286
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
5000
[31]
8-coo.
4000.
, s-coo.
2000
2o-coo.
1000.
0
l0
20
90 40 50 TIME (MINUTES)
50
70
FIo. 5. LTB4 metabolism by isolated rat hepatocytes. The reversed-phase HPLC radiochromatogram of [3H]LTB4 metabolites formed by hepatocytes (5 ml cells, 5 x 106 cells/ml) incubated with LTB4 (3/zM) for 45 min. The identity of 20-COOH-LTB4 is determined by coelution with a synthetic standard. 18-COOH-LTB4 and 16-COOH-LTB4 are identified by electron impact mass spectrometry. Reversed-phase HPLC conditions are as outlined in the text.
solvent front of the gradient reversed-phase HPLC system. Further studies on the metabolism of LTB4 by isolated rat hepatocytes are in progress. The relevance of the isolated rat hepatocyte model of LTE4 metabolism to the fate of leukotrienes in vivo has been supported by the identification of 20-COOH-LTE4 (Metabolite B) and 16-COOH-dihydro-LTE4 (Metabolite D) in rats injected with LTE4. ~o This hepatocyte system serves as a biosynthetic source of these metabolites. to p. Perrin, J. Zirrolli, D. O. Stene, J. P. Lellouche, J. P. Beaucourt, and R. C. Murphy, Prostaglandins 37, 53 (1989).
[3 1] P u r i f i c a t i o n a n d C h a r a c t e r i z a t i o n of H u m a n L u n g L e u k o t r i e n e A4 H y d r o l a s e
By N O B U Y A
O H I S H I , TAKASHI I Z U M I , YOUSUKE SEYAMA, TAKAO S H I M I Z U
and
Leukotriene (LT) A4 hydrolase (EC 3.3.2.6) catalyzes enzymatic hydration of LTA4 [5(S)-trans-5,6-oxido-7,9-trans-ll,14-cis-eicosatetraenoic acid] to LTB4 [5(S), 12 (R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid], a potent chemotactic substance. This reaction on the epoxide differs from that of epoxide hydrolases (EC 3.3.2.3). The latter METHODSIN ENZYMOLOGY,VOL. 187
Copyright© 1990by AcademicPress, Inc. All rightsof reproductionin any formreserved.
[31]
HUMAN LUNG LEUKOTRIENE A4 HYDROLASE
287
results in the synthesis of vicinal alcohols (glycol), while the former synthesizes a compound possessing two hydroxy groups at C-5 and C-12 with a conjugated triene in between. LTA4 hydrolase activity was previously reported mainly in blood cells, especially polymorphonuclear leukocytes and erythrocytes, and the enzymes from these sources were purified. 1-3 The enzyme activity is, however, ubiquitously distributed in the cytosolic fraction of various organs of the guinea pig, and higher activities are observed in small intestine, lung, and aorta. 4 To compare the lung enzyme with those found in blood cells, and for further characterization, the enzyme was purified from the human lung. 5 Recently, this enzyme has also been purified from the guinea pig liver6 and lung. 7
Preparation and Purification of LTA4 Methyl Ester LTA4 methyl esters are synthesized according to Ohkawa and Terao s and Gleason e t al. 9 These are obtained as mixtures of several geometric isomers, and can be separated by straight-phase HPLC. The HPLC conditions are as follows: columnmChemcoPak Nucleosil 50-5, 1 x 30 cm (Chemco, Osaka); solvent--n-hexane/ethyl acetate/triethylamine (100/1.2/0.4, v/v/v); flow rate--3 ml/min; column temperature--4°; UV monitor--280 nm. The column temperature is critical for good resolution and recovery. Under these conditions, four major peaks representing various isomers of LTA4 methyl ester appear at retention times of between 40 and 60 rain, the second-appearing peak being LTA4 methyl ester (Fig. 1). Gas chromatography-mass spectrometry and proton magnetic resonance studies are necessary for structural identification.
1 O. R~dmark, T. Shimizu, H. J6rnvall, and B. Samuelsson, J. BioL Chem. 259, 12339 (1984). 2 j. F. Evans, P. Dupuis, and A. W. Ford-Hutchinson, Biochim. Biophys. Acta 840, 43 (1985). 3 j. McGee and F. Fitzpatrick, J. Biol. Chem. 260, 12832 (1985). 4 T. Izumi, T. Shimizu, Y. Seyama, N. Ohishi, and F. Takaku, Biochem. Biophys. Res. Commun. 135, 139 (1986). 5 N. Ohishi, T. Izumi, M. Minami, S. Kitamura, Y. Seyama, S. Ohkawa, S. Terao, H. Yotsumoto, F. Takaku, and T. Shimizu, J. Biol. Chem. 262, 10200 (1987). 6 j. Haeggstr6m, T. Bergman, H. J6rnvaU, and O. R/idmark, Eur. J. Biochem. 174, 717 (1988). 7 H. Bito, N. Ohishi, I. Miki, M. Minami, T. Tanabe, T. Shimizu, and Y. Seyama, J. Biochem. 105, 261 (1989). 8 S. Ohkawa and S. Terao, J. Takeda Res. Lab. 42, 13 (1983). 9 j. G. Gleason, D. B. Bryan, and C. M. Kinzig, Tetrahedron Lett. 21, 1129 (1980).
