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MOLECULAR CLONING MOUSE LEUKOTRIENE

1516-1524

AND EXPRESSION OF A4 HYDROLASE CDNA

Juan F. Medina*, Olof RBdmark, Colin D. Funk and Jesper Z. Haeggstrijm Department of Physiological Chemistry, Karolinska Institutet, S-104 01 Stockholm, Sweden Received

April

1, 1991

A cDNA clone for mouseleukotriene A4 hydrolaseencoding the full sequenceof the enzyme was isolated from a mouse spleen h ZAP-II library. The identification was ascertainedby expressionof enzyme activity in Escherichiucoli. The encodedprotein has 610 amino acidsandexhibits an extensive identity (93%) with the humanleukotriene & hydrolase. A region spanningbetweenresidues233 and 340, where the zinc binding site is located, was found to be perfectly conservedbetweenthe two species. We found six sites of polymorphism in the cDNA sequence of mouse LTA4 hydrolase, one of which leads to a difference in the encoded amino acid. The polymorphism of cDNA was confirmed by reverse transcription-PCR sequencingof mousespleentotal RNA, preparedasa mixture from ten different strains. 0 1991Academic Press,

Inc.

Leukotriene A4 (LTA&

a pivotal intermediatein the biosynthesisof leukotrienes, is

derived from arachidonicacid via oxidative metabolismcatalyzed by 5lipoxygenase (1). The hydrolysis of its epoxide function by LTA4 hydrolase leads to leukotriene B4 (LTBq), which is consideredto be an important inflammatory mediator. LTA4 hydrolaseis a cytosolic enzyme, originally found in neutrophils.Characteristic featuresof this enzyme are the “suicide inactivation” occurring during catalysis, and the wide tissuedistribution (2,3). Human LTA4 hydrolase cDNAs have been cloned (45, and the human spleencDNA hasbeenexpressedasan active fusion protein in E. coli (6). A zinc binding motif hasbeenidentified in the primary structureof the enzyme (7,8), and it was recently characterized as a zinc metalloenzyme, which also possesses peptidase activity (9- 11). The availability of LTA4 hydrolasecDNAs from several speciesand the comparison of their deducedamino acid sequencescould help to elucidatethe reaction mechanism(s) of the enzyme. In the presentstudy we report the isolation, sequencingand expressionof mouseLT& hydrolasecDNA. *To whom correspondence should be sent.

Abbreuiafions: LT&, leukotriene Ad; LTB4, leukotriene B4; RT, reverse transcription; PCR, polymerase chain reaction; bp, basepairs; kb, kilobases; IPTG, isopropyl+Dthiogalactopyranoside. 0006-291X/91 Copyright All rights

$1.50 1991 by Academic Press, of reproduction in any form 0

fm. resmed.

