse IgG were from Boehringer Mannheim; trimethylphosphite was from Aldrich; organic solvents were from Burdick and Jackson Laboratories Inc. (Muskegon, MI); and phosphate-buffered saline and Hanks’ balanced salt solution containing penicillin (100 units/ml), streptomycin (0.1 mg/ml), gentamicin (0.1 mg/ml), and Hepes (25 mM) were from the University Tissue Culture Support Center. Authentic reference com- pounds included (12S)- and (12R)-hydroxy-(52,8Z,lOE,142)-eicosa- tetraenoic acid (12S and 12R-HETE); (i2S)-hydroperoxy-(5Z,&3Z, lOE,14Z)-eicosatetraenoic acid (12-HPETE); (15S)-hydroxy-(5Z,8Z, llZ,13E)-eicosatetraenoic acid (15-HETE); 8-hydroxy-11,12-ep- oxy-(5Z,9E,14Z)- and lO-hydroxy-11,12-epoxy-(5Z,8E,l4Z)-eicosa- trienoic acids (henoxilin Aa and B,); and (13S)-hvdroxv-(SZ.llE)- octadecadienoic acid (13-HODE) and (13Sj-hydroperox4-iSZ;llEj- octadecadienoic acid (13-HPODE) obtained from Biomol Research Laboratories Inc. (Plymouth Meeting, PA) and Cayman Chemical Co., Inc. (Ann Arbor, MI) and prostaglandin Bz (PGB,) and E2 (PGE,) from Upjohn (Kalamazoo, MI) or Sigma.

Cell Purification-Epithelial cells were isolated from strips of bo- vine tracheal mucosa by enzymatic dissociation with 0.02% Type I collagenase and 5 mM dithiothreitol for 2 h at 37 “C or 0.1% Pronase at 4 “C for 12-16 h in Joklik’s minimum essential medium. Cell preparations exhibited >90% exclusion of trypan blue and contained percentages of ciliated, nonciliated, and goblet cells that were similar to values reported previously (11, 12). All preparations were stained with a modified Wright’s stain, and only those with less than 1% granulocytes were analyzed.

Bovine polymorphonuclear leukocytes were prepared using a method modified from one described previously (9). Whole blood (2 liters) was mixed with a solution of 0.14 M citric acid, 0.2 M trisodium citrate, and 0.22 M dextrose (200 ml) and then centrifuged at 300 x g for 10 min. The resulting supernatant (containing platelets) was removed, the cell pellet was treated with distilled water to lyse residual erythrocytes, and the remaining leukocytes were washed and resus- pended in Hepes-buffered Hanks’ balanced salt solution (pH 7.4). Preparations contained 51% neutrophils, 31% eosinophils, and 18% mononuclear cells with a ratio of platelets to leukocytes of 2:l.

Cell Incubation Conditions-Whole cell capacity for lipoxygenation was tested by resuspending replicate aliquots of cells in Hepes- buffered Hanks’ balanced salt solution containing arachidonic acid dissolved in ethanol. Preliminary experiments demonstrated that final concentrations of 2 x lo6 cells/ml, 80 WM arachidonic acid, and 0.5% ethanol for 30 min at 37 “C resulted in maximal product gen- eration. Reagents incubated with boiled cells (100 “C for 15 min) or with cell-free media demonstrated insignificant generation of arachi- donic acid metabolites.

Subcellular Fractionation-Arachidonic acid oxygenation was also tested in subcellular fractions obtained after disruption of cells by sonication and separation of fractions by differential centrifugation at 4 “C. Suspensions of 1-5 x 10’ cells/ml were sonicated at the microtip limit (105 watts) for 30 s (three cycles of 10 s each) in 10 mM Tris-HCl (pH 7.4) containing 5 mM EDTA and 1 mM EGTA. Assay of sonicated mixtures demonstrated no loss of oxygenation activity compared with intact cells. The mixtures were centrifuged at 12,000 x g for 20 min, and the resulting supernatants were centrifuged at 100,000 X g for 1 h. Protein in each fraction was determined by the Bradford method using bovine serum albumin as a standard (Bio- Rad).

