Isolation and Characterization of Thromboxane ... - Semantic Scholar

Vol. 260,No. 28, Issue of December 5, pp. 15059-15067,1985 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 byThe American Society of Biological Chemists, Inc.

Isolation and Characterization of Thromboxane Synthase from Human Platelets as a Cytochrome P-450 Enzyme* (Received for publication, March 27, 1985)

Michael HaurandS and Volker Ullrich From the Faculty of Biology, University of Konstanz, Federal Republic of Germany

(2,3). Inaddition to its occurrence in platelets, TxBz isfound Thromboxane synthase from human platelets was purifiedto apparent homogeneity by conventional in many tissues such as lung (4), spleen (4), lymphocytes, chromatographic techniques. A 423-fold enrichment leukocytes, macrophages (&lo), brain ( l l ) , and kidney (12) over the specific content in the 100,000 x g sediment and hasbeen implicated in many biologically important procfrom platelet homogenates was obtained. The enzyme esses. It is themain component of the so-called “rabbit aorta gave a single bandon sodium dodecyl sulfate-gel elec- contracting substance’’ and causes respiratory tract and vastrophoresis corresponding to a monomeric molecular cular smooth muscle contraction (2, 13, 14). It plays a role in weight of 58,800. One heme per polypeptide chain was the maintenance of hemostasis and is a potent mediator in present, and by optical and EPR spectroscopy a close pathological conditions such asvasospasm, ulceration, thromanalogy to the group of cytochrome P-450 proteins bosis, and asthma (15, 16). The biological activity ofTxA2 was established. From its substrate prostaglandin Hz, depends on the presence of specific receptors (17-19) and the stable end product thromboxane B z is formed with a specific activity of 24.1 pmol min-l mg of protein-’ seems to involve the intracellular liberation of calcium (20) which corresponds to a molecular activity of 1628 and, as shown in platelets, the activation of phospholipase C min-l. The enzyme formed 12~-hydroxy-5,8,10-hep- (21). Several groups have attempted to isolate the enzyme tadecatrienoic acid together with thromboxane Bz in a that converts the cyclooxygenase product PGHz to TxA,; 1:l ratio. Both products were identified by gas chro- however, a separationof the cyclooxygenase activity from the matography-mass spectrometry analysis. As reported thromboxane synthaseactivity has only been achieved to date previously for platelet microsomes (Ullrich, V., and (22-24). Our interest in thromboxane synthase originated from the Haurand, M. (1983)Adv. Prostaglandin Thromboxane Leukotriene Res. 11, 105-110), the purehemoprotein peculiar endoperoxide rearrangement involved. Its endoperspectrally interacts with pyridine-or imidazole-based oxide substrate, PGH2, canundergo an alternative rearrangeinhibitors and for the potent inhibitor imidazo-(1,5- ment leading to prostacyclin, also a highly potent eicosanoid, a)pyridine-5-hexanoic acida stoichiometric binding to and in many aspects an antagonist of TxAz (25). Both rearthe heme was shown. Substrate analogs with a meth- rangements seem to require the activation of one of the ylene group replacing the oxygen in either the 9- or endoperoxide oxygen atoms since a new oxygen-carbon bond 1l-position caused difference spectrashowing spectral must be formed for TxAz and PGI, synthesis. Such reactions shifts towards387 and 407nm, respectively. are typical of cytochrome P-450-dependent monooxygenases The identification of thromboxane synthase as a P- which act as oxene transferases (26) in the essential part of 450 protein suggests that the heme-thiolate group of the hydroxylation mechanism. Since cytochrome P-450 can endo- also react with oxene donors such as organic hydroperoxides the enzyme is required to split and activate the peroxide bond of prostaglandin Hz. (27) or iodoso derivatives (28), it seemed to us that a similar mechanism could be involved in the formation of TxA2 and PGI, from PGH,. First indications in favor of this notion Thromboxane B2 was discovered in 1974 by Hamberg and were obtained from spectroscopic inhibitor studies on aortic Samuelsson (1)as a metabolite of arachidonic acid in blood microsomal prostacyclin synthase (29) and proof was preplatelets. It was soon recognized to be a biologically inactive sented by the isolation and characterization of prostacyclin compound derived from thromboxane (Txl) Az, a potent in- synthase as a cytochrome P-450-like protein (30, 31). Studies on the cytochrome P-450 protein in human platelet ducer of platelet aggregation and of platelet release reactions microsomes, first described by Cinti and Feinstein (32), re* This work was supported by the Deutsche Forschungsgemein- vealed that the cytochrome was devoid of monooxygenase schaft, Projects A4 (SFB 156) and U1 36/12-1. The costs of publica- activity, but formed ligand-type spectra with the well known tion of this article were defrayed in part by the payment of page thromboxane synthase inhibitors UK-37,248 and OKY-1581 charges. This article must therefore be hereby marked “advertiement” in accordance with 18 U.S.C. Section 1734 solely to indicate (33). In thepresent paperwe describe the isolation of thromboxthis fact. ane synthase from human platelets and give experimental $ Partial thesis at theUniversity of Saarland. The abbreviations used are: Tx, thromboxane; PG, prostaglandin; proof that it belongs to the group of cytochrome P-450 proacid; teins. U-54701, (15S)-hydroxy-lla,9a-epoxyiminoprosta-5,13-~enoic U-51605, (15S))-hydroxy-9a,lla-azoprosta-5,13-dienoic acid; Uacid; 46619, (15S)-hydroxy-9~,11~-methanoepoxyprosta-5,13-dienoic EXPERIMENTAL PROCEDURES U-44069, (15S)-hydroxy-9a,llcu-epoxymethanoprosta-5,13-dienoic acid; UK-37,248, 4-(-2-(1H-imidazol-l-yl)ethoxy)benzoicacid; OKYPreparation of Human Platelet Microsomes-Human platelet-rich 1581, sodium(E)-3-(4-(3-pyridylmethyl)phenyl)-2-methylacrylate; plasma containing citrate, dextrose, and adenine as anticoagulants CGS-13080, imidazo(l,5-a)pyridine-5-hexanoicacid HHT, 12~-hy- was centrifuged a t 20 X g for 30 min to remove leukocytes and red droxy-5,8,10-heptadecatrienoic acid. blood cells.Platelets were collected by repeated centrifugation at 250-