288
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
[3 1]
~..._~.,~,.~ CO0Cfl3 E 0 CO O4
0eC~
k~~CO°CH~
--•6
.
,~
2'0 4'0 6'0 do Retention time {min)
FIG. 1. Straight-phaseHPLCseparationof LTA4methylesterand its geometricisomers. Four majorpeaks eluteat retentiontimes around40-60 min and the structureof each peakis illustrated.
Saponification of LTA4 Methyl Ester LTA4 is unstable in aqueous buffer solutions at acidic and neutral pH, and is stabilized in alkaline organic solutions at a lower temperature. LTA4 is synthesized as the methyl ester form and is stored in alkaline organic solution such as benzene/triethylamine (100/2, v/v) in a nitrogen atmosphere at - 7 0 °. When in use, LTA4 methyl ester is saponified in tetrahydrofuran (400/~l) plus 0.1 M lithium hydroxide in water (100/.el) under conditions of vigorous mixing overnight at room temperature. The aliquot is then dried under a stream of nitrogen (complete dryness should be avoided), and dissolved with an appropriate amount of ethanol. The con-
[31]
HUMANLUNGLEUKOTRIENEA4 HYDROLASE
289
centration of LTA4 is determined from UV absorption, using a molar extinction coefficient of 40,000 M - lcm- ~ at 280 nm. Assay of LTA4 Hydrolase The standard incubation mixture (50 ~l in 1.5 ml sampling tube) contains the enzyme in 0.1 M Tris-HCl buffer (pH 7.8). After preincubation for several minutes at 37 °, 1/~g of LTA4 in 1/A of ethanol is added. After 1 rain of incubation, 117/~l of stopping solution (0.1% acetic acid in methanol containing 0.3 nmol of prostaglandin B2 as an internal standard) is added. After the mixture is kept at - 2 0 ° for at least 20 rain followed by centrifugation at l04 g for 10 min, a 50-/.d aliquot of the supcrnatant is directly injected onto HPLC. The conditions are as follows: c o l u m n n T S K - O D S 80TM, 0.46 × 15 cm (Tosoh, Tokyo); solvent--methanol/water/acetic acid (70/30/0.05, v/v/v); flow rate--1 ml/min; column temperaturen35°; UV monitorm270 nm. PGBz and LTB4 elute at about 7 and 11 min, respectively, and the amount of LTB4 formed is calculated from the peak area ratio LTB4/PGB2. Purification Procedure All procedures other than column chromatography are done at 4 ° . Column chromatography is performed using a fast protein liquid chromatography (FPLC) system (Pharmacia, Uppsala), at room temperature. All centrifugations are carried out at 10,000 g at 4° for 20 rain, unless otherwise indicated. Step 1. Human lung (about a 50 g autopsy specimen with no inflammatory and other disease processes) is minced with scissors in 3 volumes of ice-cold 20 mM potassium phosphate buffer containing 5 mM EDTA and 0.15 M NaC1 (pH 7.4). After homogenization using a Polytron-type homogenizer (output dial 3, 1 min, 3 times), the sample is centrifuged. Step 2. The 10,000 g supernatant is subjected to ammonium sulfate fractionation. The precipitates between 40 and 70% saturation are collected, dissolved in 50 ml of 20 mM Tris-HCl buffer (pH 8), and then dialyzed against two changes of 40 volumes of the same buffer. Step 3. After centrifugation and filtration through 0.45 ~m filters, the dialyzed sample (one-third or one-fourth of total volume per one cycle of chromatography) is applied to a Mono Q HR 10/10 column (1 × l0 cm, Pharmacia) preequilibrated with 20 mM Tris-HCl buffer (pH 8) at a flow rate of 2 ml/min. The column is washed with the same buffer (about 20 ml), and the adsorbed proteins are eluted with a 75 ml linear gradient between 0-0.15 M KCI, in the same buffer. The enzyme activity elutes at a KC! concentration of around 0.1 M.
