[go: up one dir, main page]

US20030040093A1 - Platelet activating factor acetylhydrolase, and gene thereof - Google Patents

Platelet activating factor acetylhydrolase, and gene thereof Download PDF

Info

Publication number
US20030040093A1
US20030040093A1 US09/961,253 US96125301A US2003040093A1 US 20030040093 A1 US20030040093 A1 US 20030040093A1 US 96125301 A US96125301 A US 96125301A US 2003040093 A1 US2003040093 A1 US 2003040093A1
Authority
US
United States
Prior art keywords
leu
gly
val
glu
phe
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US09/961,253
Inventor
Hideki Adachi
Masafumi Tsujimoto
Hiroyuki Arai
Keizo Inoue
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Leica Mikrosysteme GmbH
Asubio Pharma Co Ltd
Original Assignee
Suntory Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Assigned to LEICA MIKROSYSTEME AKTIENGESELLSCHAFT reassignment LEICA MIKROSYSTEME AKTIENGESELLSCHAFT CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: LEICA AG
Application filed by Suntory Ltd filed Critical Suntory Ltd
Priority to US09/961,253 priority Critical patent/US20030040093A1/en
Publication of US20030040093A1 publication Critical patent/US20030040093A1/en
Assigned to DAIICHI SUNTORY PHARMA CO., LTD. reassignment DAIICHI SUNTORY PHARMA CO., LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SUNTORY LIMITED
Abandoned legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)

Definitions

  • This invention relates to a novel platelet activating factor acetylhydrolase, and a gene encoding the same.
  • a platelet activating factor acetylhydrolase is an enzyme, which acts on a platelet activating factor (hereinafter abbreviated as “PAF”) and eliminates its 2-acetyl group to deprive PAF of its activity.
  • PAF platelet activating factor
  • PAF acetylhydrolase is usable as a preventive or therapeutic for various diseases caused by PAF.
  • the present invention has as a primary object the provision of PAF acetylhydrolase which can fulfill the above-described desires.
  • the present inventors chose the bovine liver as a source, and by various isolation and purification procedures, progressively increased the purity of PAF acetylhydrolase while placing a focus on its enzymatic activity. As a result, the present inventors have succeeded in obtaining bovine PAF acetylhydrolase as a pure product and further in determining its amino acid sequence. In addition, from the amino acid sequence of the PAF acetylhydrolase, a gene encoding the enzyme has been found by methods known per se in the art.
  • the present invention has been completed based on these findings, and provides a human PAF acetylhydrolase, which plays an important role as a PAF-inhibiting substance, and also a gene which encodes the enzyme and is important for the synthesis of the enzyme by genetic engineering.
  • the human PAF acetylhydrolase according to the present invention selectively degrades PAF and hence, is usable as medicines or biochemical reagents for the prevention and treatment of diseases caused by PAF, for example, diseases such as asthma, exudative tympanitis, hemorrhagic colitis and adult respiratory distress syndrome.
  • the human PAF acetylhydrolase according to the present invention can be prepared as will be described next.
  • PAF acetylhydrolase is first collected from an animal. From the PAF acetylhydrolase, the animal PAF acetylhydrolase cDNA is determined. Using the animal PAF acetylhydrolase cDNA, the human PAF acetylhydrolase cDNA is detected from a human gene library. The human PAF acetylhydrolase cDNA is inserted in an appropriate vector and then cultured in an adequate host organism, whereby the human PAF acetylhydrolase is obtained.
  • bovine liver to be used as a source one obtained from a bovine immediately after its slaughter is preferred.
  • bovine liver is first washed with an appropriate buffer (for example, 10 mM Tris-HCl buffer containing 250 mM sucrose and 1 mM EDTA and having a pH of 7.4), it is homogenized with the same buffer. The homogenate is then centrifuged to obtain a soluble fraction.
  • an appropriate buffer for example, 10 mM Tris-HCl buffer containing 250 mM sucrose and 1 mM EDTA and having a pH of 7.4
  • PAF acetylhydrolase activity which is used as an index for the selective collection of the PAF-acetylhydrolase-containing fraction can be determined, for example, by the method disclosed in Japanese Patent Application Laid-Open (Kokai) No. HEI 7-39373.
  • bovine PAF acetylhydrolase cDNA GTCGACCCACGCGTCCGAGTTGACCGTCTGGGCTGTTTCTGAGGGTCAA CGTGACTCGCCGTCAAGTTCAGCCACTGCCCAAGTCGTCGTTCAGTTCA GTTGGTTATGAG ATG GGG GTC AAC CAG TCT GTG AGC TTC CCA CCC GTC ACG GGA CCC CAC CTC GTA GGC TGT GGG GAT GTG ATG GAG GGT CAG AGC CTC CAG GGC AGC TTC TTT CGA CTG TTC TAC CCG TGC CAA GAG GCA GAG GAG ACC TCG GAG CAG CCC CTG TGG ATT C
  • the human gene library is screened by a method known per se in the art while using the bovine PAF acetylhydrolase cDNA as a template.
  • the bovine PAF acetylhydrolase cDNA is labeled, for example, by incorporating fluorescein-12-dUTP through PCR.
  • ECL Enhanced Chemiluminescence; Amersham K. K.
  • the human PAF acetylhydrolase cDNA obtained as described above is next introduced in an appropriate vector plasmid, and host cells such as mammal cells are then transformed by a commonly-employed recombinant DNA technique to express the human PAF acetylhydrolase.
  • the expression of the human PAF acetylhydrolase can be confirmed by a western blot technique which makes use of an anti-human PAF acetylhydrolase antibody.
  • the introduction into the plasmid, the establishment of the transformed strain, the culture of the strain and the like can be conducted by the general recombinant DNA technology.
  • a suitable expression system can be selected for use in the present invention. It is possible to improve the efficiency of secretion and the level of expression by adding or improving a signal sequence and/or choosing an appropriate host.
  • host cells illustrative examples include cultured cells of bacteria, yeasts, other fungi, human and other animals, and cultured cells of plants.
  • the polynucleotide according to the present invention is inserted in a suitable expression vector, for example, pUC-PL-cl vector, the expression vector is introduced in adequate host cells, for example, E. Coli W3110 or the like, and the host cells are then cultured.
  • the target human PAF acetylhydrolase can thereafter be collected as a protein from the thus-obtained cultured matter (cells or culture medium).
  • a procaryote or an eucaryote can be used as the host.
  • the procaryote include bacteria, especially Escherichia Coli and Bacillus bacteria, for example, B. subtilis .
  • the eucaryote include eucaryotic micro-organisms such as yeasts, for example, Saccharomyces yeasts, especially S. Servisiae ; insect cells such as armyworm ( Spodoptera Frugiperda ) cells and silkworm ( Bombyx mori ) cells; and animal cells such as human cells, monkey cells and mouse cells, especially monkey cells, for example, COS1 and COS 7.
  • the expression vector examples include plasmids, pharges, phargemids, viruses [baculoviruses (for insect cells), vaccinia viruses (for animal cells)].
  • the promoter in the expression vector is selected depending on the host cells. For examples, lac promoters, trp promoters, trc promoters and the like can be used as promoters for bacteria; and adh 1 promoters, pgk promoters and the like can be used as promoters for yeasts.
  • baculovirus polyhedrin promoters can be mentioned as promoters for insects; and early and late promoters of Simian virus 40 (SV40) can be mentioned as promoters for animal cells.
  • SV40 Simian virus 40
  • the enhancer of SV40 is inserted either upstream or downstream of the gene.
  • the transformation of the host by the expression vector can be conducted by a common method known per se in the art. Such methods are disclosed, for example, in “Current Protocols in Molecular Biology”, John Wiley & Sons, Inc.
  • the culture of the transformants can also be conducted by a usual method.
  • the purification of the human PAF acetylhydrolase from the cultured matter can be conducted following procedures commonly employed for the isolation and purification of proteins, for example, by ultrafiltration and/or one or more of various column chromatographic procedures, for example, chromatography making use of “Sepharose”.
  • the human PAF acetylhydrolase can be advantageously obtained.
  • the human PAF acetylhydrolase according to the present invention is represented by the following formula (I): Met Gly Val Asn Gln Ser Val Gly Phe Pro Pro Val Thr Gly Pro His Leu Val Gly Cys Gly Asp Val Met Gln Gly Gln Asn Leu Gln Gly Ser Phe Phe Arg Leu Phe Tyr Pro Cys Gln Lys Ala Glu Glu Thr Met Glu Gln Pro Leu Trp Ile Pro Arg Tyr Gln Tyr Cys Thr Gly Leu Ala Gln Tyr Leu Gln Phe Asn Lys Arg Cys Gly Gly Leu Leu Phe Asn Leu Ala Val Gly Ser Cys Arg Len Pro Val Ser Trp Asn Gly Pro Phe Lys Thr Lys Asp Ser Gly Tyr Pro Leu Ile Ile Phe Ser Gly Phe Lys Thr Lys Asp Ser Gly
  • the human PAF acetylhydrolase selectively degrades PAF and oxidized phospholipids and has physiologically active effects such anti-inflammatory effects.