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AND METHODS

A mouse spleen cDNA library in bacteriophage lcgtl 1 was purchased from Clontech (Palo Alto, CA). Another mouse spleen cDNA library in X ZAP-II vector was constructed using materials from Stratagene (La Jolla, CA) and Pharmacia (Uppsala, Sweden). T7 Sequencing and Double-stranded nested deletion kits, restriction endonucleases and nucleic acid modifying enzymes were from Pharmacia, unless otherwise indicated. Radioactive nucleotides and multiprime DNA labeling kits were from Amersham. Sea [email protected] (low gelling temperature) agarose was from FMC (Rockland, ME) and Qiagen tips were from Diagen GmhH (Dusseldorf, Germany). Oligonucleotides were synthesized by Scandinavian Gene Synthesis AB (Kiiping, Sweden). Preparation of RNA and Northern blot analysis. Total RNA was isolated from mouse spleens (obtained from 60 animals from 10 different strains: 30% MRL Ipr/lpr, 15% Balb/B and < 9% of each of the remaining strains) by the method of Chomczynski and Sacchi (12). Poly(A) RNA was obtained by chromatography on oligo (dT)-cellulose (two passages). Northern blot analysis of RNA was performed as described (13). Construction and screening of Mouse Spleen cDNA libraries. Initially, a mouse spleen cDNA library in hgtll was screened using a “P-labeled DNA fragment of human LTA4 hydrolase cDNA (nucleotides 1054-1880, ref. 4) as hybridization probe. The library was plated on E. colt’ strain Y1090 and screened as described (4). Putative positive clones were purified by two succesive rounds of screening-hybridization. One of the positive clones (hsp3L) contained the longest cDNA insert (1.7-kb). This insert was subcloned into pUC18 vector to facilitate amplification and probe preparations. A second cDNA library, made according to the method of Gubler and Hoffman (14) using a kit from Pharmacia, was screened to obtain an additional cDNA containing the region 5’ upstream of the hsp3L insert. For cDNA synthesis, 2 pg of mouse spleen poly(A) RNA was primed with 0.15 pg of random hexamers. cDNAs obtained were ligated to h ZAP-II vector and packaged with Gigapack Gold packaging extract (both from Stratagene) according to the manufacturer’s instructions. This cDNA library was plated on E. coli strain XLl-Blue, and screened with the OS-kb EcoRVBgnJ fragment of the hsp3L insert (nucleotides 206-738, Fig. 2). A positive h ZAP-II clone was obtained and helper phage R408 was used for the in vivo excision (15) of the pBluescript plasmid harbouring the cDNA insert (pZBL2 recombinant plasmid). Preparation of recombinuntplasmids. Appropriate restriction fragments were isolated by electrophoresis on a low gelling temperature (1%) agarose gel. After melting of gel slices and addition of urea to prevent the agarose from polymerizing, DNA fragments were purified with Qiagen tips and further ligated into vectors to transform competent cells E. coli strain JMlOl (16). Recombinant plasmids of high purity, suitable for double strand DNA sequencing, were obtained by the boiling method (17), followed by phenol/chloroform extraction and precipitation with ammonium acetate (as described below for precipitation of PCR products). Reverse transcription-PCR amplification. To obtain mRNA sequences (Fig. 3), reverse transcription coupled to PCR-amplification was performed according to published procedures (18-21) with some modifications. Total RNA (10 pg in 10 ~1 of water, heated at 65’ C, 10 min) was transferred to a 1.5~ml tube containing a mixture of 19 pl of water, 4 pl of 10 x PCR buffer (0.5 M KCl, 0.2 M Tris-HCl (pH 8.3), 25 mM MgCl,, 1 mg/ml of gelatin), 2 ~1 of 10 mM dNTP, 2pl of (dT)r7-adaptor (15 ng/l.tl; see ref. 20 for more details about this primer), 2 ~1 of 100 mM dithiothreitol and 1 ~1 (37 units) of ribonuclease inhibitor RNasin (Pharmacia). After addition of 200 units (1 pl) of Moloney Murine Leukemia Virus reverse transcriptase (BRL, Maryland), the sample was incubated at 37°C for 60 min. For PCR-amplification (in a siliconized 0.5-ml tube), 4 ~1 of the cDNA pool was mixed with 5 p,l of 10 x PCR buffer, 5 pl of 2 mM dNTP, 1 ~1 of 5’ primer and 1 ~1 of 3’ primer (100 n&l each) and 32 pl of water. The mixture was covered with one drop of mineral oil (Sigma) and denatured at 95’ C for 5 min. Addition of 2 f.~lof Thermus aquaticus (Taq) polymerase (1 U&l) was followed by 30-40 cycles of amplification with the step program 55”C, 2 min; 72”C, 3 min; 94”C, 1 min, and finished by 55’C, 2 min and an extension at 72°C for 10 min. To produce single-stranded DNA (21) 1 pl of the PCR product was amplified again in a volume of 100 ~1 using 30 cycles and only one primer (the primer used in each case was the one in the opposite sense to the 1517