290
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
[31]
Step 4. Active fractions from the previous step are collected (I015 ml), and the same volume of 2 M ammonium sulfate solution (adjusted to pH 7.2 with 2 M Tris) is added. The ammonium sulfate solution must be added dropwise very slowly while stirring, otherwise insoluble materials may appear. The sample (one-half of total sample per one cycle of chromatography) is then applied to a phenyl-Superose HR 5/5 column (0.5 × 5 cm, Pharmacia) preequilibrated with 0.1 M Tris-HC1 buffer containing 1 M ammonium sulfate (pH 7.2). After washing with the same buffer at a flow rate of 0.5 ml/min, the column is eluted with a 20 ml linear gradient between the starting buffer and the terminating buffer (0.1 M Tris-HC1 buffer, pH 7.6). The enzyme activity elutes at the ammonium sulfate concentration of around 0.35 M. Step 5. Active fractions from the previous step are collected (about 6 ml) and concentrated to 1 ml with a Centricon-10 (Amicon, Danvers, MA). The sample is applied to a Superose 12 HR 16/50 column (1.6 × 50 cm, Pharmacia) and eluted with 20 mM Tris-HCl buffer containing 0.1 M KC1 (pH 8.0) at a flow rate of l ml/min. The enzyme activity is eluted at a retention volume of around 55-60 ml. Step 6. The final step of the purification is hydroxyapatite column chromatography. In some cases, depending on the starting materials, this step can be omitted. After dialysis against 100 volumes of 5 mM potassium phosphate buffer (pH 6.3), active fractions from the gel filtration chromatography are applied to a hydroxyapatite column (KB column, 0.78 x 13.5 cm, KOKEN, Tokyo), preequilibrated with the same buffer, and eluted with a 50-ml linear gradient of 5 to 200 mM potassium phosphate buffer (pH 6.3). The enzyme activity elutes at around 60 mM potassium phosphate concentration. A representative result of the purification is summarized in Table I. The purified enzyme has a specific activity ranging from 0.2 to 0.4/.,mol LTB4/ nag. min under standard assay conditions. (For the comparison with Vmax, refer to "kinetic properties.") Although we initially attempted to purify the enzyme from the human lung using the method described for human leukocytes,1 a major impurity with a molecular weight of about 80,000 could not be removed. Because this impurity is successfully removed by phenyl-Superose column chromatography, this is a key step in the purification of the enzyme from the human lung. The yield in this step is, however, low (about 30-40%). Physical Properties Molecular weights of the purified LTA4 hydrolase from the human lung are determined as 68,000-71,000 on SDS-PAGE, values in good accord
[31]
HUMANLUNGLEUKOTRIENEA4 HYDROLASE
291
TABLE I PURIFICATION OF LTA4 HYDROLASE FROM HUMAN LUNG
Step 1. 2. 3. 4. 5. 6.
10,000 g Supernatant 40-70% (NH4)2SO 4 Mono Q Phenyl-Superose Superose 12 Hydroxyapatite
Total protein (rag)
Total activity (nmoi/min)
Specific activity (nmol/mg • rain)
Yield (%)
1,300 470 20 1.2 0.28 0.17
440 310 250 --~ 59 38
0.34 0.66 13 --~ 210 220
100 70 57 --~ 13 8.6
a In this step, ammonium sulfate inhibits the enzyme activity; therefore, precise determination of the activity is not feasible.
with those noted for the enzyme from other sources. 1,2,6,7 except for that from human erythrocytes (54,000 - 1000 on SDS-PAGE). 3 The pI value is determined to be 5.1-5.3 on a Mono P chromatofocusing column. The N-terminal amino acid sequence of the human lung enzyme is Pro-Glu-IleVal-Asp-Thr-Xaa-Ser-Leu-Ala-Ser-Pro-Ala-Ser-Val, 1° which is identical with findings in human leukocytes. 1 The N-terminal sequence for the guinea pig liver 6 and lung 7 enzyme is identical to that of the human enzyme, except for the conservative substitution of two amino acid residues, Ile-3 and Ser-14 (human) to Val and Thr (guinea pig), respectively. This enzyme is stable at 4° for at least 1 month, but is sensitive to freezing and thawing, one cycle of which decreases activity by about 50%. Kinetic Properties The pH optima of the enzyme reaction ranges between 7.8 and 9.0. Neither divalent cations (Ca 2÷, Mg 2+, Mn 2÷ ) nor divalent cation chelators (EDTA, EGTA) affect the enzyme activity. The enzyme activity is reduced by 20% in the presence of 1 M KC1 and by 50% in the presence of 0.2 M ammonium sulfate. 2-Mercaptoethanol (5 mM) or dithiothreitol (0.5 raM) does not affect the enzyme activity, ll On the other hand, SHblocking reagents such as N-ethylmaleimide, p-chloromercuribenzoic acid, and HgC12 inhibit the enzyme activity (ICs0 3 raM, 0.7/zM, 0.5/zM, lO Xaa at N-7 amino acid is determined as Cys by the molecular cloning of LTA4 hydrolase cDNA (see footnotes 16 and 17). i~ SH-reducing reagents affect the elution profile of the enzyme on Mono Q and Mono P column chromatography, when included in the purification procedure of the enzyme from guinea pig lung. 7
292
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
Addition of enzyme
[31]
( ~ ) ~
0.6"
0.4" .J
0.2"
S
~o
~
~
Control (o1
~
~
~
Time (min) FI~. 2. Time course of the enzymatic hydration of LTA4 by the human lung LTA4 hydrolase in the presence of albumin. The purified enzyme (0.44/zg) is incubated in 50/~1 reaction mixture containing LTA4 (80/zM), BSA (2 mg/ml), and 0.1 M Tris-HCl buffer (pH 7.8) at 37° for 5 sec to 5 rain (O). After 30-sec incubation (indicated by vertical arrow), 0.44/zg of enzyme (A) or 1.1/zg of LTA4 ( , ) is added and the reaction halted at the time indicated.
respectively, when 0.44/.~g of enzyme is preincubated with these reagents for 5 min at 37°). In determining kinetic parameters, two points should be taken into consideration. First, LTA4 is unstable in aqueous buffer solutions at neutral pH (half-life less than 10 sec at 25 ° at pH 7.8) and nonenzymatically hydrolyzed to 6-trans-LTB4 (major products) and 5,6-dihydroxy acids (minor products), In fact, under the standard assay condition, the reaction ceases within 20 sec. Second, the enzyme reaction follows a suicide-type one, namely, the enzyme is inactivated by the substrate, LTA4. Figure 2 shows a typical time course of the reaction in the presence of bovine serum albumin which stabilizes LTA4. z2 Again, the rate of reaction declines after 30 sec, and is fully recovered when the enzyme, but not the substrate, is added. Since LTB4 (8/zM) or 6-trans-LTB4 (38/zM) does not inhibit the enzyme reaction, the substrate, LTA4, may inactivate the enzyme. For the above two reasons, the initial velocity must be determined from reactions 12 F. A. Fitzpatrick, D. R. Morton, and M. A. Wynalda, J. Biol. Chem. 257, 4680 (1982).