  • the human PAF acetylhydrolase according to the present invention is not limited to the peptide of the formula (I) but includes peptides having homology therewith, namely, peptides having the same function as the peptide represented by the formula (I) despite substitution, deletion, addition or the like of amino acids at parts of their sequences.
  • the bovine PAF acetylhydrolase represented by the formula (III) may be contemplated to be available by gene manipulation in a similar manner as the human PAF acetylhydrolase. As a matter of fact, however, the bovine PAF acetylhydrolase cannot be obtained unless eucaryotic host cells are used.
  • bovine PAF acetylhydrolase by gene manipulation, it is therefore necessary to employ as host cells those derived from an eucaryote and to select and use a vector compatible with the host cells.
  • An antibody against the human PAF acetylhydrolase or bovine PAF acetylhydrolase (which may hereinafter be collectively called the “PAF acetylhydrolase”) according to the present invention can also be obtained following usual procedures.
  • the antibody can be obtained by sensitizing an animal such as a rabbit with the PAF acetylhydrolase, separating its serum and, if necessary, purifying an immunoglobulin fraction from the serum.
  • an animal such as a rabbit with the PAF acetylhydrolase
  • the enzyme in a form bound on a carrier protein such as bovine serum albumin (BSA) or methyl BSA may be used as,an immunogen.
  • BSA bovine serum albumin
  • methyl BSA may be used as,an immunogen.
  • the enzyme can also be used together with Freund's complete adjuvant (FCA) or Freund's incomplete adjuvant (FICA) to increase the production of the antibody. It is desired to conduct the sensitization of the animal twice or more. The frequency of sensitization can be determined while checking the antibody titer of the serum by test sampling of blood. The whole blood of an immune animal may be used by slaughtering it as needed. As an alternative, an immune animal may be subjected to booster sensitization as many times as needed to maintain a constant antibody titer, and blood samples may be collected in small quantities as needed for immediate use. It is also possible to obtain a monoclonal antibody in a usual manner by sensitizing a mouse with the enzyme and then forming hybridomas from spleen cells and myeloma cells of the sensitized mouse.
  • FCA Freund's complete adjuvant
  • FICA Freund's incomplete adjuvant
  • 3 H-acetyl PAF 1-O-hexadecyl-2-[ 3 H-acetyl]-sn-glycero-3-phosphocholine
  • a standard culture system for the measurement of PAF acetylhydrolase was composed of 50 mM Tris-HCl (pH 7.4), 5 mM EDTA, 5 mM 2-mercaptoethanol (2-ME) and 20 nmol 3 H-acetyl PAF. The total volume of the sample was 0.25 ml.
  • a fresh bovine liver was purchased from a slaughterhouse and was then treated within 3 hours of the slaughter. Treatments were all conducted at 0 to 4° C.
  • the liver was homogenized in a Waring blender subsequent to the addition of a homogenizing buffer [10 mM Tris-HCl (pH 7.4), 250 mM sucrose, 1 mM EDTA] in an amount 5 times as much as the liver.
  • the resulting homogenate was centrifuged for 30 minutes under 100,000 ⁇ g, followed by the removal of a solid portion.
  • the resultant supernatant was centrifuged further for 1 hour under 100,000 ⁇ g, whereby a dissolved portion was obtained (supernatant portion)
  • a plasmid DNA was prepared and the base sequence was determined.
  • the clone was a full-length clone which contained ATG encoding initiating methionine.
  • Encoding 43 N-terminal amino acids were the same as the corresponding amino acids in the sequence of the bovine PAF acetylhydrolase up to the 40th amino acid, and there was poly A at the 3′ end.
  • a more accurate determination of the base sequence was conducted.
  • the cDNA was found to consist of 2188 bp and to contain an ORF (open reading frame) consisting of 253 amino acids.
  • 140 amino acids had been deleted.
  • the segment of the deleted 140 amino acids contains a “catalytic triad” of serine, histidine and aspartic acid, which exhibits catalytic activity.
  • the cDNA is therefore not believed to have PAD acetylhydrolase activity.
  • a primer was synthesized at positions flanking the deleted region, and PCR was conducted using the library DNA as a template. From the human brain cDNA, two bands were obtained, one corresponding to the above-described cDNA with the 140 amino acids deleted, and the other to a cDNA having substantially the same length as the bovine PAF acetylhydrolase cDNA. From the foregoing, the human brain library DNA was expected to contain, in addition to the above-obtained cDNA, a human PAF acetylhydrolase cDNA which is actually equipped with PAF acetylhydrolase activity.
  • the human brain cDNA library was diluted to give 2000 clones per well, followed by incubation on 5 96-well plates. Subpools consisting of 10 wells were prepared, and positive pools were determined by PCR (Pool Nos. 10, 20, 28, and 38). With respect to these subpools, PCR was conducted well after well, so that positive pools were confirmed (Pool Nos. 10-5, 20-10, and 38-12).
  • the amino acid sequence of the human PAF acetylhydrolase was determined. It was found to be represented by the formula (I). Up to 88%, the sequence was the same as that of the bovine PAF acetylhydrolase (346/392 amino acids). On the other hand, it was 42% identical to that of the plasma human PAF acetylhydrolase (162/392 amino acids).
  • the above cDNA was incorporated in the pUC-Pl-cl vector, introduced in E. coli W3110 and then subjected to expression.
  • the protein was investigated for activity. Human PAF acetylhydrolase activity was confirmed.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Saccharide Compounds (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

Abstract

A protein having activities of a human platelet activating factor acetylhydrolase, and represented by an amino acid sequence represented by the following formula (I) or an amino acid sequence having homology therewith; and a DNA encoding the protein: Met Gly Val Asn Gln Ser Val Gly Phe Pro Pro Val Thr Gly Pro His Leu Val Gly Cys Gly Asp Val Met Glu Gly Gln Asn Leu Gln Gly Ser Phe Phe Arg Leu Phe Tyr Pro Cys Gln Lys Ala Glu Glu Thr Met Glu Gln Pro Leu Trp Ile Pro Arg Tyr Glu Tyr Cys Thr Gly Leu Ala Glu Tyr Leu Gln Phe Asn Lys Arg Cys Gly Gly Leu Leu Phe Asn Leu Ala Val Gly Ser Cys Arg Leu Pro Val Ser Trp Asn Gly Pro Phe Lys Thr Lys Asp Ser Gly Tyr Pro Leu Ile Ile Phe Ser His Gly Leu Gly Ala Phe Arg Thr Leu Tyr Ser Ala Phe Cys Met Glu Leu Ala Ser Arg Gly Phe Val Val Ala Val Pro Glu His Arg Asp Arg Ser Ala Ala Thr Thr Tyr Phe Cys Lys Gln Ala Pro Glu Glu Asn Gln Pro Thr Asn Glu Ser Leu Gln Glu Glu Trp Ile Pro Phe Arg Arg Val Glu Glu Gly Glu Lys Glu Phe His Val Arg Asn Pro Gln Val His Gln Arg Val Ser Glu Cys Leu Arg Val Leu Lys Ile Leu Gln Glu Val Thr Ala Gly Gln Thr Val Phe Asn Ile Leu Pro Gly Gly Leu Asp Leu Met Thr Leu Lys Gly Asn Ile Asp Met Ser Arg Val Ala Val Met Gly His Ser Phe Gly Gly Ala Thr Ala Ile Leu Ala Leu Ala Lys Glu Thr Gln Phe Arg Cys Ala Val Ala Leu Asp Ala Trp Met Phe Pro Leu Glu Arg Asp Phe Tyr Pro Lys Ala Arg Gly Pro Val Phe Phe Ile Asn Thr Glu Lys Phe Gln Thr Met Glu Ser Val Asn Leu Met Lys Lys Ile Cys Ala Gln His Glu Gln Ser Arg Ile Ile Thr Val Leu Gly Ser Val His Arg Ser Gln Thr Asp Phe Ala Phe Val Thr Gly Asn Leu Ile Gly Lys Phe Phe Ser Thr Glu Thr Arg Gly Ser Leu Asp Pro Tyr Glu Gly Gln Glu Val Met Val Arg Ala Met Leu Ala Phe Leu Gln Lys His Leu Asp Leu Lys Glu Asp Tyr Asn Gln Trp Asn Asn Leu Ile Glu Gly Ile Gly Pro Ser Leu Thr Pro Gly Ala Pro His His Leu Ser Ser Leu