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Thromboxane Synthase

500 X g for 15 min and after each centrifugation the pellet was resuspended in 10mM potassium phosphate buffer, pH 7.0, containing 10 mM EDTA, 5 mM glucose, 0.1 mM dithiothreitol, 1.15% KC1, 2 mg/liter of leupeptin, 2 mg/liter of pepstatin, 10 mg/liter of tryspin inhibitor, and 44 mg/liter of phenylmethylsulfonyl fluoride. The platelets were then rapidly frozen in liquid nitrogen and stored for use a t -80 "C. For subcellular fractionation platelets (60-ml aliquots, containing 10 g wet weight of platelets) were thawed slowly, gassedwith nitrogen for 5-10 min, and thensonicated in anice-water bath with a Branson sonifier (model B30, power setting at 10, Yd-inch probe) for 12 s. The sonication procedure was repeated 12 times with 1-min intervals for cooling. The sonicate (480 ml) was centrifuged at 7,000 X g for 15 min, and the supernatant was spun down a t 130,000 X g for 60 min. The resulting precipitate was homogenized with a glass-Teflon homogenizer in 480 ml of 10 mM potassium phosphate buffer, pH 7.4, containing 10 mM EDTA, 0.1 mM dithiothreitol, and 1.15% KC1, and was again centrifuged a t 130,000 X g for 60 min. The pellet was homogenized in 10 mM potassium phosphate buffer, pH 7.4, containing 20% (v/v) glycerol, 1mM dithiothreitol, and 1 mM EDTA, to give a final volume of 75 ml with a protein concentration of 13 mg/ml. Enzyme Purifkution-All manipulations were performed at 0-4 "C in buffers containing 10 mM potassium phosphate, pH 7.4, 20% (v/ v) glycerol, 0.1 mM EDTA, and 0.1 mM dithiothreitol unless noted otherwise. All buffers and gels were degassed before use. Lubrol PX was warmed up to 40 "C before pipetting into buffers. A 10% (w/v) cholate stock solution was prepared by dissolving cholic acid in water with the addition of 5 N NaOH until the pH was 8.0. For enzyme purification the microsomes were prepared, solubilized, and fractionated on DEAE-Sephacel the same day. Solubilization of thromboxane synthasewas achieved by adding to 75 ml of microsomes 10 ml of buffer containing 1.7% (v/v) Lubrol PX and4.25% (w/v) cholate with stirring to give a final concentration of 0.2% Lubrol PX and 0.5% cholate. After continued stirring in an ice-water bath for 30 min, the mixture was centrifuged at 130,000 X g for 60 min. The resulting clear brown-yellow supernatant was added to 50 ml of DEAE-Sephacel, equilibrated with buffer containing 1 mM EDTA, 0.2% (v/v) Lubrol PX, and0.5% cholate. The suspension was stirred for 30 min and filtered through a fine fritted glass funnel. The gel waswashed by resuspending it in50 ml of buffer as used for equilibration, stirring the suspension for 20 min, and filtering again. This procedure was repeated until absorption a t 280 nm could no longer be detected in the filtrate. Elution was performed by resuspending the gel several times while stirring in 50 ml of buffer containing 50 mM NaC1, 0.05% (v/v) Lubrol PX, and 0.2% cholate and filtering. Fractions containing thromboxane synthase were pooled and applied to an octylamine-Sepharose 4B column (1cm X 25 cm), equilibrated with the above elution buffer without sodium chloride. The column was washed with 100 ml of buffer containing 15% (v/v) dimethyl formamide, 0.02% (v/v) Lubrol PX, and 0.2% cholate, and the column was then eluted using a stepwise gradient of 30, 60, 90, 110, and 150 mM NaC1. Under these conditions most of the enzyme was eluted from the column a t a salt concentration of 110 mM NaC1. These fractionswere pooled and diluted 4-fold with buffer containing 0.1% (v/v) Lubrol PX and0.4% cholate. The diluted eluate was then applied to a DEAE-Sephacel column (1 cm X 20 cm). The enzyme was eluted with buffer containing 0.1% (v/v) of the nonionic detergent Lubrol PX andwithout additional salt. Enzyme-containing fractions were pooled and were diluted while stirring with buffer of pH 4.5 which contained 0.1% (v/v) Lubrol PX, to give a final pH of 6.5. This solution was then applied to a CMSepharose CL-GB column (1cm X 15 cm),equilibrated with buffer of pH 6.5 which contained 0.1% (v/v) Lubrol PX. Thromboxane synthase was not absorbed by the column and was completely eluted with equilibration buffer. In the final purification step, this eluate was applied to an Ultrogel HA column (1 cm X 20 cm), equilibrated with the same buffer as used in the previous step. The column was washed with 60 ml of equilibration buffer and thena stepwise gradient of potassium phosphate pH 7.0 (30, 60, and 100 mM) in 100-ml portions was applied. The enzyme was obtained by eluting the column with 100 mM potassium phosphate buffer, pH 7.0, containing 0.2% Lubrol PX and 0.4% cholate. Fractions having a detectable absorption at 424 nm were pooledand concentrated in anAmicon ultrafiltration cell with a PM-30 filter. The enzyme was found to be stable at 0 "C for 6-8 weeks, and no loss of enzymatic activity could be observed by freezing and thawing the enzyme once.