Enzyme Assay of Subcellular Fro&ohs-The pellets obtained by

centrifugation at 12,000 and at 100,000 x g were resuspended in 10 mM Tris-HCl (pH 7.4) with 5 mM EDTA and 1 mM EGTA for assay of oxygenation activity, and the cell sonicate and supernatant from 100,000 X g were assayed directly. In general, 100 or 200 ~1 of an enzyme-containing fraction (2-8 mg of protein/ml) was added to 800 or 900 ~1 of 125 mM Tris-HCl (pH 7.4) (final concentration of 100 mM Tris-HCI) with [3H] or [‘?]arachidonic acid or [“Cllinoleic acid and 1% ethanol. Conditions were varied (0.1-160 uM fattv acid substrate; incubation time, l-45 min; incubation temperature, 20- 37 “C; pH 6.0-9.2) to characterize enzyme-substrate interaction. In other experiments, the effects of adding fatty acid hydroperoxide (1 pM 13-HPODE) or divalent cations (5 mM CaCl*, MnC12, or MgClz) were also tested.

Extraction and Derivatization of Oxygenation Products-Incuba- tions were stopped by rapid cooling to 4 “C, an aliquot of PGBz was added to serve as an internal standard for product recovery, and in some experiments 5 ~1 of trimethylphosphite was added to reduce hydroperoxides to alcohols. Cell supernatants or enzyme mixtures were extracted with 2-propanol/chloroform/acetic acid or with diethvl ether/acetic acid as described’ previously (9, 11, 12). Recoveries of PGB, and 12.HETE were similar (75 f 3%. mean & S.E.). For some analyses, carboxyl groups of compounds were converted to methyl esters by treatment with ethereal diazomethane as described previ- ously (10).

HPLC-Extracts were reconstituted in chromatographic solvent and analyzed by HPLC on a liquid chromatograph (model 1090M; Hewlett-Packard Co., Palo Alto, CA). For reverse-phase HPLC, the chromatograph was fitted with a guard cartridge, and a 4.6 X lOO- mm analytical column packed with 3-pm octadecylsilane-coated par- ticles (Dynamax, Rainin Instrument Co., Woburn, MA). A flow rate of 1.0 ml/min was used with two solvents (A and B) set at 34% B for O-9 min. 55% B for lo-27 min. and 85% B for 28-35 min where A was water/acetic acid (lOO:O.Ol; v/v), and B was acetonitrile/acetic acid (lOO:O.Ol). Chiral-phase HPLC was carried out with a pair of 4.6 x 250-mm columns in series which were packed with 5-pm aminopro- pyl silica particles ionically bonded to dinitrobenzoylphenylglycine (J. T. Baker) at a flow rate of 0.8 ml/min. Two solvents (A and B) were used in an isocratic program of 8% B where A was hexane and B was hexane/2-propanol (100:4). The HPLC eluate was monitored using a Hewlett-Packard 1040 diode array spectrophotometer set at 270 nm for conjugated triene diols (including PGBZ) and 235 nm for conjugated dienes (including HETEs). The outflow from the spectro- photometer was routed to a Flo-One-0 detector (Radiomatic Instru- ments & Chemical Co., Tampa, FL) for concurrent measurement of ‘H or 14C. Standard molar absorption coefficients were used for quantification and were verified by measurements based on specific activity as described previously (10).

Thin Layer Chromatography (TLC&Enzyme incubations with [“Clarachidonic acid were stopped with the addition of 2.25 volumes of diethyl ether, methanol, 1 M citric acid (30&l), the mixture was centrifuged at 1500 x g, and the upper phase was spotted on 20 X 20- cm Silica Gel 60 plates that were preactivated for 1 h at 110 “C. Plates were developed in petroleum ether/diethyl ether/acetic acid (15:85:0.1) for 50 min at -10 “C and then subjected to radioscanning for detection of products.