Description

    BACKGROUND OF THE INVENTION
  • 1. Field of the Invention [0001]
  • This invention relates to a novel platelet activating factor acetylhydrolase, and a gene encoding the same. [0002]
  • 2. Description of the Related Art [0003]
  • A platelet activating factor acetylhydrolase is an enzyme, which acts on a platelet activating factor (hereinafter abbreviated as “PAF”) and eliminates its 2-acetyl group to deprive PAF of its activity. Since PAF is a mediator for inflammation which causes defluxion of tissue fluid through finer vessels, vasodilation, smooth muscle contraction, endothelial adhesion, activation of neutrophils, macrophages or eosinophilic leukocytes, or the like, PAF acetylhydrolase is usable as a preventive or therapeutic for various diseases caused by PAF. [0004]
  • Some reports have been made about PAF acetylhydrolase to date. For its use as a medicine, however, there is an outstanding desire for the provision of a PAF acetylhydrolase having higher purity and stronger action compared with conventional PAF acetylhydrolase. Further, from the viewpoint of safety, PAF acetylhydrolase derived form human being instead of an animal is desired. [0005]
    • SUMMARY OF THE INVENTION
  • With the foregoing in view, the present invention has as a primary object the provision of PAF acetylhydrolase which can fulfill the above-described desires. [0006]
  • Interested in the wide-spread distribution of PAF acetylhydrolase in animal organs such as the brain and kidneys, the present inventors chose the bovine liver as a source, and by various isolation and purification procedures, progressively increased the purity of PAF acetylhydrolase while placing a focus on its enzymatic activity. As a result, the present inventors have succeeded in obtaining bovine PAF acetylhydrolase as a pure product and further in determining its amino acid sequence. In addition, from the amino acid sequence of the PAF acetylhydrolase, a gene encoding the enzyme has been found by methods known per se in the art. [0007]
  • Moreover, using the bovine PAF acetylhydrolase cDNA, the present inventors have also succeeded in identifying the human PAF acetylhydrolase cDNA. [0008]
  • The present invention has been completed based on these findings, and provides a human PAF acetylhydrolase, which plays an important role as a PAF-inhibiting substance, and also a gene which encodes the enzyme and is important for the synthesis of the enzyme by genetic engineering. [0009]
  • The human PAF acetylhydrolase according to the present invention selectively degrades PAF and hence, is usable as medicines or biochemical reagents for the prevention and treatment of diseases caused by PAF, for example, diseases such as asthma, exudative tympanitis, hemorrhagic colitis and adult respiratory distress syndrome.[0010]
    • DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
  • The human PAF acetylhydrolase according to the present invention can be prepared as will be described next. PAF acetylhydrolase is first collected from an animal. From the PAF acetylhydrolase, the animal PAF acetylhydrolase cDNA is determined. Using the animal PAF acetylhydrolase cDNA, the human PAF acetylhydrolase cDNA is detected from a human gene library. The human PAF acetylhydrolase cDNA is inserted in an appropriate vector and then cultured in an adequate host organism, whereby the human PAF acetylhydrolase is obtained. [0011]
  • Upon practice of the present invention, it is first necessary to obtain animal PAF acetylhydrolase from an organ of an animal such as the brain, liver or kidneys by purifying it through repetitions of known isolation and purification procedures while using PAF acetylhydrolase activity as an index. A description will hereinafter be made of a process for obtaining PAF acetylhydrolase by using a bovine liver as an example. [0012]
  • As the bovine liver to be used as a source, one obtained from a bovine immediately after its slaughter is preferred. [0013]
  • After the bovine liver is first washed with an appropriate buffer (for example, 10 mM Tris-HCl buffer containing 250 mM sucrose and 1 mM EDTA and having a pH of 7.4), it is homogenized with the same buffer. The homogenate is then centrifuged to obtain a soluble fraction. [0014]
  • Making combined use-of hydrophobic chromatography, ion exchange chromatography, adsorption chromatography, gel filtration chromatography and the like, the soluble fraction is purified until a single peak is observed by Mono Q FPLC, so that PAF acetyl hydrolase can be obtained. [0015]
  • Incidentally, PAF acetylhydrolase activity which is used as an index for the selective collection of the PAF-acetylhydrolase-containing fraction can be determined, for example, by the method disclosed in Japanese Patent Application Laid-Open (Kokai) No. HEI 7-39373. [0016]
  • With respect to the bovine PAF acetylhydrolase obtained in the above-described manner, its amino acid sequence was investigated by a method known per se in the art. As a result, the amino acid sequence has been found to be represented by the following formula (III): [0017]
    Met Gly Val Asn Gln Ser Val Ser Phe Pro Pro Val
    Thr Gly Pro His Leu Val Gly Cys Gly Asp Val Met
    Glu Gly Gln Ser Leu Gln Gly Ser Phe Phe Arg Leu
    Phe Tyr Pro Cys Gln Glu Ala Glu Glu Thr Ser Glu
    Gln Pro Leu Trp Ile Pro Arg Tyr Glu Tyr Cys Ala
    Gly Leu Ala Glu Tyr Leu Lys Phe Asn Lys Arg Trp
    Gly Gly Leu Leu Phe Asn Leu Gly Val Gly Ser Cys
    Arg Leu Pro Val Ser Trp Asn Gly Pro Phe Lys Thr
    Lys Asp Ser Gly Tyr Pro Leu Ile Ile Phe Ser His
    Gly Met Gly Ala Phe Arg Thr Val Tyr Ser Ala Phe
    Cys Met Glu Leu Ala Ser Arg Gly Phe Val Val Ala
    Val Pro Glu His Arg Asp Gly Ser Ala Ala Ala Thr
    Cys Phe Cys Lys Gln Thr Pro Glu Glu Asn Gln Pro
    Asp Asn Glu Ala Leu Lys Glu Glu Trp Ile Pro His
    Arg Gln Ile Gln Glu Gly Glu Lys Gln Phe Tyr Val
    Arg Asn Tyr Gln Val His Gln Arg Val Ser Gln Cys
    Val Arg Val Leu Lys Ile Leu Gln Gln Val Thr Ala
    Gly Gln Ala Val Leu Asn Ile Leu Pro Gly Gly Leu
    Asp Leu Met Thr Leu Lys Gly Gly Ile Asp Val Ser
    Arg Val Ala Val Met Gly His Ser Phe Gly Gly Ala
    Thr Ala Ile Leu Ala Leu Ala Lys Gln Met Gln Phe
    Arg Cys Ala Val Ala Leu Asp Ala Trp Met Phe Pro
    Leu Glu His Asp Phe Tyr Pro Thr Ala Arg Gly Pro
    Ile Phe Phe Ile Asn Ala Glu Lys Phe Gln Thr Val
    Glu Thr Val Asn Leu Met Lys Lys Ile Cys Asp Gln
    His His GIn Ser Arg Ile Ile Thr Val Leu Gly Ser
    Val His Arg Ser Leu Thr Asp Phe Val Phe Val Ala
    Gly Asn Trp Ile Ser Lys The Phe Ser Ser His Thr
    Arg Gly Ser Leu Asp Pro Tyr Glu Gly Gln Glu Thr
    Val Val Arg Ala Met Leu Ala Phe Leu Gln Lys His
    Leu Asp Leu Lys Glu Asp Tyr Asp Gln Trp Asn Asn
    Phe Ile Glu Gly Ile Gly Pro Ser Leu Thr Pro Gly
    Ala Pro His His Leu Ser Ser Leu
  • Further, from the peptide sequence of the bovine PAF acetylhydrolase of the formula (III), a gene encoding the enzyme was determined by a method known per se in the art. The gene (hereinafter called the “bovine PAF acetylhydrolase cDNA”) has been found to be identified by the following formula (IV): [0018]
    GTCGACCCACGCGTCCGAGTTGACCGTCTGGGCTGTTTCTGAGGGTCAA
    CGTGACTCGCCGTCAAGTTCAGCCACTGCCCAAGTCGTCGTTCAGTTCA
    GTTGGTTATGAG ATG GGG GTC AAC CAG TCT GTG AGC TTC