Protein was estimated by using a modified Lowry method (34) and by SDS-polyacrylamide gel electrophoresis after staining with Coomassie Blue R-250 and scanning the gels with a LKB 2202 ultrascan laser densitometer. Bovine serum albumin was used as a standard; in order to correct for interfering detergents, glycerol, and salts, it was dissolved in thesame buffers as thesamples. Optical Spectroscopy-For cytochrome P-450 determinations the samples were gassed with CO for about 1min, and thena few crystals of dithionite were added to the sample cuvette. An extinction coefficient of 91 mM" cm" (tmM 450-490nm) was used for the calculation of cytochrome P-450 concentrations (35). Binding spectra of solubilized microsomes or enriched or purified enzyme preparations were obtained by adding ligands dissolved in buffer or solvent to thesample cuvette and corresponding amounts of buffer as solvent to the reference cuvette. Both measurements of binding spectra and determinations of cytochrome P-450 were performed a t 12 "C using 500-111 cuvettes. Protoheme was determined spectrophotometrically after converting the heme to thepyridine-hemochromogen in thepresence of 0.1 N NaOH and 20% (v/v) pyridine as described (35). All optical measurements were performed using a Cary 118 spectrophotometer. EPR Spectroscopy-For EPR spectra, preparations enriched in enzyme were prepared as described above byfractionating solubilized human plateletmicrosomes on DEAE-Sephacel. As shown by optical absolute spectra, the preparations used were free of other hemoproteins. Fractions containing thromboxane synthase were pooled (100 ml) and concentrated to 500 p1 by ultrafiltration. To avoid high detergent concentrations, the ultrafiltration was stopped when the volume was reduced to 5 ml and the ultrafiltrate was diluted 3-fold with buffer without detergent, before the final concentration to 500 pl. 250 pl of the ultrafiltrate, containing approximately 6 nmol of heme, were transferred to a quartz tube(4.5 mminside diameter) and frozen in liquid nitrogen. After recording the EPR spectrum the sample was carefully thawed and 15nmol of UK-37,248, dissolved in 10 pl of buffer, were added and a new EPR spectrum was recorded. All EPR measurements were performed a t X-band with a Bruker BER 420 spectrometer (Bruker Instruments, Karlsruhe, Germany) equipped with a 100 kHz modulation unit and a NMR gaussmeter. Spectra were evaluated as described in Ref. 36. The low temperature unit was a Helitran LTC-11OC system (Air Products, Allentown, PA) operating at approximately 16 K. Assay for Monooxygenase Activity-Possible monooxygenase activity of thromboxane synthase was studied using 7-ethoxycoumarin as substrate. Dealkylation of 7-ethoxycoumarin was measured as described (37) using an Eppendorf fluorimeter. Solubilizedhuman platelet microsomes were added to the incubation mixture in different amounts (0.1-1.0 mg of protein) and fluorescence was recorded for 30 min. Control experiments were performed with rat liver microsomes. Disc Gel Electrophoresis-Discontinuous polyacrylamide gel electrophoresis on 1.5-mm thick and 20-cm long gels was performed as described by Laemmli (38), except that 25 mM Tris and 192 mM glycine in theelectrophoresis buffer were used.Protein fractionswere first heated for 30 min at 56 "C and then for 2 min a t 95 "C in 62.5 mM Tris-chloride buffer, pH 6.8, containing 1% (w/v) SDS, 5% (v/v) glycerol, 5% (v/v) 2-mercaptoethanol, and 0.001% (w/v) bromphenol blue. The stacking gel contained 4% (w/v) acrylamide and theseparating gel contained 10% (w/v) acrylamide. As protein standards phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa), and a-lactalbumin (14.1 kDa) were used. Stacking and separating took place a t 20 mA/gel using a Desaphor VA200 slab gel apparatus (Desaga, Mannheim, Germany) at a temperature of 12 "C. After electrophoresis the gels were fixed in 20% (v/v) isopropanol and 10% (v/v) acetic acid. Proteins were detected by staining with Coomassie Blue R-250 (39) or by silver staining as described by Morrissey (40). Mobilities were measured relative to theband of the tracking dye. For molecular weight estimation the RF values of the protein markerswere plotted using linear regression analysis. Preparation of [l-14C]PGH2-[1-14C]Arachidonicacid (58.4 mCi/ mmol) was diluted with cold arachidonic acid to give a specific activity of 6830 dpm/nmol. [1-14C]PGHz was prepared using a method similar to that of Hammarstrom (41). 20 ml of sheep vesicular gland microsomes in 90 mM potassium phosphate buffer,pH 8.0 (8 mg ofprotein/ ml), were preincubated for 20 min in ice with 6 pmol of fresh phenol, and then 15 pmol of sodium p-hydroxymercuribenzoateand 20 nmol of heme were added and thesolution was warmed to 37 "cfor 7 min.

Thromboxane Synthase After these preincubations 8.2 pmol of arachidonic acid, dissolved in 200 plof ethanol, was added and themixture was shaken at 37 "C for 45 s. The reaction was stopped quickly by adding 1.3 ml of 2 M citric acid and 30 ml of diethyl ether/pentane (5:2, v/v), in which the reaction products were extracted. After phase separation the lower phase was removed and again extracted with ether/pentane (5:2, v/ v). The organic phases were combined, dried over 7.5 g of anhydrous MgS04, and filtered to dryness on a rotary evaporator. The residue was dissolved in 3 ml of ethyl acetate. This stock solution could be stored for several months a t -80 "C. PGHz was separated from arachidonic acid and other arachidonic acid metabolites by chromatography on silicic acid (Mallinckrodt, 100 mesh) (41). PGHZ thus obtained was checked for impurities by TLC on silica gel plates using ether/petrol ether (W80 "C)/acetic acid (8015:01, v/v/v) as solvent system. The spot corresponding to PGHz could be completely converted to PGF,, by SnC4 asdescribed in Ref. 60. PGF2, formed was identified by gas chromatography-mass spectrometry. The TLC plates were analyzed for radioactivity on a TLC linear analyzer (Isomess, Essen, Germany).The purity of PGHz with respect to total14Clabel present was 90-95%. Assay for Thromboxane Synthase Actiuity-Aliquots of [l-'*C] PGH, in dry acetone were pipetted into glass test tubes, and the acetone was removed under nitrogen in a water bath a t 20 "C. Samples containing enzyme diluted with 50 mM potassium phosphate buffer, pH 7.4, to a volume of 200 pl were added and incubated for 30 s at 20 "C with agitation. The reaction was stopped by adding 100 pl of 200 mM citric acid, yielding a pH of 3.0. The reaction products were extracted twice with 1.5 ml of ethyl acetate/pentane (103, v/v) and the extracts were dried under nitrogen. The residue was dissolved in 50 pl of chloroform/methanol (2:1, v/v) and spotted on silica gel thin layer plates (Merck, Darmstadt, Germany). The TLC plates were developed as described (42), using chloroform/ethyl acetate/methanol/acetic acid/water (703081:0.5, v/v/v/v/v) as solvent system. The TLCplates were analyzed for radioactivity by autoradiography or with a TLC linear analyzer. PGE,, PGDz, PGFz,, and TxBz were used as reference standards and were visualized by spraying with phosphomolybdic acid and heating. The recovery of 1-l4C-labeled compounds was estimated by measuring the remaining radioactivity in the water phase. The amounts of TxB, formed were determined by measuring the percentage of radioactivity found in the TxBz fraction using a TLC linear analyzer and by the amounts of [1-14C] PGHz added to the incubation. In order to determine the specific activity of the pure enzyme, 0.3 pg of thromboxane synthase was incubated with 32.95 nmol of PGH2 in a total volume of 200 p1 of buffer and the incubation was stopped after 15 s. Under these conditions only small amounts of the substrate were converted into TxBZ. Gas Chromatography-Mass Spectrometry--164 nmol of [ 1-14C] PGHZ (2225 dpm/nmol) dissolved in 20 p1 of acetone were incubated 3 min a t room temperature with 4 pg of purified TxB2 synthase in 400 pl of 50 mM potassium phosphate buffer, pH 7.4. The reaction was stopped by addition of200 p1 of 200 mM citric acid, and the reaction products were extracted twice with 1 ml of diethyl ether. The free acids were converted to methyl esters with diazomethane and the products were then separated by TLC on silica gel plates. The silica gel in the regions corresponding to TxBz-Me and HHTMe was scraped from the plate and was eluted with methanol. The trimethylsilyl ether derivatives were prepared and analyzed by gas chromatography-mass spectrometry using a LKB 2091 gas chromatograph-mass spectrometer equipped with a J & W Durabond DB-5 fused silica capillary column (30 m X 0.32 mm). Synthetic TxB, was used as a standard. Materials-The PGHz analogs, U-54701, U-51605, U-46619, and U-44069, and TxB, were kindly provided by The Upjohn Co. UK37,248-01 was a gift from Pfizer, Central Research, England, OKY1581 was a gift from Ono Pharmaceutical Co. Ltd, Japan, and CGS13080 wasa gift from Ciba-Geigy Co., Ardsley, NY. DEAE-Sephacel, CM-Sepharose CL-GB, octylamine-Sepharose 4B, and low molecular weight gel standards were obtained from Pharmacia, Freiburg, Germany, and Ultrogel HA wasobtained from LKB, Miinchen, Germany. Arachidonic acid was purchased from Larodan, Malmo, Sweden, and [l-'4C]arachidonic acid (58.4 mCi/mmol) was from Amersham Buchler, Braunschweig, Germany. The following products were obtained from Sigma: PGEz, PGD,, PGF&., TxB,, soybean trypsin inhibitor, hemin,p-hydroxymercuribenzoate,leupeptin, pepstatin, phenylmethylsulfonyl fluoride, and Lubrol PX. Glycerol and cholic acid were obtained from Roth, Karlsruhe, Germany. Organic solvents and thin layer plates (Silica Gel60,0.20 mm) were obtained from Merck,