Zmmunoprecipitation of 12-Lipoxygenase-A monoclonal antibody directed against porcine leukocyte 12-lipoxygenase (10x-2), which cross-reacted with bovine leukocyte l2-lipoxygenase, and one directed against the human platelet 12-lipoxygenase (HPLO-3), which cross- reacted with bovine platelet lZIlip&ygenase, were prepared as de- scribed nreviouslv (9.13). Varving amounts of lox-2 or HPLO-3 (0.03- 30 Gg) were incubated with p&e&i A-bearing S. aureus for 10 min at 24 “C. washed in 50 mM Tris (DH 8.0). and then incubated with aliqudts from the 100,000 X g supernatant of sonicated epithelial cells or leukocvtes for 30 min at 4 “C in 100 mM Tris-HCl (DH 7.4). For HPLO-3,“10% S. aureu.s was preincubated with 160 rg/$ anti-mouse IgG for 10 min at 24 “C. Immune complexes were centrifuged at 1,500 x g for 10 min, and resulting supernatants and pellets were assayed for enzymatic activity. Supernatants were assayed by direct addition of 25 pM arachidonic acid and 1 pM 13-HPODE, and pellets were assayed after resuspension in phosphate-buffered saline (pH 7.4) with 0.05% Tween 20, reprecipitation, and addition of arachidonic acid and 13-HPODE in 100 mM Tris-HCl (pH 7.4).

Zmmunoaffinity Chromatography-Lox-2 (6 mg) was conjugated to 3 ml of Affi-Prep 10 (Bio-Rad), and the material was used to pack a 1 x 4-cm Econo-Column (Bio-Rad). After equilibration with 50 mM diethanolamine HCl (pH 7.8) containing 0.05% Triton X-305, 0.15 M NaCl, and 20% glycerol, the cytosolic fraction from sonicated epithe-

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12-Lipoxygenme in Tracheal Epithelial Cells 1773

ha1 cells or leukocytes (5-15 ml) was applied. The column was washed with equilibration buffer for 10 min at 1 ml/min, and then enzyme was eluted with 50 mM diethanolamine (pH 10.4) containing 0.3% Triton X-305, 0.5 M NaCl, 20% glycerol, 2 mM 2-mercaptoethanol, and 20 j.tM ferrous ammonium sulfate. Fractions of 1 ml each were collected in tubes containing 0.2 ml of 1.0 M Tris-HCl (pH 7.4), assayed for 12-lipoxygenase activity and protein concentration, and stored at -70 “C.

Immunoblotting-Aliquots of the cytosol fraction from sonicated epithelial cells and leukocytes and from immunoaffnity-purified 12- lipoxygenase from leukocytes and epithelial cells were subjected to 10% polyacrylamide gel electrophoresis (PAGE, l-mm thickness) in the presence of 0.1% sodium dodecyl sulfate (SDS) (14). Protein bands on the gel were detected by silver staining, The gels also underwent electrophoretic transfer to nitrocellulose membranes (15), which were then stained with a polyclonal rabbit antibody against the porcine leukocyte 12-lipoxygenase (16). Binding of primary anti- body was detected using a biotinylated anti-rabbit IgG secondary antibody and a preformed avidin-biotinylated horseradish peroxidase complex (Vectastain ABC kit, Vector Laboratories). The membrane was washed and subjected to the peroxidase reaction by exposure to a mixture of 0.6 mM 3,3’-diaminobenzidine HCl, 3 mM hydrogen peroxide, and 50 mM Tris-HCl (pH 7.6).

Spectrophotometric Assay of Purified Enzyme-An aliquot (40-100 ~1) of the eluate from immunoaffinity chromatography containing leukocyte or epithelial 12-lipoxygenase activity was added to 120-160 ~1 or 900-950 J of 200 mM Tris-HCl (pH 7.4) (final reaction volume of 200 ~1 or 1 ml) containing 25 pM fatty acid substrate, 0.02% Tween 20, and 1 pM 13-HPODE at 37 “C. The reaction time course of the purified 12-lipoxygenase was monitored continuously by detection of conjugated dienes at 240 nm in a Beckman DU-64 spectrophotometer, and initial velocity of the reaction was calculated for each substrate using a molar absorption coefficient of 22,000. Assays were monitored at 240 nm (rather than 235 nm) because of a decrease in background absorbance and consequent improvement in signal/noise.

RESULTS

Identification of Whole Cell and Cytosol Products-Bovine tracheal epithelial cells and the 100,000 X g supernatant fraction from disrupted cells invariably converted arachidonic acid to a single predominant metabolite comigrating with 12- HETE during reverse-phase HPLC (Fig. 1). Analysis of the product by gas chromatography and electron-impact mass spectrometry (11) and by chiral-phase HPLC (Fig. 2) dem- onstrated that the compound was (lBS)-HETE. Generation of 12-HPETE was detected only when metabolites were proc- essed and analyzed at -10 “C by TLC (Fig. 3). In parallel experiments (not shown), bovine polymorphonuclear leuko- cytes and the 100,000 X g supernatant fraction from disrupted leukocytes generated a nearly identical profile of metabolites during analysis by reverse-phase HPLC. Mean levels of 12- HETE were 601 and 158 pmol/106 epithelial cells and leuko- cytes, respectively (n = 3). Both epithelial and leukocyte cytosol fractions incubated with arachidonic acid also resulted in the generation of a low level of (15S)-HETE (Figs. 1 and 2).