    CCA CCC GTC ACG GGA CCC CAC CTC GTA GGC TGT GGG
    GAT GTG ATG GAG GGT CAG AGC CTC CAG GGC AGC TTC
    TTT CGA CTG TTC TAC CCG TGC CAA GAG GCA GAG GAG
    ACC TCG GAG CAG CCC CTG TGG ATT CCC CGC TAT GAG
    TAC TGC GCT GGC CTG GCC GAA TAC CTA AAG TTT AAT
    AAG CGC TGG GGG GGG TTA CTG TTC AAC CTG GGT GTG
    GGA TCT TGT CGC CTG CCT GTT AGC TGG AAT GGC CCC
    TTT AAA ACA AAG GAC TCT GGA TAC CCC TTG ATC ATC
    TTC TCT CAT GGC ATG GGA GCC TTG AGG ACA GTG TAT
    TCA GCC TTC TGC ATG GAG CTG GCT TCT CGT GGC TTT
    GTG GTT GCT GTA CCA GAG CAC AGG GAT GGG TCA GCT
    GCG GCC ACC TGT TTC TGC AAG CAG ACC CCA GAG GAG
    AAC CAG CCT GAC AAT GAG GCC CTG AAG GAG GAA TGG
    ATC CCC CAC CGT CAA ATT GAG GAA GGG GAG AAG GAA
    TTC TAT GTT CGG AAC TAC CAG GTG CAT CAG AGG GTG
    AGC GAG TGT GTG AGG GTG TTG AAG ATC CTA CAA GAG
    GTC ACT GCT GGG CAG GCC GTT CTC AAC ATC TTG CCT
    GGC GGA TTG GAT CTG ATG ACC TTG AAG GGC GGC ATT
    GAC GTG AGC CGT GTG GCT GTA ATG GGA CAT TCA TTT
    GGA GGG GCC ACA GCT ATT CTG GCC TTG GCC AAG GAG
    ATG CAA TTT AGG TGT GCT GTG GCT TTG GAC GCT TGG
    ATG TTT CCT CTG GAG CAT GAC TTT TAC CCC ACG GCC
    CGA GGC CCT ATC TTC TTT ATC AAT GCT GAG AAG TTC
    CAG ACA GTG GAG ACT GTC AAC TTG ATG AAA AAG ATT
    TGT GAC CAG CAC CAC CAA TCC AGG ATC ATA ACT GTC
    CTT GGT TCT GTT CAT CGG AGT CTA ACC GAC TTT GTT
    TTT GTG GCT GGT AAC TGG ATT AGT AAA TTC TTC TCC
    AGT CAC ACC CGT GGA AGC TTG GAC CCC TAT GAA GGT
    CAG GAG ACC GTG GTG CGG GCC ATG TTG GCC TTC CTG
    CAG AAG CAT CTT GAC CTG AAA GAG GAC TAT GAC CAG
    TGG AAC AAC TTC ATT GAA GGC ATT GGC CCA TCA CTG
    ACC CCA GGG GCC CCA CAC CAT CTG TCC AGC CTG TAG
    GCACAACTGGTCATCTTGTGGAAGGTCCCTGAGCTGAGTTCCCGTGTGGG
    GCCTGCCCAGGGATACCCTTGGCCTCCTATCAGGAAGTGATTGCCATGAC
    CCTTCTGTGTTGATTGAGAGGATATAATCACACTGCTGATTGGTAACGGG
    GTACTTGGATTCTCAGACTTGTCGATCTTAAACTCATGTTGGGACTTGGG
    TTCACTTACTGATGGGCAAACGGGCATTCTGAGGACTGAGCCTTAATGGT
    ATGGAGAACAAACAGTGGGATGGGGCTGGGGAAGATCTAAGCCCTAAGCT
    GGGCACTATGAGCCCTATAAACCCAACCAGCCAACACCCTCACCTTGGGC
    AAGTATGACTTCTGCAGGTCGACTCT
  • To obtain human PAF acetylhydrolase from the bovine PAF acetylhydrolase cDNA obtained as described above, the human gene library is screened by a method known per se in the art while using the bovine PAF acetylhydrolase cDNA as a template. [0019]
  • Described specifically, the bovine PAF acetylhydrolase cDNA is labeled, for example, by incorporating fluorescein-12-dUTP through PCR. By the colony hybridization technique that selects each positive colony by ECL (Enhanced Chemiluminescence; Amersham K. K.), colonies containing the human PAF acetylhydrolase cDNA can be obtained. [0020]
  • The human PAF acetylhydrolase cDNA obtained as described above has been found to be identified by the following formula (II): [0021]
    GCAGGTCTCGACCCACGCGTCCGCGGACGCGTGGGCGAGAAGTGCTTCCA
    AGCGTCCATTTTGAGCCTTGGAAACTACGACGACCAAAGGGCCACGGGTT
    CCTGGGTCGTTTCTCATTTCCGTCGAGTTAAACGTCTGGGGCTGCTTCTG
    AGGAATCAGCTTGGCTGGCCAGCAAGTTCAGCTCCGGCAAGTCATTTGAT
    TCACCCGGTGATGAA ATG GGG GTC AAC CAG TCT GTG GGC
    TTT CCA CCT GTC ACA GGA CCC CAC CTC GTA GGC TGT
    GGG GAT GTG ATG GAG GGT CAG AAT CTC CAG GGG AGC
    TTC TTT CGA CTC TTC TAC CCC TGC CAA AAG GCA GAG
    GAG ACC ATG GAG CAG CCC CTG TGG ATT CCC CGC TAT
    GAG TAC TGC ACT GGC CTG GCC GAG TAC CTG CAG TTT
    AAT AAG CGC TGC GGG GGC TTG AAT GGC CCC TTT AAG
    ACA AAG GAC TCT GGA TAC CCC TTG ATC ATC TTC TCC
    CAT GGC CTA GGA GCC TTC AGG ACT TTG TAT TCA GCC
    TTC TGC ATG GAG CTG GCC TCA CGT GGC TTT GTG GTT
    GCT GTG CCA GAG CAC AGG GAC CGG TCA GCG GCA ACC
    ACC TAT TTC TGC AAG CAG GCC CCA GAA GAG AAC CAG
    CCC ACC AAT GAA TCG CTG CAG GAG GAA TGG ATC CCT
    TTC CGT CGA GTT GAG GAA GGG GAG AAG GAA TTT CAT
    GTT CGG AAT CCC CAG GTG CAT CAG CGG GTA AGC GAG
    TGT TTA CGG GTG TTG AAG ATC CTG CAA GAG GTC ACT
    GCT GGG CAG ACT GTC TTC AAC ATC TTG CCT GGT GGC
    TTG GAT CTG ATG ACT TTG AAG GGC AAC ATT GAC ATG
    AGC CGT GTG GCT GTG ATG GGA CAT TCA TTT GGA GGG
    GCC ACA GCT ATT CTG GCT TTG GCC AAG GAG ACC CAA
    TTT CGG TGT GCG GTG GCT CTG GAT GCT TGG ATG TTT
    CCT CTG GAA CGT GAC TTT TAC CCC AAG GCC CGA GGA
    CCT GTG TTC TTT ATC AAT ACT GAG AAA TTC CAG ACA
    ATG GAG AGT GTC AAT TTG ATG AAG AAG ATA TGT GCC
    CAG CAT GAA CAG TCT AGG ATC ATA ACC GTT CTT GGT
    TCT GTT CAT CGG AGT CAA ACT GAC TTT GCT TTT GTG
    ACT GGC AAC TTG ATT GGT AAA TTC TTC TCC ACT GAA
    ACC CGT GGG AGC CTG GAC CCC TAT GAA GGG CAG GAG
    GTT ATG GTA CGG GCC ATG TTG GCC TTC CTG CAG AAG
    CAC CTC GAC CTG AAA GAA GAC TAT AAT CAA TGG AAC
    AAC CTT ATT GAA GGC ATT GGA CCG TCG CTC ACC CCA
    GGG GCC CCC CAC CAT CTG TCC AGC CTG TAG GCACAACTGG
    CCATTTGTAAAGTCACTTCAGCCAAGTTTTCATTTGGGAGCTACCCAAGG
    GCACCCATGAGCTCCTATCAAGAAGTGATCAACGTGACCCCTTTTCACAG
    ATTGAAAGGTGTAATCACACTGCTGCTTGGATAACTGGGTACTTTGATCT
    TAGATTTGATCTTAAAATCACTTTGGGACTGGGATCCCTTGCTGATTGAC
    AAACAGACTTTCTGGGACCTTGATGGAGTGGGGAACAAGCAGTAGAGTGG
    GACTGGGGGAGACCCAGGCCCCGGGCTGAGCACTGTGAGGCCTGGATGTG
    AAGACTCAGCCCAGCGAAGCTCATTCCCTTACCCCCGGCCAGTGCTGCTG
    CTTCAGTGGAAGAGATGAAGCCAAAGGACAGAATGAAAATCCCTACCTTC
    AGAGACTCTAGCCCAGCCCAACACCATCTCTTCCTACCTCTCAGCCTTCT
    CCCTCCCCAGGGCCACTTGTTGAAGTCTGAGCACTTTATGTAAATTTCTA
    GGTGTGAGCCGTGATCACATTTTCTATTTATTTCCAAGTCTTCTCATTGT
    ATGGAACATAGTACTACTTATACTTACAGTAGTAAGTTATACTTGTGAGC
    CCACAGAGTGGCAGACAGCATGGCTCTCACAGCACAGGGAGAAAAACTGA
    GGTACACAGAGGTACCTCAGAAGCTCTGGATGTCTTTGGGGGTTTTGCTA
    AGTGTATCTTGATAGGAAACAACAAAAGCACGTTGAGATGGGGAAGATGA
    CAGAACAACAGTGTTAAATGGCCATTTGCACAGGCCTTTGCCACAACAGA
    GAAGTAGTTTGGTCAGCTAAAACTCAGCTGCAGCCTGGACAGTAGAGCGA
    GACCCCATCTTAAAAATAAAGAAGGCTGGGCGTGGTGGCTCATGCCTGTA
    ATCCCAGCACTTTGGGAGGCCAAGGCAGGCAGATCACTTAAGGCCAGGAG
    TTCAAGACCACCTGGCCAACATGGTGAAACCCCGTCTCTACTAAAAATAC
    AAAAAATTAGCCTGGCGTAATGGCAGGCGCCTATAATCCCAGCTACTCAG
    GAGGCTGAAGCAGAAGAATCACTTGAACCTAGGAGGCGGAGGTTGCAGTG
    AGTCAAGATCGCGCCACTGCACTCCAGCCTGGGTGACAGAGCAAGACTCT
    GTCTT
  • Following conventional procedures, the human PAF acetylhydrolase cDNA obtained as described above is next introduced in an appropriate vector plasmid, and host cells such as mammal cells are then transformed by a commonly-employed recombinant DNA technique to express the human PAF acetylhydrolase. The expression of the human PAF acetylhydrolase can be confirmed by a western blot technique which makes use of an anti-human PAF acetylhydrolase antibody. The introduction into the plasmid, the establishment of the transformed strain, the culture of the strain and the like can be conducted by the general recombinant DNA technology. [0022]
  • From expression systems known to artisans, a suitable expression system can be selected for use in the present invention. It is possible to improve the efficiency of secretion and the level of expression by adding or improving a signal sequence and/or choosing an appropriate host. Although no particular limitation is imposed on host cells, illustrative examples include cultured cells of bacteria, yeasts, other fungi, human and other animals, and cultured cells of plants. Namely, the polynucleotide according to the present invention is inserted in a suitable expression vector, for example, pUC-PL-cl vector, the expression vector is introduced in adequate host cells, for example, [0023] E. Coli W3110 or the like, and the host cells are then cultured. The target human PAF acetylhydrolase can thereafter be collected as a protein from the thus-obtained cultured matter (cells or culture medium).