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Darmstadt, Germany. Rat liver microsomes were prepared as described in Ref. 49. NADPH was obtained from Boehringer Mannheim, Mannheim, Germany. Other reagents were purchased from common commercial sources. Platelet-rich plasma was kindly donated by the Deutsches Rotes Kreuz, Ulm and Bad Kreuznach, Germany. Sheep vesicular gland microsomes were prepared as described in Ref. 41 and 7-ethoxycoumarin was prepared as described in Ref. 37.

RESULTS Spectral and Enzymatic Properties of Human Platelet Microsomes-In agreement with earlier results reported by Cinti

andFeinstein(32), the 100,000 X g sediment of platelet homogenates gave rise to a difference spectrum with bands at 427 and 450 nm when carbon monoxide was present in both cuvettes and sodium dithionite in the sample cuvette (data not shown). If the 450 nm band is considered to be cytochrome P-450 and thecorresponding extinction coefficient of 91 mM" cm-' for 450-490 nm (35) is assumed, on an average a specific content of 35 f 5 pmol/mg of protein was obtained. Compared to microsomal preparations from organs with drug monooxygenase activities, several differences could be observed. The kinetics of formation of the 450 nm absorption band were rather slow and took about 30 min for completion, and no conversion to cytochrome P-420 was detectable even after 12 h. If dithionite was replaced by NADPH asa reductant and the solution was gassed for 1 min with CO, the band at 427 nm appeared but not the 450 nm band. This indicates that platelet P-450 is not reduced enzymatically by NADPH, although NADPH-dependent cytochrome c reductase activity was present. Also, with cyclohexane (47) and benzphetamine (48), the typical substrate-binding spectra of the unspecific drug monooxygenase cytochrome P-450 in liver were not observed. Finally, 7-ethoxycoumarin, a widely used substrate for the unspecific monooxygenase system, was not O-dealkylated. Together these results support the assumption that platelet cytochrome P-450 is not partof a drug monooxygenase system. Our hypothesis that this cytochrome is associated with the formation of TxA, from PGH, was supported mainly by the spectral interaction of typical TxA2 synthase inhibitors like UK-37,248,OKY-1581, or 1-benzylimidazole with platelet microsomes (33). Recently, a new potentinhibitor, CGS13080, has been described which was, therefore, tested forits spectral effects on platelet microsomes (Fig. 1). This compound gave qualitatively and quantitatively results similar to UK-37,248 and OKY-1581, exhibiting a minimum at 412 nm anda peak at 423 nm.That thischromophore is identical to cytochrome P-450 was deduced from the fact that when CGS-13080 and dithionite were present in both cuvettes and CO in thesample cuvette, no 450 nm absorption band could be detected. Control experiments with sheep vesicular gland microsomes clearly showed that no spectral interaction of the thromboxane synthase inhibitors was obtained with cyclooxygenase, another hemoprotein present in human plateletmicrosomes. The conversion of PGH, to TxB, by platelet microsomes was inhibited byCGS-13080 in a concentration-dependent manner (results not shown). It is therefore tempting to assume that thespectral andinhibitory interactions are intimately linked. When human platelet microsomes were incubated with [1-14C]PGHz,a maximum of 33% of the added substrate was converted into TxB, and 42% was converted into HHT, giving a TxB2:HHT ratioof 1:1.27. The specific thromboxane synthaseactivity of the microsomal fraction was calculated as 57 nmol of TxBz min-l mg protein-'. Preincubations of the microsomes with UK-37,248 diminished the yield of TxB2 in a concentration-dependent

Synthase

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I

360

I

Thromboxane

I

400 440 Wavelength nm

I

I

480

FIG. 1. Titration of human platelet microsomes with CGS13080. The difference spectra were performed as described in the text. The titrationcurves correspond approximately to the following ligand concentrations: a, 69 nM; b, 98 nM; c, 127 nM; d, 155 nM; e, 184 nM; f, 213nM; g, 242nM; h, 270nM; i, 299nM. The microsomes contained 330 pmol of cytochrome P-450/ml corresponding to 10.5 mg of protein/ml.