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sequencing primer; see below). After the PCK reaction, the product was diluted with an equal volume of water and precipitated by the addition of 1 volume of 7.5 M ammonium acetate and 2.5 volumes of ethanol, followed by chilling at -70°C for 20 min and centrifugation (15 min). The pellet was carefully washed with cold 80% ethanol, dried under vacuum and resuspended in 10 pl of water. After addition of annealing buffer, this single-stranded DNA was ready for the annealing to the sequencing primer. DNA sequencing. Both single and double stranded DNAs were sequenced by the dideoxy chain termination method (22). In the case of Xgtl 1 inserts, they were previously subcloned in Ml 3mp 18 vector. For the relatively long cDNA insert from hsp3L, a series of overlapping and unidirectional deletions were made according to the method of Henikoff (23), using a kit from Pharmacia. Hi&II produced recessed 3’ ends, which were filled in with thionucleotides, rendering ends resistant to exonuclease III. A further digestion with BamHI produced a new 5’ end with overhang, allowing for unidirectional digestion with exonuclease III (in 75 mM NaCl at 30’ C). Aliquots were removed at timed intervals (110 seconds), treated with Sl nuclease and recircularized with T4 DNA ligase. Recircularized DNAs were used to transform highly competent JMlOl cells (16). Single-stranded DNAs were isolated and the Ml3 -40 primer was used in the sequencing reactions. Deletions of the insert subcloned in both orientations allowed its sequence to be determined for both strands. Single-stranded DNAs produced by PCR were sequenced following the same procedure as for single-stranded template from Ml3 recombinants. The primers employed were those in the opposite sense to the primer used for the single strand production. End labelling of primers was not required, since good results were obtained by the usual sequencing procedure with addition of [3sS]dATPcxS to the reaction mixture. For sequencing of recombinant plasmids obtained by the boiling method, double strand DNA was denatured in 0.4 M NaOH, at room temperature for 10 min. After precipitation with ethanol, this DNA was generally suitable for the dideoxy termination sequencing. The primers employed were synthetic oligonucleotides (20-22 bases long) designed on the basis of known sequences. Expression of LTA4 hydrolase cDNA. pZBL2 insert was subclonedinto pUC9 (Fig. 4) in a similar way as describedfor expressionof humanLTA4 hydrolase cDNA (6). Thus, digestion with AvaI allowed the isolation of the insert, which was treated with Klenow fragment to create blunt ends.Ligation to pUC9 (previously cut with NincII) gave the recombinant pULTA4. Cells (E. coli JMlOl) transformedwith pULTA4 were cultured at 37” C, in M9 medium (50 mM Na2HP04, 22 mM KH2PO4,0.5 g/l NaCI, 20 mM N&Cl) containing 0.2% casaminoacids, 2mM MgS04 and 75 pg/ml ampicillin. IPTG was addedto a final concentration of 0.5 mM when absorbance, “,,,~0.2, and the incubation wascontinued typically for 2 11~ h. Aliquots of culture were collected every 30 min. After centrifugation, cells were resuspendedin homogenizationbuffer (50 mM TrisHCI (pH 8), 5 mM EDTA, 2 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride and 60 pg/ml soybean trypsin inhibitor, 1 ml of the buffer/25 ml of culture), and homogenizedby sonication. The homogenatewas centrifuged for 10 min at 10 000 x g and aliquots of supernatant (100 pl) were incubated with LTA4 (40 pM, room temperature, 30 seconds). Incubations were stopped by addition of 1 volume of methanol, and prostaglandinBr was addedas internal standard.Sampleswere extracted on [email protected] columns(Macherey-Nagel, Diiren, Germany). After washingwith water and methanol/water (25:75), the adsorbedproducts were eluted with 2 ml of methanol and LTB4 formation was analyzed by reverse phase HPLC. The column (Nova-Pak C18,4 pm, Radial Pakmcartridge, 5 mm x 100mm, Waters) waseluted with methanol/water/trifluoroacetic acid (72:28:0.007), at 1.2 ml/min, as described by Steinhilber et al. (24). RESULTS

AND DISCUSSION

Isolation and characterization of mouseLTA4 hydrolase clones.This study describes the molecular cloning and expressionof mouseLTA4 hydrolase cDNA. Screening of a hgtl 1 library from mousespleen,using a fragment of humanLTA4 hydrolase cDNA as hybridization probe, gave several positive clones. hsp3L clone contained the longest insert and its entire nucleotide sequencewas determined (1682-bp long, Figs. 1 and 2). 1518

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pzBL2

Frc. Partial restriction enzyme map and sequencing strategy of the two major cDNAs encoding mouse LT& hydrolase. Direction and extent of sequence determinations are indicated by arrows. The thick bar shows the protein coding region.