[31]
293
HUMAN LUNG LEUKOTRIENE A4 HYDROLASE T A B L E II
SUBSTRATE AND INACTIVATORSPECIFICITIESOF ETA4 HYDROLASE R e q u i r e d Structures: Substrate
Inactivator
COOH
Compound
Structure
~ LTA B
LTA 4
LTA 5
~ ~ ~
Substrate
Inactivator
-
+
+
+
COOH
O
0
O
COOH
COOH
COOH
+ +
0
COOCH3
LTA4-Me
+
~ ~
COOH
COOH
(continued)
294
BIOSYNTHESIS, ENZYMOLOGY,
AND CHEMICAL SYNTHESIS
[31]
TABLE II (continued) Compound
Structure
Substrate
Inactivator
,,O
~
COOH
•
t 6 C / O " O x H ~ - - CO ~OH 14,15-LTA4 1 1
II,12-EET
~
-
+
OH
0
14,15-EET O o Styreneoxide
with as short an incubation time as practically possible. Thus, Vm~xand Km values obtained from 10-sec incubation are 2-3/.~mol LTB4/mg.min and 12-13/xM, respectively. This Vma~value is about 10-fold greater than that obtained with a 1-min incubation of the standard assay condition (0.20.4 ttmol/mg" min). Substrate and Inactivator Specificities LTA4 methyl ester, geometric isomers of LTA4 (7,11-trans-9-cis form and 7-trans-9,11-cis form), and 14,15-LTA4 do not serve as a substrate but inactivate the lung enzyme, the concentration of 50% inactivation with 0.8/zg of enzyme being less than 10 ~M. On the other hand, styrene oxide
[3 II
HUMAN LUNG LEUKOTRIENE A4 HYDROLASE
295
and 5(S)-trans-5,6-oxido-8,10,14-cis-12-trans-eicosatetraenoic acid (an isomer of LTA4 without allylic epoxide, see Fig. 1) neither serve as a substrate nor do they inactivate the lung enzyme. Neutrophil LTA4 hydrolase also converts LTA5 to LTBs, albeit less efficiently than LTA4 to LTB4.13 5(S)-trans-5,6-oxido-7,9-trans-Eicosadienoic acid and LTA3 fail to serve as a substrate, but they inactivate the neutrophil enzyme. 14.~5 Finally, 11, 12-oxide-5,8,14-cis-eicosatrienoic acid (EET) and 14,15-EET do not inactivate the erythrocyte e n z y m e ) All these results are summarized in Table II. Taken together, LTA4 hydrolase shows a strict substrate specificity, only for LTA4 and for LTAs, and may be susceptible to inactivation by 1,2-oxide-3,5-hexadiene structure which includes highly reactive allylic epoxide. The covalent binding of LTA4 to the enzyme has been reported, 15 but the relationship between this binding and suicide-type inactivation is not well understood. Human LTA4 hydrolase cDNA has been cloned and the complete primary structure elucidated. 16A7 This clone is expressed in Escherichia coli as a fusion protein, with full enzyme activity. ~8 Acknowledgment This work is supported by grant-in-aid from the Ministry of Education, Science and Culture of Japan.
t3 D. J. Nathaniel, J. F. Evans, Y. Leblanc, C. Lrveillr, B. J. Fitzsimmons, and A. W. Ford-Hutchinson, Biochem. Biophys. Res. Commun. 131, 827 (1985). 14 j. F. Evans, D. J. Nathaniel, S. Charleson, C. Lrveillr, R. Zamboni, Y. Leblanc, R. Frenette, B. J. Fitzsimmons, S. Leger, P. Hamel, and A. W. Ford-Hutchinson, Prostaglandins Leukotrienes Med. 2,3, 167 (1986). 15 j. F. Evans, D. J. Nathaniel, R. J. Zamboni, and A. W. Ford-Hutchinson, J. Biol. Chem. 260, 10966 (1985). 16 M. Minami, S. Ohno, H. Kawasaki, O. R~dmark, B. Samuelsson, H. J6rnvall, T. Shimizu, Y. Seyama, and K. Suzuki, J. Biol. Chem. 262, 13873 (1987). 17 C. D. Funk, O. R~tdmark, J. Y. Fu, T. Matsumoto, H. Jrrnvall, T. Shimizu, and B. Samuelsson, Proc. Natl. Acad. Sci. U.S.A. 84, 6677 (1987). 18 M. Minami, Y. Minami, Y. Emori, H. Kawasaki, S. Ohno, K. Suzuki, N. Ohishi, T. Shimizu, and Y. Seyama, FEBS Lett. 229, 279 (1988).