manner and simultaneously slightly increased the amounts of PGEz andPGD,. Solubilization and Purification of Human Platelet Thromboxane Synthase-Solubilization of thromboxane synthase from human platelet microsomes was achieved with several nonionic detergents (TritonX-100, Triton N-101, Lubrol PX, Lubrol WX, or Tween 20) a t concentrations of about 0.5% (v/v) in the presence of 20-30% (v/v) glycerol, giving yields of solubilized enzyme of between 80-95%. No solubilization of thromboxane synthase occurred with cholate alone at a concentration of 0.6% (w/v). When the enzyme was solubilized with mixtures of cholate and nonionic detergents, the amount of nonionic detergent could be decreased by 60%. For the purification of thromboxane synthase a combination of 0.2% (v/v) Lubrol PX and 0.5% (w/v) cholate in thepresence of 20% (v/v) glycerol was used, because the enzyme seemed to be stabilized by Lubrol PX. Theyield of solubilized enzyme under these conditions was 90-95% and anenrichment of the specific cytochrome P-450 content of up to 66 pmol/mg of protein was obtained. The solubilized enzyme was bound to DEAE-Sephacel, but large amounts of gel (60 ml of gel for 24 nmol of enzyme) were necessary. The concentration of the ionic detergent was found to be critical for the binding of the enzyme to thegel. Enzyme solubilized in thepresence of only nonionic detergent (0.5% (v/v) Lubrol PX) did not bind to the gel. The batch technique led to a greater enrichment of thromboxane synthasewhen compared to column chromatography. The specific cytochrome P-450 content of the eluate

was 910pmol/mg of protein, and theeluate was free of other cytochromes, as suggested by the absolute spectra. The yields of enzyme in this stepwere about 60%. The concentration of the nonionic detergent also proved to be critical for binding the enzyme to octylamine-Sepharose. High concentrations of Lubrol P X prevented binding to this gel. When the enzyme did not bind to the column, treatment with Bio-Beads SM-2 was used to remove an excess of detergent. After binding of thromboxane synthase to the column, washing with 15% (v/ v) dimethyl formamide elutedproteins with weak hydrophobic interactions. The recovery of thromboxane synthase by chromatography on Octylamine-Sepharose was 54% and the specific cytochrome P-450 content increased to 1.89 nmol/mg of protein. A further enrichment of thromboxane synthase was obtained by a second DEAE-Sephacel column step. In thiscase, the enzyme waseluted with cholate-free buffer while the NaCl concentration remained constant. In addition, the enzyme eluate contained only small amounts of cholate and the pH of the eluate could be adjusted to pH 6.5 directly without dialysis. Chromatography on CM-Sephacel CL-GB resulted in a specific content of 5.37 nmol of cytochrome P-450/mg of protein and as the enzyme did not bind to the column, the yields of enzyme were about 95% in this purification step. In the final step, the enzymewas bound to an Ultrogel HA column. Since the capacity of this gel for binding thromboxane synthasewas low, it turnedout to be advantageous to run a small column twice. Thromboxane synthase could not be eluted from this column with 100 mM potassium phosphate buffer, pH 7.0, containing 0.2% (v/v) Lubrol PX. Theaddition of 0.4% cholate to the buffer was required for elution of the enzyme. The yield of enzyme was 40% in the main fraction, and a total of 50% could be eluted from the column. The specific cytochrome P-450 content of the main fraction after ultrafiltration was 14.8 nmol/mg of protein. Purification of thromboxane synthasewas accompanied by a 423-fold enrichment of the specific cytochrome P-450 content (Table I), and the purified enzyme showed a single band on SDS-gel electrophoresis (Fig. 2). Several preparations were performed in the same way and the purification procedure was found to be reproducible within a narrow range. The total yield of a single preparation as described was routinely about 2.1 nmol of hemoprotein. TABLE I Purification of thromboxane synthase from human platelets Thromboxane synthase was purified as described under "Experimental Procedures." Cytochrome P-450, protein, and thromboxane synthase activity were assayed as described in the text. Specific Purification step

Volume

Total protein content

ml

mg

nmol'mg protein

754 72 0.035 1. Microsomes 0.066 75 368 2. Solubilized microsomes 0.91 3. I. DEAE-Sephacel 123 16.3 1.89 119 4.3 4. OctylamineSepharose 5. 11. DEAE-Sephacel 39 2.92 1.9 43 6. CM-Sepharose 5.37 0.98 7.7 0.14314.8 7. Ultrogel HA

Total

cytochrome p-450

Purification"

nmol

-fold

26.4 24.3

%

1 100 1.9 92

14.8 8.1

26 54

5.6 5.3 2.12

83 153 423

56 30.7

21.2 20 8 Specific activity: microsomes: 57 nmol of TxBz mg of protein" min" (20 "C);purified enzyme: 24.1 pmolof TxB2 mgprotein" min" (20 "C).

Thromboxai%eSynthase

15063

Properties of Human PlateletThromboxane Synthase-The into HHT, giving a TxB2:HHT ratioof 1. The identity of TxB, and HHT formed by the enzyme molecularweight of thromboxane synthase was estimated from 20-cm SDS-polyacrylamide gels as M , = 58,800 using purified here was verified by gas chromatography-mass specthe following RFvalues of the protein markers:94 kDa (0.13), trometry. Following fractionation of the products by TLC the 67 kDa (0.197), 43 kDa (0.51), 20.1 kDa (0.686), and 14.11 gel in theregions corresponding to the methyl esters of TxB2 kDa (0.823) and anRFvalue of 0.26 for thromboxane synthase. and HHT was scraped from the TLC plate, the compounds The absolute optical spectrum of the pure hemoprotein is were eluted with methanol andconverted to their trimethylshown in Fig. 3. The Soret band of the oxidized form was silyl ether derivatives. The mass spectrum of the TxB2derivlocated a t 424 nm. In the UV region the d band appeared a t ative showed ions at mle: 600, 585, 529, 510, 495, 440, 439, 357 nm and the aromatic amino acids absorbed a t 280 nm 420, 366, 323, 301, 295, 256 (base peak), 225, 217, 211, 199, with a ratio of A2RO:A424 = 1.2. 191,173,155, and129. The same spectrum was obtained using As seen from the spectrum, the absorption band a t 280 nm synthetic TxB, as a standard. For the HHT derivative the is not only due to the absorption of aromatic aminoacids but mass spectrum showed ions at mle: 366, 351, 335, 295, 276, also to light scattering as a result of micelles in solution. and 225. The mass spectra obtained corresponded closely to When the spectrum of the purified enzyme was recorded at those published in Ref. 1. higher sensitivity before concentration by ultrafiltration, the Spectral Propertiesof Thromboxane Synthase-During the ratio between the absorbance a t 280 and 424 nm was 0.7. purification of thromboxane synthase we noticed that the Since we noticed that the Soret band at424 nm was shifted spectral properties of the enzyme changed slightly. Likewise, of about 6 nm to the red compared to the spectrum seen after we could not observespectrophotometricallyabinding a final dialysis was employed thromboxane synthase inhibitors such as UK-37,248, CGSthe first DEAE chromatography, to remove residual detergents. This caused a reversal of the 13080,OKY-1581, or 1-benzylimidazole withthe pureenzyme, Soret absorption back to 418 nm with correspondingCY and /3 although the enzyme obviously had reacted as seen by the bands at 570 and 537 nm, respectively (Fig. 4). After gassing inhibition of TxBz synthesis. This could be explained by the fact that theimidazol and pyridine ligand complexes formed with CO and addition of sodium dithionite, the Soret band of both enzyme forms shifted to450 nm, proving that the he-by the inhibitors absorb at the same wavelength as the pure moprotein thromboxane synthase is a cytochrome P-450 pro- enzyme. Since in platelet microsomes as well as in enriched tein.Thepresence of the specific thromboxanesynthase enzyme preparations the binding spectra could be observed, a inhibitor CGS-13080 inhibited the formationof the CO com- modification of the enzymeseemed to have taken place. plex a t 450 nm. Indeed, afterfractionation of solubilized microsomes on The specific activity of thromboxane synthase was deter- DEAE-Sephacel, the cytochrome showed its Soret band at mined by incubating the enzyme with [1-14C]PGH2 and sep- 418 nm and thereforewas able to produce a difference specarating the reaction products by TLC. In order to keep the trum with the inhibitors shifting the Soret band to 424 nm. reaction in a linear dependence on time at the chosen sub- Titrations with these compounds of thromboxane synthase strate concentration, the incubation was stopped after 15 s partially purified by chromatography onDEAE-Sephacel and when only about 6% of the substrate was converted to TxB,. concentrated by ultrafiltration resulted in difference spectra The pure enzyme exhibited a high specific activity of about qualitatively similar to those obtained with platelet micro24.1 pmol of TxB2/min.mg of protein. On a heme basis a somes (33).The analysis of the spectral changes ina binding diagram, plotting the free ligand against the bound ligand molecular activity of 1628 was calculated. When the enzyme was incubated for 1min below saturating (data not shown), revealed a titration curve indicating that amounts of substrate (15 p~ PGH,), a maximum of 41% of the compounds used reacted stoichiometrically with the cythe substratewas converted into TxB2 and 39% was converted tochrome P-450 protein. During theenzyme preparation the