Matching of the obtained sequence with the sequence of human LTA4 hydrolase cDNA (4) revealed a high identity (87%), with no gaps. The 3’ region possessed a TAA stop codon followed by 51 bp. The last 10 residues were adenosine, but no polyadenylation signals (25) could be identified. The 5’ end of the hsp3L insert matched the residue 206 in the coding region of human LTA4 hydrolase cDNA, and did not contain any ATG initiation codon. Thus, about 300 bp were apparently missing at the 5’ end of ksp3L cDNA. An additional cDNA (pZBL2 insert, 1936-bp long excluding the flanking EcoRI/NotI adaptors, Fig. 2), which contains the complete protein coding region, was finally obtained from another cDNA library (made from mouse spleen RNA in the vector h ZAPII), using a fragment of hsp3L insert as probe. The nucleotide sequence shows a putative ATG initiation codon followed by a continuous open reading frame 1830-bp long. This first ATG triplet of the pZBL2 insert was considered to be the initiation codon because: i) it is in the sense frame (the other frames are unacceptable as coding frames) and matches perfecly with the initiation codon from the human LTA4 hydrolase cDNA , and ii) there is an ACC triplet at position -3, -2, and -1 upstream from the putative ATG initiation codon, which is a well conserved feature in most eukaryotic messages (26). The 5’ noncoding region in pZBL2 is 80-bp long, and the 3’ noncoding region is identical to the one in hsp3L insert, but lacks the last 31 bp. About 200 bp of noncoding sequence (probably at the 3’ end) is still missing, as estimated by the size of the mouse LT& (approx. 2.2-kb) in Northern blot analysis.

hydrolase mRNA

The open reading frame in pZBL2 insert (1830-bp excluding ATG; Fig. 2), as well as the encoded protein (610 amino acids, the initiation methionine excluded, with a calculated molecular weight of 68,917), has the same length as the human counterpart (4,5). A striking sequence identity (93% at the amino acid level and 87% at the nucleotide level) was also observed. Particularly, the region between amino acid residues 233 and 340 is identical. A portion of 45 amino acids, within this completely conserved region, contains 6 histidine residues. Two of these, at positions 295 and 299, together with Giu318 conform to a catalytic zinc site (27). His-295 and His-299, which presumably anchor the zinc atom to the protein, are located in a portion of the sequence (eight amino acids 1519

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FIG. 2. Nucleotide sequence of mouse LT& hydrolase cDNAs and the deduced amino acidsequence. Nucleotides are numbered beginning with the first residue of the ATG initiation codon. Nucleotides 5’ of ATG are designated by negative numbers. Polymorphisms in six positions are indicated by underlined triplets; R is A/G; Y is C/T. Amino acids are numbered from the N-terminal proline residue. The alternative amino acid at position 592 is indicated. The residues which differ from the human enzyme sequence are shown in italics. Underlining of amino acids represents the putative zinc binding site.

long) human

with a moderate LT&

hyclrolase

enzyme was compared thermolysin)

prediction

for a helical

was recently

observed

structure

(28). The zinc binding

site of

when the amino acid sequence

of this

to those of certain peptidases

(7,8). In accordance

and neutral proteases (for example

with this sequence homology, 1520

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TABLE I

Polymorphisms

in mouse LTA4 hydrolase cDNA (and mRNA)

840 pZBL2 cDNA hsp3L cDNA RT-PCR of total RNA

Nucleotide positions 1167 1734 1778 1797

A G GATAGG G R

1803

C

G

A

A

Y

A

G

G

The six positions with nucleotide differences between the sequences of pZBL2 and hsp3L cDNAs are indicated. The bottom line shows the results of the reverse transcription-PCR sequencing of mouse spleen total RNA prepared as a mixture fmm 10 different strains. R is A+G and Y is C+T (or C+U in corresponding mRNAs). Numbering begins with the fist base of the initiation codon.