  • As the host, a procaryote or an eucaryote can be used. Usable examples of the procaryote include bacteria, especially [0024] Escherichia Coli and Bacillus bacteria, for example, B. subtilis. On the other hand, usable examples of the eucaryote include eucaryotic micro-organisms such as yeasts, for example, Saccharomyces yeasts, especially S. Servisiae; insect cells such as armyworm (Spodoptera Frugiperda) cells and silkworm (Bombyx mori) cells; and animal cells such as human cells, monkey cells and mouse cells, especially monkey cells, for example, COS1 and COS 7.
  • Usable examples of the expression vector include plasmids, pharges, phargemids, viruses [baculoviruses (for insect cells), vaccinia viruses (for animal cells)]. The promoter in the expression vector is selected depending on the host cells. For examples, lac promoters, trp promoters, trc promoters and the like can be used as promoters for bacteria; and adh 1 promoters, pgk promoters and the like can be used as promoters for yeasts. Further, baculovirus polyhedrin promoters can be mentioned as promoters for insects; and early and late promoters of [0025] Simian virus 40 (SV40) can be mentioned as promoters for animal cells.
  • When an enhancer is used, for example, the enhancer of SV40 is inserted either upstream or downstream of the gene. [0026]
  • The transformation of the host by the expression vector can be conducted by a common method known per se in the art. Such methods are disclosed, for example, in “Current Protocols in Molecular Biology”, John Wiley & Sons, Inc. [0027]
  • The culture of the transformants can also be conducted by a usual method. The purification of the human PAF acetylhydrolase from the cultured matter can be conducted following procedures commonly employed for the isolation and purification of proteins, for example, by ultrafiltration and/or one or more of various column chromatographic procedures, for example, chromatography making use of “Sepharose”. [0028]
  • In the above-described manner, the human PAF acetylhydrolase can be advantageously obtained. The human PAF acetylhydrolase according to the present invention is represented by the following formula (I): [0029]
    Met Gly Val Asn Gln Ser Val Gly Phe Pro Pro Val
    Thr Gly Pro His Leu Val Gly Cys Gly Asp Val Met
    Gln Gly Gln Asn Leu Gln Gly Ser Phe Phe Arg Leu
    Phe Tyr Pro Cys Gln Lys Ala Glu Glu Thr Met Glu
    Gln Pro Leu Trp Ile Pro Arg Tyr Gln Tyr Cys Thr
    Gly Leu Ala Gln Tyr Leu Gln Phe Asn Lys Arg Cys
    Gly Gly Leu Leu Phe Asn Leu Ala Val Gly Ser Cys
    Arg Len Pro Val Ser Trp Asn Gly Pro Phe Lys Thr
    Lys Asp Ser Gly Tyr Pro Leu Ile Ile Phe Ser His
    Gly Leu Gly Ala Phe Arg Thr Leu Tyr Ser Ala Phe
    Cys Met Glu Leu Ala Ser Arg Gly Phe Val Val Ala
    Val Pro Glu His Arg Asp Arg Ser Ala Ala Thr Thr
    Tyr Phe Cys Lys Gln Ala Pro Gln Gln Asn Gln Pro
    Thr Asn Gln Ser Leu Gln Gln Gln Trp Ile Pro Phe
    Arg Arg Val Glu Glu Gly Glu Lys Glu Phe His Val
    Arg Asn Pro Gln Val His Gln Arg Val Ser Glu Cys
    Leu Arg Val Leu Lys Ile Leu Gln Gln Val Thr Ala
    Gly Gln Thr Val Phe Asn Ile Leu Pro Gly Gly Leu
    Asp Leu Met Thr Leu Lys Gly Asn Ile Asp Met Ser
    Arg Val Ala Val Met Gly His Ser Phe Gly Gly Ala
    Thr Ala Ile Leu Ala Leu Ala Lys Glu Thr Gln Phe
    Arg Cys Ala Val Ala Leu Asp Ala Trp Met Phe Pro
    Leu Glu Arg Asp Phe Tyr Pro Lys Ala Arg Gly Pro
    Val Phe Phe Ile Asn Thr Glu Lys Phe Gln Thr Met
    Glu Ser Val Asn Leu Met Lys Lys Ile Cys Ala Gln
    His Glu Gln Ser Arg Ile Ile Thr Val Leu Gly Ser
    Val His Arg Ser Gln Thr Asp Phe Ala Phe Val Thr
    Gly Asn Leu Ile Gly Lys Phe Phe Ser Thr Glu Thr
    Arg Gly Ser Leu Asp Pro Tyr Glu Gly Gln Glu Val
    Met Val Arg Ala Met Leu Ala Phe Leu Gln Lys His
    Leu Asp Leu Lys Glu Asp Tyr Asn Gln Trp Asn Asn
    Leu Ile Glu Gly Ile Gly Pro Ser Leu Thr Pro Gly
    Ala Pro His His Leu Ser Ser Leu
  • The human PAF acetylhydrolase selectively degrades PAF and oxidized phospholipids and has physiologically active effects such anti-inflammatory effects. [0030]
  • Needless to say, the human PAF acetylhydrolase according to the present invention is not limited to the peptide of the formula (I) but includes peptides having homology therewith, namely, peptides having the same function as the peptide represented by the formula (I) despite substitution, deletion, addition or the like of amino acids at parts of their sequences. [0031]
  • The bovine PAF acetylhydrolase represented by the formula (III) may be contemplated to be available by gene manipulation in a similar manner as the human PAF acetylhydrolase. As a matter of fact, however, the bovine PAF acetylhydrolase cannot be obtained unless eucaryotic host cells are used. [0032]
  • To obtain the bovine PAF acetylhydrolase by gene manipulation, it is therefore necessary to employ as host cells those derived from an eucaryote and to select and use a vector compatible with the host cells. [0033]
  • An antibody against the human PAF acetylhydrolase or bovine PAF acetylhydrolase (which may hereinafter be collectively called the “PAF acetylhydrolase”) according to the present invention can also be obtained following usual procedures. [0034]
  • Described specifically, the antibody can be obtained by sensitizing an animal such as a rabbit with the PAF acetylhydrolase, separating its serum and, if necessary, purifying an immunoglobulin fraction from the serum. To enhance the sensitizing ability of the enzyme in this case, the enzyme in a form bound on a carrier protein such as bovine serum albumin (BSA) or methyl BSA may be used as,an immunogen. [0035]
  • Upon sensitizing an animal, the enzyme can also be used together with Freund's complete adjuvant (FCA) or Freund's incomplete adjuvant (FICA) to increase the production of the antibody. It is desired to conduct the sensitization of the animal twice or more. The frequency of sensitization can be determined while checking the antibody titer of the serum by test sampling of blood. The whole blood of an immune animal may be used by slaughtering it as needed. As an alternative, an immune animal may be subjected to booster sensitization as many times as needed to maintain a constant antibody titer, and blood samples may be collected in small quantities as needed for immediate use. It is also possible to obtain a monoclonal antibody in a usual manner by sensitizing a mouse with the enzyme and then forming hybridomas from spleen cells and myeloma cells of the sensitized mouse. [0036]
  • The present invention will hereinafter be described in further detail by the following examples and reference examples. It is however to be noted that the present invention are by no means limited by or to these examples. [0037]
    • Referential Examen 1
  • Measurement of PAF Acetylhydrolase Activity [0038]
  • (1) Using unlabeled lyso PAF (product of Bachem Fein-chemikalien AG), 1-O-[1-[0039] 14C]hexadecyl-lyso PAF (product of New England Nuclear Company; hereinafter called the “labeled lyso PAF”) was diluted to 4,000 dpm/nmol.