A

B MW

MW FIG. 2. SDS-polyacrylamide gel electrophoresis of thromboxane synthase from different column fractions. Thromboxane synthase was purified as described under “Experimental Procedures.” Solubilized human platelet microsomes (200 pg of protein, lane a);first DEAE eluate (15pg of protein, lane b);60 mM potassium phosphate eluate of the Ultrogel HA column (each 1 pg of protein, lane c ) ; purified thromboxane synthase from different enzyme preparations (3.5 and 2.3 pg of protein, respectively, lanes d and e ) ; purified thromboxane synthase (1.48, 1.04, 0.46, and 0.16 pg of protein, respectively, lanes f-i). The gel in A was stained with Coomassie Blue R-250 and the gel in B was silver-stained.

94.000 67.000

-

-

- 67.000

43.000

-

- 43 .OOO

20.100 14.400 -

30.000

- 30 .OOO - 20 .loo

- 14 .OOO a

b

c

c

d

e

f

g

h

i

15064

Thromboxane Synthase

.040

.16

424 1

*12 FIG. 3. UV-VIS absorption spectrum of thromboxane synthase. Thromboxane synthase was purified as decribed under “Experimental Procedures.” The spectrum was recorded before the enzyme was dialysed and converted to the 418 nm form. The concentration of cytochrome P-450 was 385 pmol/ml corresponding to 26 pg of protein/ml.

-.025

J

1.020 1

.06t

\

A

1.015

, -02L

I

260

300

I

d

340

l

I

I

I

380

420

46C

Wavelength nm

.045

FIG. 4. Absorption spectrum of thromboxane synthase from 670 to 370 nm. Thromboxane synthase was purified as described under “Experimental Procedures.” The concentration of cytochrome P-450 was 330 pmol/ml corresponding to 22 p g of protein/ml. -, oxidizedenzyme after dialysis; -----,after reduction with sodium dithionite in the presence of CO.

-

418

i

-025b / 4 2 41/- y .020

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1.0 12 1.010

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537

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I

+dithionite+CO

.005 I

I

550400500

450

I

I

600

I

650

Wavelength nm conversion to the 424 nm form obviously occurred after hydrophobic chromatography. The purified enzyme could be converted back by dialysis to the original enzyme with a maximum of the Soret band at 418 nm. In this form, the enzyme showed again binding spectra with the inhibitors mentioned above. In addition, spectral interactions with PGH, analogs such as U-54701, U-51605, U-46619, and U-44069 were obtained (Fig. 5). With U-44069 the enzyme showed a binding spectrum with a minimum at 424 nm and a peak a t 407 nm, which is closely similar to thedifference spectrum observed with oxygen ethers like tetrahydrofuran in liver microsomes (54). In the case of liver cytochrome P-450 this has been interpreted as a conversion of the native 418 nm form of the oxidized enzyme to a ligand complex with the oxygen bound at the sixth ligand position. Another form of the enzyme became apparent when the

interaction with U-46619 was studied. The pure enzyme reacted with this PGH, analog by formation of a band at 387 nm and a trough at 417 nm. This resembles closely the substrate binding spectra observed with monooxygenase cytochrome P-450 (56). The underlying mechanism is thought to be the conversion of a six-coordinated low spin form absorbing a t 418 nm to a high spin five-coordinated hemoprotein. The PGHz analogs U-54701 and U-51605 gave rise to difference spectra which werequalitatively similar to each other. With both analogs spectra were measured with a peak at 427 nm and a trough at 410 nm. These data again demonstrated that the ligand at C-9 of the substrate analog showed interactions with the iron of the heme, whereas the ligand at C-11 of the substrate was not involved. After chromatography on DEAE-Sephacel, the optical absolute spectra of the enriched enzyme were identical to the

Thromboxane Synthase

15065

A

2,4 2746,7

2,249 2946,7 13

1,900 3488,3

2,039 3250 I 417

C

1Ez.002

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1

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427

1,919 3453,3

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7

G’

3600 3200 2800 2400 2,252 2943,3

B 410 I

,

,

,

,

,

,

.