was found to contain one zinc atom per enzyme molecule, and furthermore it also displayed a peptidase activity in addition to the epoxide hydrolase activity, i.e. the hydrolysis of LT& to LTB4 (9- 11). Polymorphism of LTA4 hydrolase. Six differences in the sequencebetween pZBL2 and hsp3L cDNAs were found (Table I). The differences are at the third baseof the respective codons, except the one at position 1778, and always within the families of pukes or pyrimidines (i.e., from A to G and C to T or vice versa). Only the nucleotide difference at position 1778leadsto different amino acids: pZBL2 cDNA encodesArg592 while hsp3L cDNA encodesLys-592. Reversetranscription-PCR sequencingof the mixture of total RNA (from mouse spleensfrom 10 different strains) confirmed the polymorphism of LTb hydrolase mRNA (Fig. 3). The sequencingautoradiographies showed bands corresponding to both purine derivatives at position 1167 and both pyrimidine derivatives at position 1734 (Fig. 3). Positions 840, 1778, 1797 and 1803 mainly showedbandscorrespondingto G, A, G and G, respectively, (identical to hsp3L insert, Table I). The counterpartsA, G, A and A, respectively (as in the pZBL2 insert), could only be detected after longer exposure. Thus, it seemsthat the mRNA species correspondingto pZBL2 cDNA waslessabundantin the mixture of total RNA (fig . 4). Expression of mouseLTA4 hydrolase. The identity of pZBL2 cDNA as an LTA4 hydrolase cDNA was finally confirmed by expressionof LT& hydrolase activity in E. coli. Upon induction with IPTG, a lo-fold increase in the LTA4 hydrolase activity displayed by JMlOl cells harbouring the expressionconstruct pULTA4 was observed (Fig. 5). No enzyme activity was found in non-transformed JMlOl cells or in cells transformedwith pUC9 (irrespectiveof induction with JPTG). The expressionsystem usedfor mouseLT& hydrolase gave a high yield of active enzyme (a fusion protein with 10 additional aminoacid residuesat its N-terminal; cf. ref. 6), which hasbeenpurified and characterizedasa zinc metalloenzyme with two enzyme activities (Wetterholm, A., J.F.M., O.R., Shapiro, R., J.Z.H., Vallee, B.L. and 1521

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FIG. 3. Reverse transcription-PCR sequencing of mouse spleen total RNA, prepared as a mixture from 60 animals (10 different strains). An aliquot of this RNA preparation was transcribed to cDNA by reverse transcriptase and a selected region of cDNA was amplified by PCR. Single-strand templates were produced (also by PCR) and sequenced by the dideoxy chain termination method. Upper panel: sequencing strategy of the regions where the differences between hsp3L and pZBL2 inserts are located. Arrows indicate direction and extent of sequence determinations. Lower panel: autoradiograms from RT-PCR sequence analyses, with polymorphism at positions 1167 and 1734 indicated in I and II, respectively. The sequence shown in III is the reverse of the sequence shown in II.

AvaI digestion Klenow treatment

m

HincII

digestion

0

FIG. 4. Strategy for the construction of recombinant pULTA4 for expression of LTA4 hydrolase cDNA (pZBL2 insert) in E. co/i (see materials and methods). 1522

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300 -

pULTA4

b

pULTA4 cells - IPTG

cells + IPTG

--O--

pUC9 cells +/- IPTG

200 ;i; 5 5 72 E ‘0

100

*

0

60

120

Time

1 SO

(min)

FIG. 5. Time course of the expression of LTA4 hydrolase activity in E. coli. The cell growth was similar in all cultures, as judged by measurements of the absorbance (at 600 nm) every 30 min. IPTG was added at the time indicated as zero. Aliquots of culture (25 ml) were removed at 30 min intervals, starting at absorbanceboo Nn ~0.2. Cells, resuspended in 1 ml of homogenization buffer (described in materials and methods) were homogenized by sonication. After centrifugation for 10 min at 10 000 x g, 100 ~1 of each supematant was incubated with LT& (40 f.tM), and analyzed for LTB4 formation.

Samuelsson, B.; manuscript in preparation). Thus, together with site-directed mutagenesis,the systemis a useful tool for further studiesregarding zinc binding and catalytic mechanism(s)for the epoxide hydrolase and peptidase activities of LTA4 hydrolase. Acknowledgments. We are grateful to Drs. S. Hoshiko, C. Ib%iez and D. Steinhilber for valuable discussions.We thank Ms. Agneta Nordberg for excellent assistance.The study was supportedby funds from the Swedish Medical ResearchCouncil (03X-217 and 03X-07467), from Hedlunds Stiftelse, and from O.E. & Edta Johanssons VetenskapligaStiftelse. JEM had a grant from the Wenner-Gren Foundation.