  • On the other hand, 1-O-hexadecyl-2-[[0040] 3H-acetyl]-sn-glycero-3-phosphocholine (hereinafter called “3H-acetyl PAF”) was diluted to 3,200 dpm/nmol with the un-labeled lyso PAF.
  • A standard culture system for the measurement of PAF acetylhydrolase was composed of 50 mM Tris-HCl (pH 7.4), 5 mM EDTA, 5 mM 2-mercaptoethanol (2-ME) and 20 nmol [0041] 3H-acetyl PAF. The total volume of the sample was 0.25 ml.
  • (2) Measurement of PAF acetylhydrolase activity was conducted by culturing a test sample in the above-described standard culture system at 37° C. for 30 minutes, adding 2.5 ml of chloroform/methanol (4:1 V/V) and 0.25 ml of water to terminate the reaction, and then measuring the radioactivity of a small amount (0.6 ml) of each upper layer to determine the amount of the acetate liberated from the [0042] 3H-acetyl PAF.
    • EXAMPLE 1
  • Obtainment of Bovine PAF Acetylhydrolase [0043]
  • (1) A fresh bovine liver was purchased from a slaughterhouse and was then treated within 3 hours of the slaughter. Treatments were all conducted at 0 to 4° C. The liver was homogenized in a Waring blender subsequent to the addition of a homogenizing buffer [10 mM Tris-HCl (pH 7.4), 250 mM sucrose, 1 mM EDTA] in an amount 5 times as much as the liver. The resulting homogenate was centrifuged for 30 minutes under 100,000×g, followed by the removal of a solid portion. The resultant supernatant was centrifuged further for 1 hour under 100,000×g, whereby a dissolved portion was obtained (supernatant portion) [0044]
  • (2) The supernatant portion obtained through the procedures (1) was adjusted to 1 M with NaCl. Subsequent to stirring for 15 minutes, the solution was loaded on a “BUTYL TOYOPEARL 650 M” column which had been equilibrated beforehand with a buffer composed of 50 mM Tris-HCl (pH 7.4), 1 mM EDTA and 1 M NaCl. After the column was washed with the same buffer, proteins were eluted with a linear gradient of NaCl (1 to 0 M). PAF acetylhydrolase activity was eluted as a single peak in 1 to 0 M NaCl fractions. [0045]
  • (3) Active fractions from the “BUTYL TOYOPEARL” column were loaded on a “Q-Sepharose” column which had been equilibrated with 10 mM Tris-HCl (pH 7.4), 1 mM EDTA and 20% (V/V) glycerol (buffer A). The column was washed with the buffer A. Proteins were eluted with a linear gradient of NaCl (0 to 500 mM) in the buffer A. The activity was observed in a fraction eluted with about 300 mM NaCl. [0046]
  • (4) The active fraction from the “Q-Sepharose” column was concentrated to about 6 ml in an “Amicon ultrafiltration cell” in which “YM-10” membranes were used. The thus-concentrated fraction was loaded on a “Biogel A-1.5 m” gel filtration column which had been equilibrated beforehand with 10 mM Tris-HCl (pH 7.4), 200 mM NaCl, 5 mM 2-ME, 20% (V/V) glycerol and 0.5% (W/V) “CHAPS” (buffer B). The activity was eluted as a single peak in a fraction corresponding to a molecular weight of about 40 kDa. [0047]
  • (5) The active fraction from the “Biogel-A 1.5 m” column was loaded on a hydroxyapatite column which had been equilibrated beforehand with 10 mM Tris-HCl (pH 7.4), 5 mM 2-ME, 20% (V/V) glycerol and 0.5% (W/V) “CHAPS” (buffer C). Proteins were eluted with a linear gradient which ranged from the buffer C alone to a buffer C containing 150 mM KH[0048] 2PO4. The activity was observed in a fraction which was eluted with about 50 mM KH2PO4.
  • (6) The active fraction from the hydroxyapatite column was dialyzed against the buffer C, and was then loaded on an “FPLC Mono Q HR 5/5” column which had been equilibrated beforehand with the buffer C. Proteins were eluted by a linear gradient of NaCl (0 to 500 mM) in the buffer C. The activity was observed in a fraction which was eluted with 250 mM NaCl, and a protein in the fraction was obtained as purified bovine PAF acetylhydrolase. [0049]
  • The total proteins, total activities, purification degrees (in terms of times) and the like in the individual purification steps described above are tabulated below: [0050]
    Activity Degree of
    Total proteins Total activity per weight purification Yield
    Step (mg) (μmol/min) (nmon/min/mg) (times) (%)
    Cytoplasm 46000 73.5 1.6 1 100
    BUTYL TOYOPEAL 680 16.3 24 15 22
    Q Sepharose FF 72.4 8.96 124 78 12
    Biogel A-1.5 m 6.93 7.38 1060 670 10
    Hydroxyapatite 3.45 5.29 1530 960 7.2
    Mono Q FPLC 0.3 2.16 7200 4500 2.9
    • EXAMPLE 2
  • Determination of Amino Acid Sequence of Bovine PAF Acetylhydrolase [0051]
  • (1) About 0.2 mg of the purified PAF acetylhydrolase obtained in Example 1 was reduced with 1 mg of dithiothreitol at room temperature for 2 hours, followed by the S-alkylation with 0.6% (W/V) 4-vinylpyridine at room temperature for 2 hours. [0052]
  • Using a 4.6 mm×250 mm “Vydak 304-1251 C[0053] 4” column which had been equilibrated beforehand with 20% (V/V) acetonitrile containing 0.1% (V/V) trifluoroacetic acid, the reaction mixture was subjected to reverse phase high-performance liquid chromatography (HPLC). Proteins were then eluted with a linear gradient of acetonitrile (20 to 85% V/V) which contained 0.1% (V/V) trifluoroacetic acid.
  • (2) 40 kDa polypeptide, which had been purified by the HPLC, was dialyzed against a lysylendopeptidase digestive buffer [0.5 M Tris-HCl (pH 8.5) and 4 M urea]. Next, 1 μg of a lysylendopeptidase was added to the sample. After the reaction mixture was incubated for 18 hours at 37° C., the reaction mixture was fractionated by reverse phase HPLC through a 4.6 mm×250 mm “Vydak 304-1251 C[0054] 4” column while using a linear gradient of acetonitrile (5 to 70% V/V) which contained 0.1% (V/V) trifluoroacetic acid.