2,478 2675

1

I60 400440480 360 400440480 Wavelength nm FIG.5. Difference spectra of thromboxane synthase with different PGHz analogs. Thromboxane synthase was purified as described under “Experimental Procedures.” Difference spectra were recorded following treatment of the purified enzyme with U-46619 ( A ) ,U-44069 ( B ) ,U-54701 (C), and U-51605 (D). The PGHZ analogs were dissolved in methanoland thecorresponding amounts of solvent were added tothe reference cuvette. The concentrations of the substrate analogs added to the enzyme were approximately 300 p~ and the concentration of cytochrome P-450 was 290 pmol/ml corresponding to 20 pg of protein/ml. 1

1 spectra of the pure enzyme. In addition, the Soret absorption 3600 3200 2800 2400 G band could be transformed almost to 90% to a 450 nm abFIG. 6. EPR spectra of thromboxane syhthase. Enzyme consorption band in the presence of CO and sodium dithionite. taining fractions, after chromatography on DEAE-Sephacel, free of Furthermore, the formation of this chromophor was inhibited other heme proteins, were concentrated by ultrafiltration. The EPR in the presence of specific thromboxane synthase inhibitors spectra were performed as described under “Experimental Procelike CGS-13080 or UK-37,248. This clearly shows that no dures.” A , spectrum of the enriched enzyme, showing the Soret band hemoproteins other than.$hromboxane synthase were present. at.424 nm. The sample contained approximately 6 nmol of cytochrome Therefore, these preparations were suitable for EPR spec- P-450, corresponding to 6.6 mg of protein. B, spectrum of the same troscopy and for identification of the prosthetic group of sample after additionof 15 nmol of UK-37,248. thromboxane synthase. Fig. 6 presents the EPR spectrum of meric molecular weight of 58,800. the untreated oxidized enzyme exhibiting the Soret band at From its optical spectra, it is apparent that thromboxane 424 nm. Clearly, two differently coordinated forms of the synthase is a hemoprotein. A value of 0.9 mol of heme/mol of enzyme were present: one with g values at 2.461, 2.249, and polypeptide chain was determined. Because of an observable 1.900; the second at 2.413, 2.249, and 1.919. Upon addition of loss of free hemin during enzyme purification and a rather the thromboxane synthase inhibitorUK-37,248, the narrower large method-dependent variation in protein determinations, signal was converted to a species with g values at 2.478, 2.252, this resultsuggests the presence of 1heme moiety/polypeptide and 1.894. This signal is almost identical with the more chain. When the hemoprotein is slowly reduced by sodium rhombic species of the untreated enzyme, so that this form dithionite in the presence of carbon monoxide, a band at 450 may correspond to thered-shifted form in Fig. 3. nm & observed, suggesting a cytochrome P-450 nature. EPR T o identify the prosthetic group of the hemoprotein, the spectra of the oxidized enzyme were almost identical with the heme of the enzyme was converted to itspyridine hemochro- corresponding spectra of cytochrome P-450 from Pseudomogen. The visible spectrum of the pyridine hemochromogen, m o m putidu and very similar to those of the liver microsomal shown in Fig. 7, is identical with that of protoporphyrin IX, monooxygenases. However,in contrast to these monooxygenexhibiting peaks a t 418 and 557 nm and a minimum at 542 ases, thromboxane synthase could not be reduced enzymaticnm (59). ally by NADPH or NADH in its membranous state, even though functionally active cytochrome P-450 reductase was DISCUSSION present. The very slow reduction of thromboxane synthase Thromboxane synthase from human platelets was purified observed in the presence of dithionite also serves to differento homogeneity by conventional biochemical methods using tiate thisenzyme from cytochrome P-450-dependent monooxionic and nonionic detergents. The purified protein migrated ygenases. and indicates that the heme iron in thromboxane as a single band on SDS-polyacrylamide gels with a mono- synthase operates in its oxidized ferric state. Neither molec-

15066

Thromboxane Synthase

9-position is a methylene group lacking the ability to coordinate with the iron. The fact that both the azo derivative and -.40 the ll,9-epoxyimino derivative gave rise to binding spectra which are due to a nitrogen at the sixthposition of the iron ,-.35 is additional proof that the ligand at C-9 of the substrate interacts with the heme iron. For the physiological substrate PGH2,therefore, we propose the formation of an enzyme-substrate complex in which the peroxide oxygen atom of the 9-position coordinates with the a ferric iron. In analogy to theoxygen activation by microsomal monooxygenases which leads to anoxygen atom stabilized by the ferric thiolate entity, one can assume a corresponding stabilization of an oxonium structure.Thus,a heterolytic .05 542 cleavage of the peroxide bondwould occur which may be .2! .1 followedby arearrangement to TxAz in a yet undefined 400 440 480 520 560 mechanism. Such a mechanism would be different from other Wavelength nm proposed mechanisms in which the oxygen at C-11 is considFIG. 7. Pyridine hemochromogenspectrum of the prosthetic ered to carry a positive charge (43,44). group of thromboxane synthase. Enzyme containing fractions, Our results gave clear evidence that even the purified enafter chromatography on DEAE-Sephacel, free of other heme pro- zyme not only formed TxBz from PGHz but also HHT. HHT teins, were concentrated by ultrafiltration and converted to the pyr- was generated in the same amounts as TxBz so that it is idine hemochromogen complex by addition of 0.1 N NaOH and 20% pyridine. The cytochrome P-450 content of the concentrated sample certainly not a product of a TxAz degradation process, but rather arises from an intermediate or transition state, which was approximately 1.47 nmol/ml corresponding to 1.27 mg ofprotein/ can rearrange to either TxAz or HHT. ml. Since it is shown that arachidonic acid interacts with the hydroxylating cytochrome P-450 species from liver (50, 57, ular oxygen nor electrons are required for the conversion of 58), we investigated whether aTxBz formation couldbe PGHz to TxA2. In the oxidized state, the Soret band of the solubilized as well as thepurified enzyme is located at around observed in liver microsomes and whether a spectral interaction with substrate analogs and enzyme inhibitors might 418 nm as found with various monooxygenase cytochrome P450’s. It is now generally assumed that the coordination of occur. The difference spectra obtained with U-51605 and U46619 clearly showed that these substances interact with the the ferric heme iron in this stateinvolves binding to a thiolate heme iron of cytochrome P-450 from rat liver, but the binding ligand in the fifth position and probably to a hydroxyl group spectra were qualitatively and, with regard to their affinities, in the sixth position (51). During the purification procedure, also quantitatively different from those with thromboxane a red shift of the 418 nmband to 424 nm was noticed, synthase. This was particularly apparent for U-46619, for accompanied by an increase of anisotropy in the EPR specwhich a spectrum with a peak at 422 nm and atrough at 390 trum. Since extensive dialysis converted this enzymatically nm was measured, indicating that anoxygen atom of U-46619 still active form back to the 418 nm absorbing species, we interacts with a high spin species of this cytochrome P-450. believe that higher detergent concentrationscause a reversible No spectral interaction was observed with the thromboxane conformational change which brings a nitrogen-containing synthase inhibitor UK-37,248 at the low concentrations that ligand into the sixthcoordination position. inhibit thromboxane synthase. Concentrations 5-6 orders of Complexes with nitaogen at the sixthligand position of the magnitude higher were needed to induce a ligand-type spectral heme are also obtained by the addition of pyridine- or imidainteraction with this compound. Moreover, no TxBz formazole-based inhibitors of thromboxane synthase. Such comtion was observed in incubations of PGHz with liver micropounds block TxBzformation by platelets and have IC,, somes. We have shown, therefore, that thromboxane synthase values around 2-20 nmol/liter (42, 45, 46). When added to is not reducible enzymatically and in accordance lacks 7the membrane-bound enzyme as well as to enriched enzyme ethoxycoumarin monooxygenase activity. preparations, astoichiometric binding of the inhibitors to the On the other hand, liver microsomal monooxygenases do hemoprotein was observed as calculated from the difference not catalyze TxBz formation. Nevertheless, they belong specspectra. Under these circumstances, the EPR spectrum inditrally to thesame group of cytochrome P-450 proteins. Since cates a shift to higher anisotropy, a result similar to those we previously have presented evidence that prostacyclin synpreviously reported for the binding of nitrogen-containing thase is also a cytochrome P-450 protein, it follows that a ligands like metyrapone to microsomal cytochrome P-450 (52) new class of P-450-enzymes exists in which the heme iron or cytochrome p - 4 5 0 (53). ~~~ does not undergo a redox cycle between the ferrous and ferric A different spectral change was observed upon addition of form. For these hemoproteins, the term“cytochrome”is theresubstrate analogs to thromboxane synthase. The 9,ll-epoxyfore not appropriate. methano derivative of PGHz shiftsthe 418 nm Soret bandto a shorter wavelength as indicated by its difference spectrum. thank Prof. Kubanek, Ulm, for a continuThis resembles the binding of tetrahydrofuran to microsomal ousAcknowledgments-We supply of platelet rich plasma and Dr. John Pike, (The Upjohn cytochrome P-450 (55) and is in agreement with results on Co.) for the samples of PGHz analogs. Gas chromatography-mass the corresponding oxygen ether model complexes (54). The spectrometry analysis was done in cooperation with Dr. S. Hammarsuggestion that the 9-oxygen position can coordinate to the strom and U. Diczfalusy, who also contributed with discussion and ferric heme iron is supported by the binding of the 11,9- helpful advice. EPR spectra were kindly recorded by Dr. P. Kroneck. epoxymethano derivative of PGHz. In thiscase, a shift to 387 REFERENCES nm results, which, in P450 coordination chemistry, is thought to reflect the formation of a five-coordinated complex (56). 1. Hamberg, M., and Samuelsson, B. (1974) Proc. Natl. Acad. Sci. U. S. A. 71,3400-3404 This suggests that for the 11,9-epoxymethano compound, the