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
5000
[31]
8-coo.
4000.
, s-coo.
2000
2o-coo.
1000.
0
l0
20
90 40 50 TIME (MINUTES)
50
70
FIo. 5. LTB4 metabolism by isolated rat hepatocytes. The reversed-phase HPLC radiochromatogram of [3H]LTB4 metabolites formed by hepatocytes (5 ml cells, 5 x 106 cells/ml) incubated with LTB4 (3/zM) for 45 min. The identity of 20-COOH-LTB4 is determined by coelution with a synthetic standard. 18-COOH-LTB4 and 16-COOH-LTB4 are identified by electron impact mass spectrometry. Reversed-phase HPLC conditions are as outlined in the text.
solvent front of the gradient reversed-phase HPLC system. Further studies on the metabolism of LTB4 by isolated rat hepatocytes are in progress. The relevance of the isolated rat hepatocyte model of LTE4 metabolism to the fate of leukotrienes in vivo has been supported by the identification of 20-COOH-LTE4 (Metabolite B) and 16-COOH-dihydro-LTE4 (Metabolite D) in rats injected with LTE4. ~o This hepatocyte system serves as a biosynthetic source of these metabolites. to p. Perrin, J. Zirrolli, D. O. Stene, J. P. Lellouche, J. P. Beaucourt, and R. C. Murphy, Prostaglandins 37, 53 (1989).
[3 1] P u r i f i c a t i o n a n d C h a r a c t e r i z a t i o n of H u m a n L u n g L e u k o t r i e n e A4 H y d r o l a s e
By N O B U Y A
O H I S H I , TAKASHI I Z U M I , YOUSUKE SEYAMA, TAKAO S H I M I Z U
and
Leukotriene (LT) A4 hydrolase (EC 3.3.2.6) catalyzes enzymatic hydration of LTA4 [5(S)-trans-5,6-oxido-7,9-trans-ll,14-cis-eicosatetraenoic acid] to LTB4 [5(S), 12 (R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid], a potent chemotactic substance. This reaction on the epoxide differs from that of epoxide hydrolases (EC 3.3.2.3). The latter METHODSIN ENZYMOLOGY,VOL. 187
Copyright© 1990by AcademicPress, Inc. All rightsof reproductionin any formreserved.
[31]
HUMAN LUNG LEUKOTRIENE A4 HYDROLASE
287
results in the synthesis of vicinal alcohols (glycol), while the former synthesizes a compound possessing two hydroxy groups at C-5 and C-12 with a conjugated triene in between. LTA4 hydrolase activity was previously reported mainly in blood cells, especially polymorphonuclear leukocytes and erythrocytes, and the enzymes from these sources were purified. 1-3 The enzyme activity is, however, ubiquitously distributed in the cytosolic fraction of various organs of the guinea pig, and higher activities are observed in small intestine, lung, and aorta. 4 To compare the lung enzyme with those found in blood cells, and for further characterization, the enzyme was purified from the human lung. 5 Recently, this enzyme has also been purified from the guinea pig liver6 and lung. 7
Preparation and Purification of LTA4 Methyl Ester LTA4 methyl esters are synthesized according to Ohkawa and Terao s and Gleason e t al. 9 These are obtained as mixtures of several geometric isomers, and can be separated by straight-phase HPLC. The HPLC conditions are as follows: columnmChemcoPak Nucleosil 50-5, 1 x 30 cm (Chemco, Osaka); solvent--n-hexane/ethyl acetate/triethylamine (100/1.2/0.4, v/v/v); flow rate--3 ml/min; column temperature--4°; UV monitor--280 nm. The column temperature is critical for good resolution and recovery. Under these conditions, four major peaks representing various isomers of LTA4 methyl ester appear at retention times of between 40 and 60 rain, the second-appearing peak being LTA4 methyl ester (Fig. 1). Gas chromatography-mass spectrometry and proton magnetic resonance studies are necessary for structural identification.
1 O. R~dmark, T. Shimizu, H. J6rnvall, and B. Samuelsson, J. BioL Chem. 259, 12339 (1984). 2 j. F. Evans, P. Dupuis, and A. W. Ford-Hutchinson, Biochim. Biophys. Acta 840, 43 (1985). 3 j. McGee and F. Fitzpatrick, J. Biol. Chem. 260, 12832 (1985). 4 T. Izumi, T. Shimizu, Y. Seyama, N. Ohishi, and F. Takaku, Biochem. Biophys. Res. Commun. 135, 139 (1986). 5 N. Ohishi, T. Izumi, M. Minami, S. Kitamura, Y. Seyama, S. Ohkawa, S. Terao, H. Yotsumoto, F. Takaku, and T. Shimizu, J. Biol. Chem. 262, 10200 (1987). 6 j. Haeggstr6m, T. Bergman, H. J6rnvaU, and O. R/idmark, Eur. J. Biochem. 174, 717 (1988). 7 H. Bito, N. Ohishi, I. Miki, M. Minami, T. Tanabe, T. Shimizu, and Y. Seyama, J. Biochem. 105, 261 (1989). 8 S. Ohkawa and S. Terao, J. Takeda Res. Lab. 42, 13 (1983). 9 j. G. Gleason, D. B. Bryan, and C. M. Kinzig, Tetrahedron Lett. 21, 1129 (1980).