Characterization of Enzyme Preparation-Initial experi- ments demonstrated that the epithelial12-lipoxygenase activ- ity was relatively confined to the 100,000 x g supernatant (cytosol fraction) of disrupted cells. Mean specific activity for the cytosolic fraction incubated with a concentration of ara- chidonic acid (80 PM) resulting in maximal 12-HETE forma- tion was 13.8 + 4.9 nmol/30 min/mg of protein (n = 8). Enzymatic activity in the 12,000 X g pellet (mitochondrial fraction) and the 100,000 X g pellet (microsomal fraction) was insignificant (<0.5 nmol/30 min/mg of protein). This pattern of subcellular localization is similar to that reported previ- ously for the bovine leukocyte and platelet 12-lipoxygenases that were isolated and assayed under similar conditions (5, 9).

Other characteristics of the epithelial lipoxygenase evident

1PHETE AA 12.HETE AA

111 I/ Is ii 3-

T PGE2 15HETE x 2- 6

x

0 M. L.1 , 0 10 xl 30 0 10 20 30

l-

Retention Ttme (min) FIG. 1. Reverse-phase HPLC analysis of radiolabeled ara-

chidonic acid products released from bovine tracheal epithe- lial cells (A) and from 100,000 x g supernatant (cytosol) fractions from disrupted epithelial cells (I?). Whole cells (6 ml of 2 x lo6 cells/ml of Hepes-buffered Hanks’ balanced salt solution, pH 7.4) or the cytosol fraction (2 ml of 0.3 mg of protein/ml of 100 mM Tris-HCl, pH 7.4) were incubated with [3H]arachidonic acid diluted with unlabeled arachidonic acid (final concentrations of 80 jtM arachidonic acid and 2.2 X lo5 dpm/ml) for 30 min at 37 “C. Products were extracted into acidic chloroform or diethyl ether and then resolved using the acetonitrile/water/acetic acid system de- scribed under “Experimental Procedures.” Major peaks of radioactiv- ity coeluted with 12-HETE and arachidonic acid (AA) and smaller peaks with PGE, and 15-HETE.

k 60

‘R

c 15s Q

x

I

15R go.g ,o

3 1

1: 2 !R

15s

1

15R

A-- clM..k -x--- 50 60 70 Retention Time (min)

FIG. 2. Chiral-phase HPLC analysis of UV-absorbing (A) and radiolabeled (B) arachidonate products released from the cytosol fraction of bovine tracheal epithelial cells. The cytosol fraction (diluted in 2 ml of 100 mM Tris-HCl to a final concentration of 0.3 mg of protein/ml) was incubated with 20 pM [“Clarachidonic acid for 30 min at 37 “C. Products were extracted into acidified diethyl ether, converted to the corresponding methyl esters, and analyzed by chiral-phase HPLC using the hexane/2-propanol solvent system de- scribed under “Experimental Procedures.”

from assay of the cytosol fraction of disrupted cells included: (i) the lack of dependence on divalent cations; (ii) the presence of a lag phase that was eliminated by the addition of hydro- peroxide; and (iii) maximum enzymatic activity at pH 7.4- 8.0. Enzymatic activity as assessed by 12-HETE generation was not increased by the addition of 5 mM calcium, magne-

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1774 12-Lipoxygenase in Tracheal Epithelial Cells

1P~HPETE AA 12.HETE7 1 I

6 4 8 12 16 Distance (cm)

FIG. 3. TLC of radiolabeled arachidonic acid products re- leased from the cytosol fraction of bovine tracheal epithelial cells. The cytosol fraction was incubated in 100 mM Tris-HCl with 25 pM [Wlarachidonic acid for 15 min at 25 “C. Products were extracted into acidified diethyl ether and analyzed by TLC at -10°C using the solvent system described under “Experimental Procedures.” Peaks of radioactivity correspond to trioxilin Aa and B, (peak I ), hepoxilin A, and B3 (peak 2), 12-HETE, 12-HPETE, and arachidonic acid (AA).