““c\

Synthase

Thromboxane

2. Hamberg, M., Svensson, J., and Samuelsson, B. (1975) Proc. Natl. Acad. Sci. U. S. A. 72,2994-2998 3. Sveasson,J., Hamberg, M., and Samuelsson, B. (1976) Acta Physiol. S c a d . 98,285-294 4. Hamberg, M. (1976) Biochim. Biophys. Acta 431,651-654 5. Davidson, E. M.,Doig,M.V., Ford-Hutchinson, A. W., and Smith, M. J. (1980) Adu. Prostaglandin Thromboxane Res. 8, 1661-1663 6. Goldstein, I. M., Malmsten, C. L., Kindahl, H., Kaplan, H. B., Radmark, O., Samuelsson, B., and Weissman, G. (1978)J. Exp. Med. 148,787-792 7. Morley, J., Bray, M. A., Jones, R. W., Nugteren, D. H., and van Dorp, D. A. (1979) Prostaglandins 17, 729-746 8. Murota, S., Kawamura, M., and Morita, I. (1978) Biochim. Biophys. Acta 528, 507-511 37. 9. Weidmann, M. J., Peskar, B. A., Wrogemann, K., Rietschel, E. T., Staudinger, Hj., and Fischer, H. (1978) FEBS Lett. 89, 39.136-140 10. McGuire, J. C., and Sun, F. F. (1980) Adu. Prostaglandin Thromboxane Res. 8,1665-1667 11. Wolfe, L. S., Rostworoski, W., and Marion, J. (1976) Biochem. Biophys. Res. Commun. 70,907-913 12. Zenser, T. V., Herman, C.A., Gorman, R. R., and Davis, B. B. (1977) Biochem. Biophys. Res. Commun. 79,357-363 13. Svensson, J., and Hamberg, M. (1976) Prostaglandins 12, 943950 14. Svensson, J., Strandberg, K., Tuvemo, T., and Hamberg, M. (1977) Prostaglandins 14,425-436 15. Moncada, S., and Vane, J. R. (1979) Pharmacol. Reu. 30, 293331 16. Gryglewski, R. J., Dembinska-Kiec, A., and Korbut, R. (1978) Acta Biol. Med. Ger. 37, 715-723 17. Nicolaou, K. C., Magolda, R. I., Smith, J. B., Aharony, D., Smith, X., and Lefer, A. M. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 2566-2570 18. Le Breton, G. C., Venton, D. L., Enke, S. E., and Halushka, P. V. (1979) Proc. Natl. Acad. Sci. U. S. A. 76,4097-4101 19. Stegmeier, K., Pill, J., Muller-Beckmann, B., Schmidt, F. H., White, E. C., Wolff, H. P., and Patscheke, H. (1984) Thromb. Res. 35,379-395 20. Parise, K. V., Venton, D. L., and LeBreton, G.C.(1978) J. Phnrmacol. Exp. Ther. 228, 240-244 21. Siess, W., Siegel, F. L., and Lapetina, E. G. (1983) J. Biol. Chem. 258,11236-11242 22. Hammarstrom, S., and Falardeau, P. (1977) Proc. Natl. Acud. Sci. U. S. A. 74,3691-3695 23. Yoshimoto, T., Yamamoto, S., Okuma, M., and Hayaishi, 0. (1977) J. Biol. Chem. 2 5 2 , 5871-5874 24. Hall, E. R., and Tai, H.-H. (1981) Biochim. Biophys. Acta 665, 498-503 25. Moncada, S., andvane, J. R. (1979) N. Engl. J. Med. 300,11421147 26. Ullrich, V. (1980) J. Mol. Catal. 7,159-167 27. Rahimtula, A.D., and O’Brien, P. J. (1974) Biochem. Biophys. Res. Commun. 60,440-447 28. Lichtenberger, F., Nastainczyk, W., and Ullrich, V. (1976) Biochem. Biophys. Res. Commun. 70,939-946 29. Ullrich, V., Castle, L., and Weber, P. (1981) Biochem. Pharmacol. 30,2033-2036

15067

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