288
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
[3 1]
~..._~.,~,.~ CO0Cfl3 E 0 CO O4
0eC~
k~~CO°CH~
--•6
.
,~
2'0 4'0 6'0 do Retention time {min)
FIG. 1. Straight-phaseHPLCseparationof LTA4methylesterand its geometricisomers. Four majorpeaks eluteat retentiontimes around40-60 min and the structureof each peakis illustrated.
Saponification of LTA4 Methyl Ester LTA4 is unstable in aqueous buffer solutions at acidic and neutral pH, and is stabilized in alkaline organic solutions at a lower temperature. LTA4 is synthesized as the methyl ester form and is stored in alkaline organic solution such as benzene/triethylamine (100/2, v/v) in a nitrogen atmosphere at - 7 0 °. When in use, LTA4 methyl ester is saponified in tetrahydrofuran (400/~l) plus 0.1 M lithium hydroxide in water (100/.el) under conditions of vigorous mixing overnight at room temperature. The aliquot is then dried under a stream of nitrogen (complete dryness should be avoided), and dissolved with an appropriate amount of ethanol. The con-
[31]
HUMANLUNGLEUKOTRIENEA4 HYDROLASE
289
centration of LTA4 is determined from UV absorption, using a molar extinction coefficient of 40,000 M - lcm- ~ at 280 nm. Assay of LTA4 Hydrolase The standard incubation mixture (50 ~l in 1.5 ml sampling tube) contains the enzyme in 0.1 M Tris-HCl buffer (pH 7.8). After preincubation for several minutes at 37 °, 1/~g of LTA4 in 1/A of ethanol is added. After 1 rain of incubation, 117/~l of stopping solution (0.1% acetic acid in methanol containing 0.3 nmol of prostaglandin B2 as an internal standard) is added. After the mixture is kept at - 2 0 ° for at least 20 rain followed by centrifugation at l04 g for 10 min, a 50-/.d aliquot of the supcrnatant is directly injected onto HPLC. The conditions are as follows: c o l u m n n T S K - O D S 80TM, 0.46 × 15 cm (Tosoh, Tokyo); solvent--methanol/water/acetic acid (70/30/0.05, v/v/v); flow rate--1 ml/min; column temperaturen35°; UV monitorm270 nm. PGBz and LTB4 elute at about 7 and 11 min, respectively, and the amount of LTB4 formed is calculated from the peak area ratio LTB4/PGB2. Purification Procedure All procedures other than column chromatography are done at 4 ° . Column chromatography is performed using a fast protein liquid chromatography (FPLC) system (Pharmacia, Uppsala), at room temperature. All centrifugations are carried out at 10,000 g at 4° for 20 rain, unless otherwise indicated. Step 1. Human lung (about a 50 g autopsy specimen with no inflammatory and other disease processes) is minced with scissors in 3 volumes of ice-cold 20 mM potassium phosphate buffer containing 5 mM EDTA and 0.15 M NaC1 (pH 7.4). After homogenization using a Polytron-type homogenizer (output dial 3, 1 min, 3 times), the sample is centrifuged. Step 2. The 10,000 g supernatant is subjected to ammonium sulfate fractionation. The precipitates between 40 and 70% saturation are collected, dissolved in 50 ml of 20 mM Tris-HCl buffer (pH 8), and then dialyzed against two changes of 40 volumes of the same buffer. Step 3. After centrifugation and filtration through 0.45 ~m filters, the dialyzed sample (one-third or one-fourth of total volume per one cycle of chromatography) is applied to a Mono Q HR 10/10 column (1 × l0 cm, Pharmacia) preequilibrated with 20 mM Tris-HCl buffer (pH 8) at a flow rate of 2 ml/min. The column is washed with the same buffer (about 20 ml), and the adsorbed proteins are eluted with a 75 ml linear gradient between 0-0.15 M KCI, in the same buffer. The enzyme activity elutes at a KC! concentration of around 0.1 M.