4r

0' ' -2

1 I I I -1 0 1 2

Amount of Antibody (log pg)

FIG. 4. Immunoprecipitation of bovine epithelial 12-lipox- ygenase using a monoclonal antibody raised against the por- cine leukocyte 12-lipoxygenase lox-2 and one against the human platelet 12-lipoxygenase HPLOJ. Antibodies were linked to protein A-bearing S. aureus for lox-2 and to protein A plus anti-mouse IgG for HPLO-3. Varying amounts of antibody were incubated with the 100,000 x g supernatant (cytosol fraction) from sonicated epithelial cells (200 ~1 of 2 mg protein/ml of Tris-HCl (pH 7.4)). The immune complex was precipitated, and the supernatant was assayed for enzymatic activity using TLC as described in Fig. 3. Assay of resuspended lox-2 immune complexes revealed 12-lipoxygen- ase activity that varied inversely with the corresponding supernatant activity and increased quantitatively with amount of lox-2 used (data not shown). Control experiments confirmed that HPLO-3 quantita- tively immunoprecipitated bovine platelet l2-lipoxygenase.

sium, or manganese chloride, but the addition of 1 PM 13- HPODE eliminated the 5min lag phase in the generation of 12-HETE at 20 “C (data not shown). These properties have been reported for the platelet and leukocyte 12-lipoxygenases including those isolated from bovine cells (5, 9, 17).

Immunoprecipitation-Immunoreactivity of epithelial 12- lipoxygenase was tested with two anti-12-lipoxygenase mono- clonal antibodies that selectively precipitate bovine leukocyte and platelet enzymes. The epithelial lipoxygenase reacted exclusively with the antileukocyte lipoxygenase antibody (Fig. 4) and in fact was immunoprecipitated in a manner quanti- tatively identical to the bovine leukocyte enzyme (9). On the basis of this selective antibody reactivity, immunoaffnity purification was carried out using lox-2 antibody as ligand.

Zmmunoaffinity Purification-The cytosol fraction from disrupted epithelial cells was applied directly to a column

JO

0 10 20 Fraction Number

FIG. 5. Immunoaffinity chromatography of epithelial 12- lipoxygenase. The 100,000 x g supernatant fraction from sonicated epithelial cells (10 ml of 3.5 mg of protein/ml) was applied to a 1 x 4-cm column containing Affi-Prep 10 linked to lox-2 antibody. Chro- matography was carried out as described under “Experimental Pro- cedures.” The column was washed with equilibration buffer up to fraction 10 before applying the elution buffer. Protein concentration (open circles) and 12-lipoxygenase activity (closed circles) were de- termined for each fraction using the assay conditions described in Fig. 3.

containing lox-2 antibody linked to Affi-Prep 10, and the chromatography resulted in marked purification of the epi- thelial 12-lipoxygenase. The enzyme was eluted by increased pH, detergent, and salt concentrations, and evidence of puri- fication was obtained by detection of enzymatic activity in only selected fractions (Fig. 5). Yields of purified epithelial enzyme were 4-6% of initial activity applied to the column; the corresponding yield for leukocyte enzyme was 15%. Total protein recovered from the column in the fractions containing 12-lipoxygenase activity was 1% of the initial total protein applied for both leukocyte and epithelial cell preparations. The specific activities of the purified epithelial and leukocyte enzymes based on initial reaction velocity with arachidonic acid were 0.3 and 0.7 pmol/min/mg of protein at 37 “C, respectively, and were comparable to values reported previ- ously (9).

Analysis of the epithelial lipoxygenase-containing fractions by SDS-PAGE and silver staining revealed a single predomi- nant protein band (Mr = 72,000) (Fig. 6). The molecular mass was identical to leukocyte 12-lipoxygenase purified under identical conditions. Furthermore, Western blot analysis of the immunoaffinity-purified epithelial and leukocyte enzymes using a polyclonal anti-12-lipoxygenase also stained a single protein band of identical mass (Fig. 7).