  • (3) The amino acid sequence of a peptide fragment obtained by the reverse phase HPLC was determined by an automated sequencer (“Model 477A”, trade name; manufactured by Applied Biosystems, Inc.). [0055]
  • The base sequence of the bovine PAF acetylhydrolase, which was determined from the amino acid sequence of the peptide fragment, was as shown above by the formula (III). [0056]
  • Further, from the peptide sequence (III) of the bovine PAF acetylhydrolase, a gene encoding the enzyme was determined by a method known per se in the art. The gene was found to be represented by the formula (IV). [0057]
    • EXAMPLE 3
  • Cloning of Non-active Human PAF Acetylhydrolase cDNA [0058]
  • Using as a template the bovine PAF acetylhydrolase cDNA obtained in Example 2, fluorescein-12-dUTP was incorporated in 500,000 clones of each of a fetal human liver cDNA library (pRc/CMV) and a human brain cDNA library (pCMV SPORTS) by PCR. The clones were then subjected to colony hybridization while detecting the labeling reagent by ECL, whereby cloning was conducted. As a result, a single positive clone was obtained from the human brain library. [0059]
  • A plasmid DNA was prepared and the base sequence was determined. The clone was a full-length clone which contained ATG encoding initiating methionine. Encoding 43 N-terminal amino acids were the same as the corresponding amino acids in the sequence of the bovine PAF acetylhydrolase up to the 40th amino acid, and there was poly A at the 3′ end. A more accurate determination of the base sequence was conducted. As a result, the cDNA was found to consist of 2188 bp and to contain an ORF (open reading frame) consisting of 253 amino acids. Compared with the bovine PAF acetylhydrolase cDNA, 140 amino acids had been deleted. The segment of the deleted 140 amino acids contains a “catalytic triad” of serine, histidine and aspartic acid, which exhibits catalytic activity. The cDNA is therefore not believed to have PAD acetylhydrolase activity. [0060]
  • Hence, a primer was synthesized at positions flanking the deleted region, and PCR was conducted using the library DNA as a template. From the human brain cDNA, two bands were obtained, one corresponding to the above-described cDNA with the 140 amino acids deleted, and the other to a cDNA having substantially the same length as the bovine PAF acetylhydrolase cDNA. From the foregoing, the human brain library DNA was expected to contain, in addition to the above-obtained cDNA, a human PAF acetylhydrolase cDNA which is actually equipped with PAF acetylhydrolase activity. [0061]
    • EXAMPLE 4
  • Cloning of Human PAF Acetylhydrolase cDNA [0062]
  • The human brain cDNA library was diluted to give 2000 clones per well, followed by incubation on 5 96-well plates. Subpools consisting of 10 wells were prepared, and positive pools were determined by PCR (Pool Nos. 10, 20, 28, and 38). With respect to these subpools, PCR was conducted well after well, so that positive pools were confirmed (Pool Nos. 10-5, 20-10, and 38-12). [0063]
  • Concerning these pools, incubation was conducted on plates subsequent to dilution. Using the non-active human PAF acetylhydrolase cDNA as a probe, cloning was attempted by hybridization. Labeling of the DNA was conducted with fluorescein 12-dUTP by PCR, and detection was carried out by ECL. Positive colonies were obtained from Pool Nos. 10-5 and 20-10. Plasmid DNAs of these clones were replicated, and their base sequences were then determined. As a result, a human PAF acetylhydrolase cDNA represented by the formula (II) was obtained from the clones of Pool Nos. 10-5. [0064]
  • Based on the resultant cDNA, the amino acid sequence of the human PAF acetylhydrolase was determined. It was found to be represented by the formula (I). Up to 88%, the sequence was the same as that of the bovine PAF acetylhydrolase (346/392 amino acids). On the other hand, it was 42% identical to that of the plasma human PAF acetylhydrolase (162/392 amino acids). [0065]
  • Further, the above cDNA was incorporated in the pUC-Pl-cl vector, introduced in [0066] E. coli W3110 and then subjected to expression. A band, which corresponded to a protein having a molecular weight of 42 kDa, was detected by SDS-PAGE.
  • The protein was investigated for activity. Human PAF acetylhydrolase activity was confirmed. [0067]
  • 1 4 392 amino acids amino acid single linear peptide BOVINE (Bos taurus) 1 Met Gly Val Asn Gln Ser Val Ser Phe Pro Pro Val Thr Gly Pro His 1 5 10 15 Leu Val Gly Cys Gly Asp Val Met Glu Gly Gln Ser Leu Gln Gly Ser 20 25 30 Phe Phe Arg Leu Phe Tyr Pro Cys Gln Glu Ala Glu Glu Thr Ser Glu 35 40 45 Gln Pro Leu Trp Ile Pro Arg Tyr Glu Tyr Cys Ala Gly Leu Ala Glu 50 55 60 Tyr Leu Lys Phe Asn Lys Arg Trp Gly Gly Leu Leu Phe Asn Leu Gly 65 70 75 80 Val Gly Ser Cys Arg Leu Pro Val Ser Trp Asn Gly Pro Phe Lys Thr 85 90 95 Lys Asp Ser Gly Tyr Pro Leu Ile Ile Phe Ser His Gly Met Gly Ala 100 105 110 Phe Arg Thr Val Tyr Ser Ala Phe Cys Met Glu Leu Ala Ser Arg Gly 115 120 125 Phe Val Val Ala Val Pro Glu His Arg Asp Gly Ser Ala Ala Ala Thr 130 135 140 Cys Phe Cys Lys Gln Thr Pro Glu Glu Asn Gln Pro Asp Asn Glu Ala 145 150 155 160 Leu Lys Glu Glu Trp Ile Pro His Arg Gln Ile Glu Glu Gly Glu Lys 165 170 175 Glu Phe Tyr Val Arg Asn Tyr Gln Val His Gln Arg Val Ser Glu Cys 180 185 190 Val Arg Val Leu Lys Ile Leu Gln Glu Val Thr Ala Gly Gln Ala Val 195 200 205 Leu Asn Ile Leu Pro Gly Gly Leu Asp Leu Met Thr Leu Lys Gly Gly 210 215 220 Ile Asp Val Ser Arg Val Ala Val Met Gly His Ser Phe Gly Gly Ala 225 230 235 240 Thr Ala Ile Leu Ala Leu Ala Lys Glu Met Gln Phe Arg Cys Ala Val 245 250 255 Ala Leu Asp Ala Trp Met Phe Pro Leu Glu His Asp Phe Tyr Pro Thr 260 265 270 Ala Arg Gly Pro Ile Phe Phe Ile Asn Ala Glu Lys Phe Gln Thr Val 275 280 285 Glu Thr Val Asn Leu Met Lys Lys Ile Cys Asp Gln His His Gln Ser 290 295 300 Arg Ile Ile Thr Val Leu Gly Ser Val His Arg Ser Leu Thr Asp Phe 305 310 315 320 Val Phe Val Ala Gly Asn Trp Ile Ser Lys Phe Phe Ser Ser His Thr 325 330 335 Arg Gly Ser Leu Asp Pro Tyr Glu Gly Gln Glu Thr Val Val Arg Ala 340 345 350 Met Leu Ala Phe Leu Gln Lys His Leu Asp Leu Lys Glu Asp Tyr Asp 355 360 365 Gln Trp Asn Asn Phe Ile Glu Gly Ile Gly Pro Ser Leu Thr Pro Gly 370 375 380 Ala Pro His His Leu Ser Ser Leu 385 390 1665 base pairs nucleic acid single linear cDNA BOVINE (Bos taurus) CDS 111..1286 2 GTCGACCCAC GCGTCCGAGT TGACCGTCTG