290
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
[31]
Step 4. Active fractions from the previous step are collected (I015 ml), and the same volume of 2 M ammonium sulfate solution (adjusted to pH 7.2 with 2 M Tris) is added. The ammonium sulfate solution must be added dropwise very slowly while stirring, otherwise insoluble materials may appear. The sample (one-half of total sample per one cycle of chromatography) is then applied to a phenyl-Superose HR 5/5 column (0.5 × 5 cm, Pharmacia) preequilibrated with 0.1 M Tris-HC1 buffer containing 1 M ammonium sulfate (pH 7.2). After washing with the same buffer at a flow rate of 0.5 ml/min, the column is eluted with a 20 ml linear gradient between the starting buffer and the terminating buffer (0.1 M Tris-HC1 buffer, pH 7.6). The enzyme activity elutes at the ammonium sulfate concentration of around 0.35 M. Step 5. Active fractions from the previous step are collected (about 6 ml) and concentrated to 1 ml with a Centricon-10 (Amicon, Danvers, MA). The sample is applied to a Superose 12 HR 16/50 column (1.6 × 50 cm, Pharmacia) and eluted with 20 mM Tris-HCl buffer containing 0.1 M KC1 (pH 8.0) at a flow rate of l ml/min. The enzyme activity is eluted at a retention volume of around 55-60 ml. Step 6. The final step of the purification is hydroxyapatite column chromatography. In some cases, depending on the starting materials, this step can be omitted. After dialysis against 100 volumes of 5 mM potassium phosphate buffer (pH 6.3), active fractions from the gel filtration chromatography are applied to a hydroxyapatite column (KB column, 0.78 x 13.5 cm, KOKEN, Tokyo), preequilibrated with the same buffer, and eluted with a 50-ml linear gradient of 5 to 200 mM potassium phosphate buffer (pH 6.3). The enzyme activity elutes at around 60 mM potassium phosphate concentration. A representative result of the purification is summarized in Table I. The purified enzyme has a specific activity ranging from 0.2 to 0.4/.,mol LTB4/ nag. min under standard assay conditions. (For the comparison with Vmax, refer to "kinetic properties.") Although we initially attempted to purify the enzyme from the human lung using the method described for human leukocytes,1 a major impurity with a molecular weight of about 80,000 could not be removed. Because this impurity is successfully removed by phenyl-Superose column chromatography, this is a key step in the purification of the enzyme from the human lung. The yield in this step is, however, low (about 30-40%). Physical Properties Molecular weights of the purified LTA4 hydrolase from the human lung are determined as 68,000-71,000 on SDS-PAGE, values in good accord
[31]
HUMANLUNGLEUKOTRIENEA4 HYDROLASE
291
TABLE I PURIFICATION OF LTA4 HYDROLASE FROM HUMAN LUNG
Step 1. 2. 3. 4. 5. 6.
10,000 g Supernatant 40-70% (NH4)2SO 4 Mono Q Phenyl-Superose Superose 12 Hydroxyapatite
Total protein (rag)
Total activity (nmoi/min)
Specific activity (nmol/mg • rain)
Yield (%)
1,300 470 20 1.2 0.28 0.17
440 310 250 --~ 59 38
0.34 0.66 13 --~ 210 220
100 70 57 --~ 13 8.6
a In this step, ammonium sulfate inhibits the enzyme activity; therefore, precise determination of the activity is not feasible.
with those noted for the enzyme from other sources. 1,2,6,7 except for that from human erythrocytes (54,000 - 1000 on SDS-PAGE). 3 The pI value is determined to be 5.1-5.3 on a Mono P chromatofocusing column. The N-terminal amino acid sequence of the human lung enzyme is Pro-Glu-IleVal-Asp-Thr-Xaa-Ser-Leu-Ala-Ser-Pro-Ala-Ser-Val, 1° which is identical with findings in human leukocytes. 1 The N-terminal sequence for the guinea pig liver 6 and lung 7 enzyme is identical to that of the human enzyme, except for the conservative substitution of two amino acid residues, Ile-3 and Ser-14 (human) to Val and Thr (guinea pig), respectively. This enzyme is stable at 4° for at least 1 month, but is sensitive to freezing and thawing, one cycle of which decreases activity by about 50%. Kinetic Properties The pH optima of the enzyme reaction ranges between 7.8 and 9.0. Neither divalent cations (Ca 2÷, Mg 2+, Mn 2÷ ) nor divalent cation chelators (EDTA, EGTA) affect the enzyme activity. The enzyme activity is reduced by 20% in the presence of 1 M KC1 and by 50% in the presence of 0.2 M ammonium sulfate. 2-Mercaptoethanol (5 mM) or dithiothreitol (0.5 raM) does not affect the enzyme activity, ll On the other hand, SHblocking reagents such as N-ethylmaleimide, p-chloromercuribenzoic acid, and HgC12 inhibit the enzyme activity (ICs0 3 raM, 0.7/zM, 0.5/zM, lO Xaa at N-7 amino acid is determined as Cys by the molecular cloning of LTA4 hydrolase cDNA (see footnotes 16 and 17). i~ SH-reducing reagents affect the elution profile of the enzyme on Mono Q and Mono P column chromatography, when included in the purification procedure of the enzyme from guinea pig lung. 7
292
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
Addition of enzyme
[31]
( ~ ) ~
0.6"
0.4" .J
0.2"
S
~o
~
~
Control (o1
~
~
~
Time (min) FI~. 2. Time course of the enzymatic hydration of LTA4 by the human lung LTA4 hydrolase in the presence of albumin. The purified enzyme (0.44/zg) is incubated in 50/~1 reaction mixture containing LTA4 (80/zM), BSA (2 mg/ml), and 0.1 M Tris-HCl buffer (pH 7.8) at 37° for 5 sec to 5 rain (O). After 30-sec incubation (indicated by vertical arrow), 0.44/zg of enzyme (A) or 1.1/zg of LTA4 ( , ) is added and the reaction halted at the time indicated.
respectively, when 0.44/.~g of enzyme is preincubated with these reagents for 5 min at 37°). In determining kinetic parameters, two points should be taken into consideration. First, LTA4 is unstable in aqueous buffer solutions at neutral pH (half-life less than 10 sec at 25 ° at pH 7.8) and nonenzymatically hydrolyzed to 6-trans-LTB4 (major products) and 5,6-dihydroxy acids (minor products), In fact, under the standard assay condition, the reaction ceases within 20 sec. Second, the enzyme reaction follows a suicide-type one, namely, the enzyme is inactivated by the substrate, LTA4. Figure 2 shows a typical time course of the reaction in the presence of bovine serum albumin which stabilizes LTA4. z2 Again, the rate of reaction declines after 30 sec, and is fully recovered when the enzyme, but not the substrate, is added. Since LTB4 (8/zM) or 6-trans-LTB4 (38/zM) does not inhibit the enzyme reaction, the substrate, LTA4, may inactivate the enzyme. For the above two reasons, the initial velocity must be determined from reactions 12 F. A. Fitzpatrick, D. R. Morton, and M. A. Wynalda, J. Biol. Chem. 257, 4680 (1982).