Substrate Utilization-Initial reaction velocities of immu- noaffinity-purified leukocyte and epithelial enzyme were tested in parallel to compare substrate specificity of the two enzymes. Reaction rates of the purified enzyme were deter- mined by continuous spectrophotometric assay in the pres- ence of Tween 20 and a hydroperoxy acid to normalize kinetic properties of the 12-lipoxygenase (17). In general, the two enzymes were active with a similar range of fatty acid sub- strates. Both enzymes exhibited significant activity for a variety of eicosapolyenoic acid substrates; however, the epi- thelial enzyme exhibited even greater activity than the leu- kocyte lipoxygenase for M-carbon fatty acid substrates (Table I).

To confirm that characteristics of the purified enzymes were representative of the 12-lipoxygenase activities con- tained in epithelial cells and leukocytes, the cytosolic fraction from each cell type was assayed for activity with a radiolabeled

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12-Lipoxygenase in Tracheal Epithelial Cells 1775

Epithelial Leukocyte

W -72kDa

FIG. 6. SDS-PAGE of immunoaffinity-purified 12-lipoxy- genase from bovine tracheal epithelial cells and polymorpho- nuclear leukocytes. Enzyme purification was carried out as de- scribed in Fig. 5. The immunoaffinity-purified enzymes were sub- jected to SDS-PAGE in 10% polyacrylamide, and a predominant protein band at 72 kDa was detected by silver staining.

- 72 kDa

FIG. 7. Western blot of bovine leukocyte and epithelial cell 12-lipoxygenase. SDS-PAGE was carried out as described in Fig. 6, and samples then underwent electrophoretic transfer to nitrocel- lulose and polyclonal anti-12-lipoxygenase antibody staining with 3,3’-diaminobenzidine as a peroxidase substrate. Lanes 2 and 3 con- tain purified epithelial cell lipoxygenase (1 x and 0.5 X, respectively), and lanes 2 and 4 contain purified leukocyte lipoxygenase.

18-carbon fatty acid substrate (linoleic acid) relative to ara- chidonic acid. Over a wide range of substrate concentrations, the cytosolic fraction prepared from epithelial cells exhibited markedly higher activity for linoleic acid than did the corre-

TABLE I

Substrate speclficit~~ of epithelial and leukocyte 12-lipoxygenases lmmunoaffinity-purified enzyme (40-100 ~1) was incubated in 200

mM Tris-HCl (pH 7.4) (final reaction volume of 200 ~1 or 1 ml) containing 25 pM fatty acid substrate, 0.02% Tween 20, and 1 ,uM 13- HPODE at :37 “C, and initial velocity was calculated from the rate of change in UV absorbance at 240 nm.

Initial velocity” Fatty acrd substrate

Epithelial Leukocyte

%

8,11,14,17-Eicosatetraenoic (arachi- lOOh 100 donic)

9,12-Octadecadienoic (linoleic) 51& 11 10 f 4 6,9,12-Octadecatrienoic (r-linolenic) 72 k 18 17 + 7 9,12,15-Octadecatrienoic (tu-lino- 46 -t 9 21 sz 5

lenic) 11,14-Eicosadienoic 29 f 5 24 + 6 8,11,14-Eicosatrienoic (dihomo-r- 98 + 8 94+ 14

linolenic) 11,14,17-Eicosatrienoic 32 + 5 18 f 7 5,8,11,14,17-Eicosapentaenoic 57 + 4 53 f 8 4,7,10,13,16,19-Docosahexaenoic 91 f 14 88 f 8

I’ Values represent percent of activity for arachidonic acid (mean + S.E., n = 3-5 experiments).

h 100% activity was equivalent to 4.6 and 5.0 nmol/min for epithe- lial and leukocyte enzymes, respectively.

15r A B

c-

0 SO 160 Fatty Acid Substrate Concentration (PM)

FK. 8. Concentration-response curves for 12-lipoxygenase activity in cytosol fractions from bovine tracheal epithelial cells (A) and bovine leukocytes (B). Fractions (diluted in 100 mM Tris-HCI and 0.02% Tween 20 to final protein concentrations of 0.6- 0.8 mg of protein/ml) were incubated with [“Clarachidonic (0) or linoleic acid (0) for 30 min at 37 “C, extracted with acidic diethyl ether, and analyzed by HPLC as described in Fig. 1. Products iden- tified as 12-HETE (from arachidonic acid) and 13-HODE (from linoleic acid) were quantified using specific activity. Each value represents the mean of duplicate samples.

sponding fraction from leukocytes (Fig. 8). Relative linoleic to arachidonic acid utilization by cytosolic 12-lipoxygenases was in close quantitative agreement with the relative amounts used by the immunoaffinity-purified enzymes.