REFERENCES 1. Samuelsson,B., DahlCn, S-E., Lindgren, J-A., Rouzer, C.A. and Serhan, C.N. (1987) Science 237, 1171-l 176. Samuelsson,B. and Funk, C.D. (1989) J. Biol. Chem. 264, 19469-19472. t : RAdmark, 0. andHaeggstrom,J. (1990) Adv. Prostaglandin ThromboxaneLeukotriene Res. 20, 35-45. 4. Funk, C.D., RAdmark, O., Ji-Yi, F., Matsumoto, T., Jornvall, H., Shimizu, T. a Samuelsson,B. (1987) Proc. Nat1Acad. Sci. USA 84,6677-6681. 5. Minami, M., Ohno, S., Kawasaki, H., Rtimark, O., Samuelsson,B., Jot-mall, H., Shimizu, T., Seyama,Y. and Suzuki, K. (1987) J. Biol. Chem. 262, 13873-13876. 6. Minami, M., Minami, Y., Emori, Y., Kawasaki, H., Ohno, S., Suzuki, K., Ohishi, N., Shimizu, T. and Seyama, Y. (1988) FEBS letters 229,279-282. 7. Malfroy, B., Kado-Fong, H., Gros, C., Giros, B., Schwartz, J.C. and Hellmiss, R. (1989) Biochem. Biophys. Res. Commun. 161, 236-241. Vallee, B. L. and Auld, D.S. (1990) Biochemistry 29,5647-5659. ;: Haeggstrom,J.Z., Wetterholm, A., Shapiro, R., Vallee, B.L. and Samuelsson,B. (1990) Biochem. Biophys. Res. Commun. 172, 965-970.

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10. Haeggstrom, J.Z., Wetterholm, A., Vallee, B.L. and Samuelsson, B. (1990) Biochem. Biophys. Res. Commun. 173, 431-437. 11. Minami, M., Ohishi, N., Mutoh, H., Izumi, T., Bito, H., Wada, H., Seyama, Y., Toh, H. and Shimizu, T. (1990) Biochem. Biophys. Res. Commun. 173,620-626. 12. Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, 156-159. 13. Medina, J.F., Barrios, C., Funk, C.D., Larsson, 0. Haeggstrom, J. and Radmark, 0. (1990) Eur. J. Biochem. 191,27-31. Gubler, U. and Hoffman, B.J. (1983) Gene 25,263-269. ::: Short, J.M., Fernandez, J.M., Sorge, J.A. and Huse, W.D. (1988) Nucl. Acids Res. 16, 7583-7600. 16. Hanahan, D. (1985) in DNA Cloning (Glover, D.M., ed) Vol. 1, pp. 109-135, IRL Press, Oxford. Holmes, D.S. and Quigley, M. (1981) Anal. Biochem. 114, 193- 197. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A. (1988) Science 239,487-491. Rappolee, D.A., Wang, A., Mark, D. and Werb, Z. (1989) J. Cell. Biochem. 39, l-11. :;: Frohman, M.A., Dush, M.K. and Martin, G.R. (1988) Proc. Natl. Acad. Sci. USA 85, 8998-9002. 21. Gibbs, R.A., Nguyen, P.-N., McBride, L.J., Koepf, S.M. and Caskey, C.T. (1989) Proc. Natl. Acad. Sci. USA 86, 1919-1923. 22. Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. S. (1984) Gene 28, 351-359. 23. He&off, 24. Steinhilber, D., Herrmann, T. and Roth, H.J. (1989) J. Chromatogr. 493, 361-366. 25. Wickens, M. (1990) TZBS 15, 277-281. 26. Kozak, M. (1986) Cell 44,283-292. Vallee, B.L. and Auld, D.S. (1990). Proc. Natl. Acad. Sci. USA 87, 220-224. Z: Chou, P.Y. and Fasman, G.D. (1974) Biochemistry 13,222-245.

1524

Molecular cloning and expression of mouse leukotriene A4 hydrolase cDNA.

A cDNA clone for mouse leukotriene A4 hydrolase encoding the full sequence of the enzyme was isolated from a mouse spleen lambda ZAP-II library. The i...
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