[31]
293
HUMAN LUNG LEUKOTRIENE A4 HYDROLASE T A B L E II
SUBSTRATE AND INACTIVATORSPECIFICITIESOF ETA4 HYDROLASE R e q u i r e d Structures: Substrate
Inactivator
COOH
Compound
Structure
~ LTA B
LTA 4
LTA 5
~ ~ ~
Substrate
Inactivator
-
+
+
+
COOH
O
0
O
COOH
COOH
COOH
+ +
0
COOCH3
LTA4-Me
+
~ ~
COOH
COOH
(continued)
294
BIOSYNTHESIS, ENZYMOLOGY,
AND CHEMICAL SYNTHESIS
[31]
TABLE II (continued) Compound
Structure
Substrate
Inactivator
,,O
~
COOH
•
t 6 C / O " O x H ~ - - CO ~OH 14,15-LTA4 1 1
II,12-EET
~
-
+
OH
0
14,15-EET O o Styreneoxide
with as short an incubation time as practically possible. Thus, Vm~xand Km values obtained from 10-sec incubation are 2-3/.~mol LTB4/mg.min and 12-13/xM, respectively. This Vma~value is about 10-fold greater than that obtained with a 1-min incubation of the standard assay condition (0.20.4 ttmol/mg" min). Substrate and Inactivator Specificities LTA4 methyl ester, geometric isomers of LTA4 (7,11-trans-9-cis form and 7-trans-9,11-cis form), and 14,15-LTA4 do not serve as a substrate but inactivate the lung enzyme, the concentration of 50% inactivation with 0.8/zg of enzyme being less than 10 ~M. On the other hand, styrene oxide
[3 II
HUMAN LUNG LEUKOTRIENE A4 HYDROLASE
295
and 5(S)-trans-5,6-oxido-8,10,14-cis-12-trans-eicosatetraenoic acid (an isomer of LTA4 without allylic epoxide, see Fig. 1) neither serve as a substrate nor do they inactivate the lung enzyme. Neutrophil LTA4 hydrolase also converts LTA5 to LTBs, albeit less efficiently than LTA4 to LTB4.13 5(S)-trans-5,6-oxido-7,9-trans-Eicosadienoic acid and LTA3 fail to serve as a substrate, but they inactivate the neutrophil enzyme. 14.~5 Finally, 11, 12-oxide-5,8,14-cis-eicosatrienoic acid (EET) and 14,15-EET do not inactivate the erythrocyte e n z y m e ) All these results are summarized in Table II. Taken together, LTA4 hydrolase shows a strict substrate specificity, only for LTA4 and for LTAs, and may be susceptible to inactivation by 1,2-oxide-3,5-hexadiene structure which includes highly reactive allylic epoxide. The covalent binding of LTA4 to the enzyme has been reported, 15 but the relationship between this binding and suicide-type inactivation is not well understood. Human LTA4 hydrolase cDNA has been cloned and the complete primary structure elucidated. 16A7 This clone is expressed in Escherichia coli as a fusion protein, with full enzyme activity. ~8 Acknowledgment This work is supported by grant-in-aid from the Ministry of Education, Science and Culture of Japan.
t3 D. J. Nathaniel, J. F. Evans, Y. Leblanc, C. Lrveillr, B. J. Fitzsimmons, and A. W. Ford-Hutchinson, Biochem. Biophys. Res. Commun. 131, 827 (1985). 14 j. F. Evans, D. J. Nathaniel, S. Charleson, C. Lrveillr, R. Zamboni, Y. Leblanc, R. Frenette, B. J. Fitzsimmons, S. Leger, P. Hamel, and A. W. Ford-Hutchinson, Prostaglandins Leukotrienes Med. 2,3, 167 (1986). 15 j. F. Evans, D. J. Nathaniel, R. J. Zamboni, and A. W. Ford-Hutchinson, J. Biol. Chem. 260, 10966 (1985). 16 M. Minami, S. Ohno, H. Kawasaki, O. R~dmark, B. Samuelsson, H. J6rnvall, T. Shimizu, Y. Seyama, and K. Suzuki, J. Biol. Chem. 262, 13873 (1987). 17 C. D. Funk, O. R~tdmark, J. Y. Fu, T. Matsumoto, H. Jrrnvall, T. Shimizu, and B. Samuelsson, Proc. Natl. Acad. Sci. U.S.A. 84, 6677 (1987). 18 M. Minami, Y. Minami, Y. Emori, H. Kawasaki, S. Ohno, K. Suzuki, N. Ohishi, T. Shimizu, and Y. Seyama, FEBS Lett. 229, 279 (1988).