DISCUSSION

The present data demonstrate that bovine tracheal epithe- lial cells contain an archidonate 12-lipoxygenase that exhibits immunological reactivity and kinetic characteristics similar to the leukocyte and distinct from the platelet form of the enzyme. In particular, the epithelial enzyme was quantita- tively immunoprecipitated and immunoaffinity purified using the same monoclonal antibody (lox-2) as for the bovine leu- kocyte 12-lipoxygenase. By contrast, epithelial lipoxygenase showed no reactivity with an antiplatelet 12-lipoxygenase (HPLO-3). Furthermore, characterization of substrate lipox- ygenation of the purified enzymes demonstrated that epithe- lial and leukocyte 12-lipoxygenase exhibit relatively broad substrate specificity in comparison with the platelet form of the enzyme (2, 5, 8, 9). In fact, the epithelial enzyme showed

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1776 12-Lipoxygenase in Tracheal Epithelial Cells

further differences in rates of substrate lipoxygenation even from the leukocyte form of the enzyme. It is therefore possible that subtle differences in enzyme structure are present be- tween even the leukocyte and epithelial12-lipoxygenases, but definitive proof of further heterogeneity will require charac- terization of enzyme structures.

The presence of an active 12-lipoxygenase in epithelial cells was unexpected. Immunoassay and immunohistochemistry of 12-lipoxygenase in porcine tissues demonstrate the enzyme in leukocytes of peripheral blood (13) and various organs (16) but not in resident parenchymal cells (16). Previous studies of isolated tracheal (and nontracheal) epithelial cells from other species have also failed to detect predominant 12- lipoxygenase activity (10, 12, 18). Of note is the presence of a highly active arachidonate 15-lipoxygenase in epithelial cells from human trachea, and this enzyme shares many of the biochemical features of the bovine tracheal epithelial 12- lipoxygenase (10-12). Both lipoxygenases convert arachidonic acid to 15 and 12-HETE (although in differing proportions), and this dual capability enables both to generate an identical series of dihydroxylated conjugated triene derivatives (3, 9, 10, 12, 17, 19). For other fatty acid substrates, even the predominant product of the two lipoxygenases is the same. For example, both enzymes convert linoleic acid to its 13- hydroperoxy analogue (8, 20). It is therefore of interest that linoleic acid is the most abundant substrate for oxygenation in tracheal epithelial cells (21) and might serve as a major fatty acid source of oxygenated mediators of cell function. The high concentrations of the 12- and Slipoxygenases in airway epithelial cells and granulocytes (13, 19, 22) continue to suggest a role for the lipoxygenases in the development of allergic and inflammatory hypersensitivity (7).

The present characterization of a distinct epithelial 12- lipoxygenase may have other implications for lipoxygenase function. Of particular interest are the findings of relatively broad substrate acceptability for the epithelial form of the 12- lipoxygenase and the relatively evanescent detection for some of its lipoxygenation products (e.g. hydroperoxy and hydrox- yepoxy acids). These findings suggest that the enzyme may play a much broader role in fatty acid modification than generation of only arachidonate-derived mediators. Further- more, even for arachidonic acid, oxygenation products other than the stable mono- and diHETEs might be expected to have biological effects. It is also possible that differences in enzyme-substrate interaction between lipoxygenases from dif- ferent cell types may confer a degree of specificity which is critical to their role in cellular function. For example, the capacity of epithelial lipoxygenase to utilize a variety of fatty acid substrates might make the enzyme a candidate to serve as a safeguard against the array of fatty acids released by

epithelial phospholipase activity (10, 21). This function may not be as vital in leukocytes or platelets if they have little release of 1% or 20-carbon polyenoic fatty acids or if the cells possess alternative mechanisms for elimination of excess or unsuitable fatty acid substrates.

Ackno&dgment-We gratefully acknowledge the expert technical assistance of Barbara Ferdman and Laurie Pryde.

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J R Hansbrough, Y Takahashi, N Ueda, S Yamamoto and M J Holtzmancells distinct from leukocyte and platelet forms of the enzyme.

Identification of a novel arachidonate 12-lipoxygenase in bovine tracheal epithelial

1990, 265:1771-1776.J. Biol. Chem. 

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