GGCTGTTTCT GAGGGTCAAC GTGACTCGCC 60 GTCAAGTTCA GCCACTGCCC AAGTCGTCGT TCAGTTCAGT TGGTTATGAG ATG GGG 116 Met Gly 1 GTC AAC CAG TCT GTG AGC TTC CCA CCC GTC ACG GGA CCC CAC CTC GTA 164 Val Asn Gln Ser Val Ser Phe Pro Pro Val Thr Gly Pro His Leu Val 5 10 15 GGC TGT GGG GAT GTG ATG GAG GGT CAG AGC CTC CAG GGC AGC TTC TTT 212 Gly Cys Gly Asp Val Met Glu Gly Gln Ser Leu Gln Gly Ser Phe Phe 20 25 30 CGA CTG TTC TAC CCG TGC CAA GAG GCA GAG GAG ACC TCG GAG CAG CCC 260 Arg Leu Phe Tyr Pro Cys Gln Glu Ala Glu Glu Thr Ser Glu Gln Pro 35 40 45 50 CTG TGG ATT CCC CGC TAT GAG TAC TGC GCT GGC CTG GCC GAA TAC CTA 308 Leu Trp Ile Pro Arg Tyr Glu Tyr Cys Ala Gly Leu Ala Glu Tyr Leu 55 60 65 AAG TTT AAT AAG CGC TGG GGG GGG TTA CTG TTC AAC CTG GGT GTG GGA 356 Lys Phe Asn Lys Arg Trp Gly Gly Leu Leu Phe Asn Leu Gly Val Gly 70 75 80 TCT TGT CGC CTG CCT GTT AGC TGG AAT GGC CCC TTT AAA ACA AAG GAC 404 Ser Cys Arg Leu Pro Val Ser Trp Asn Gly Pro Phe Lys Thr Lys Asp 85 90 95 TCT GGA TAC CCC TTG ATC ATC TTC TCT CAT GGC ATG GGA GCC TTC AGG 452 Ser Gly Tyr Pro Leu Ile Ile Phe Ser His Gly Met Gly Ala Phe Arg 100 105 110 ACA GTG TAT TCA GCC TTC TGC ATG GAG CTG GCT TCT CGT GGC TTT GTG 500 Thr Val Tyr Ser Ala Phe Cys Met Glu Leu Ala Ser Arg Gly Phe Val 115 120 125 130 GTT GCT GTA CCA GAG CAC AGG GAT GGG TCA GCT GCG GCC ACC TGT TTC 548 Val Ala Val Pro Glu His Arg Asp Gly Ser Ala Ala Ala Thr Cys Phe 135 140 145 TGC AAG CAG ACC CCA GAG GAG AAC CAG CCT GAC AAT GAG GCC CTG AAG 596 Cys Lys Gln Thr Pro Glu Glu Asn Gln Pro Asp Asn Glu Ala Leu Lys 150 155 160 GAG GAA TGG ATC CCC CAC CGT CAA ATT GAG GAA GGG GAG AAG GAA TTC 644 Glu Glu Trp Ile Pro His Arg Gln Ile Glu Glu Gly Glu Lys Glu Phe 165 170 175 TAT GTT CGG AAC TAC CAG GTG CAT CAG AGG GTG AGC GAG TGT GTG AGG 692 Tyr Val Arg Asn Tyr Gln Val His Gln Arg Val Ser Glu Cys Val Arg 180 185 190 GTG TTG AAG ATC CTA CAA GAG GTC ACT GCT GGG CAG GCC GTT CTC AAC 740 Val Leu Lys Ile Leu Gln Glu Val Thr Ala Gly Gln Ala Val Leu Asn 195 200 205 210 ATC TTG CCT GGC GGA TTG GAT CTG ATG ACC TTG AAG GGC GGC ATT GAC 788 Ile Leu Pro Gly Gly Leu Asp Leu Met Thr Leu Lys Gly Gly Ile Asp 215 220 225 GTG AGC CGT GTG GCT GTA ATG GGA CAT TCA TTT GGA GGG GCC ACA GCT 836 Val Ser Arg Val Ala Val Met Gly His Ser Phe Gly Gly Ala Thr Ala 230 235 240 ATT CTG GCC TTG GCC AAG GAG ATG CAA TTT AGG TGT GCT GTG GCT TTG 884 Ile Leu Ala Leu Ala Lys Glu Met Gln Phe Arg Cys Ala Val Ala Leu 245 250 255 GAC GCT TGG ATG TTT CCT CTG GAG CAT GAC TTT TAC CCC ACG GCC CGA 932 Asp Ala Trp Met Phe Pro Leu Glu His Asp Phe Tyr Pro Thr Ala Arg 260 265 270 GGC CCT ATC TTC TTT ATC AAT GCT GAG AAG TTC CAG ACA GTG GAG ACT 980 Gly Pro Ile Phe Phe Ile Asn Ala Glu Lys Phe Gln Thr Val Glu Thr 275 280 285 290 GTC AAC TTG ATG AAA AAG ATT TGT GAC CAG CAC CAC CAA TCC AGG ATC 1028 Val Asn Leu Met Lys Lys Ile Cys Asp Gln His His Gln Ser Arg Ile 295 300 305 ATA ACT GTC CTT GGT TCT GTT CAT CGG AGT CTA ACC GAC TTT GTT TTT 1076 Ile Thr Val Leu Gly Ser Val His Arg Ser Leu Thr Asp Phe Val Phe 310 315 320 GTG GCT GGT AAC TGG ATT AGT AAA TTC TTC TCC AGT CAC ACC CGT GGA 1124 Val Ala Gly Asn Trp Ile Ser Lys Phe Phe Ser Ser His Thr Arg Gly 325 330 335 AGC TTG GAC CCC TAT GAA GGT CAG GAG ACC GTG GTG CGG GCC ATG TTG 1172 Ser Leu Asp Pro Tyr Glu Gly Gln Glu Thr Val Val Arg Ala Met Leu 340 345 350 GCC TTC CTG CAG AAG CAT CTT GAC CTG AAA GAG GAC TAT GAC CAG TGG 1220 Ala Phe Leu Gln Lys His Leu Asp Leu Lys Glu Asp Tyr Asp Gln Trp 355 360 365 370 AAC AAC TTC ATT GAA GGC ATT GGC CCA TCA CTG ACC CCA GGG GCC CCA 1268 Asn Asn Phe Ile Glu Gly Ile Gly Pro Ser Leu Thr Pro Gly Ala Pro 375 380 385 CAC CAT CTG TCC AGC CTG TAGGCACAAC TGGTCATCTT GTGGAAGGTC 1316 His His Leu Ser Ser Leu 390 CCTGAGCTGA GTTCCCGTGT GGGGCCTGCC CAGGGATACC CTTGGCCTCC TATCAGGAAG 1376 TGATTGCCAT GACCCTTCTG TGTTGATTGA GAGGATATAA TCACACTGCT GATTGGTAAC 1436 GGGGTACTTG GATTCTCAGA CTTGTCGATC TTAAACTCAT GTTGGGACTT GGGTTCACTT 1496 ACTGATGGGC AAACGGGCAT TCTGAGGACT GAGCCTTAAT GGTATGGAGA ACAAACAGTG 1556 GGATGGGGCT GGGGAAGATC TAAGCCCTAA GCTGGGCACT ATGAGCCCTA TAAACCCAAC 1616 CAGCCAACAC CCTCACCTTG GGCAAGTATG ACTTCTGCAG GTCGACTCT 1665 392 amino acids amino acid single linear peptide HUMAN 3 Met Gly Val Asn Gln Ser Val Gly Phe Pro Pro Val Thr Gly Pro His 1 5 10 15 Leu Val Gly Cys Gly Asp Val Met Glu Gly Gln Asn Leu Gln Gly Ser 20 25 30 Phe Phe Arg Leu Phe Tyr Pro Cys Gln Lys Ala Glu Glu Thr Met Glu 35 40 45 Gln Pro Leu Trp Ile Pro Arg Tyr Glu Tyr Cys Thr Gly Leu Ala Glu 50 55 60 Tyr Leu Gln Phe Asn Lys Arg Cys Gly Gly Leu Leu Phe Asn Leu Ala 65 70 75 80 Val Gly Ser Cys Arg Leu Pro Val Ser Trp Asn Gly Pro Phe Lys Thr 85 90 95 Lys Asp Ser Gly Tyr Pro Leu Ile Ile Phe Ser His Gly Leu Gly Ala 100 105 110 Phe Arg Thr Leu Tyr Ser Ala Phe Cys Met Glu Leu Ala Ser Arg Gly 115 120 125 Phe Val Val Ala Val Pro Glu His Arg Asp Arg Ser Ala Ala Thr Thr 130 135 140 Tyr Phe Cys Lys Gln Ala Pro Glu Glu Asn Gln Pro Thr Asn Glu Ser 145 150 155 160 Leu Gln Glu Glu Trp Ile Pro Phe Arg Arg Val Glu Glu Gly Glu Lys 165 170 175 Glu Phe His Val Arg Asn Pro Gln Val His Gln Arg Val Ser Glu Cys 180 185 190 Leu Arg Val Leu Lys Ile Leu Gln Glu Val Thr Ala Gly Gln Thr Val 195 200 205 Phe Asn Ile Leu Pro Gly Gly Leu Asp Leu Met Thr Leu Lys Gly Asn 210 215 220 Ile Asp Met Ser Arg Val Ala Val Met Gly His Ser Phe Gly Gly Ala 225 230 235 240 Thr Ala Ile Leu Ala Leu Ala Lys Glu Thr Gln Phe Arg Cys Ala Val 245 250 255 Ala Leu Asp Ala Trp Met Phe Pro Leu Glu Arg Asp Phe Tyr Pro Lys 260 265 270 Ala Arg Gly Pro Val Phe Phe Ile Asn Thr Glu Lys Phe Gln Thr Met 275 280 285 Glu Ser Val Asn Leu Met Lys Lys Ile Cys Ala Gln His Glu Gln Ser 290 295 300 Arg Ile Ile Thr Val Leu Gly Ser Val His Arg Ser Gln Thr Asp Phe 305 310 315 320 Ala Phe Val Thr Gly Asn Leu Ile Gly Lys Phe Phe Ser Thr Glu Thr 325 330 335 Arg Gly Ser Leu Asp Pro Tyr Glu Gly Gln Glu Val Met Val Arg Ala 340 345 350 Met Leu Ala Phe Leu Gln Lys His Leu Asp Leu Lys Glu Asp Tyr Asn 355 360 365 Gln Trp Asn Asn Leu Ile Glu Gly Ile Gly Pro Ser Leu Thr Pro Gly 370 375 380 Ala Pro His His Leu Ser Ser Leu 385 390 2559 base pairs nucleic acid single linear cDNA HUMAN CDS 216..1392 4 GCAGGTCTCG ACCCACGCGT CCGCGGACGC GTGGGCGAGA AGTGCTTCCA AGCGTCCATT 60 TTGAGCCTTG GAAACTACGA CGACCAAAGG GCCACGGGTT CCTGGGTCGT TTCTCATTTC 120 CGTCGAGTTA AACGTCTGGG GCTGCTTCTG AGGAATCAGC TTGGCTGGCC AGCAAGTTCA 180 GCTCCGGCAA GTCATTTGAT TCACCCGGTG ATGAA ATG GGG GTC AAC CAG TCT 233 Met Gly Val Asn Gln Ser 1 5 GTG GGC TTT CCA CCT GTC ACA GGA CCC CAC CTC GTA GGC TGT GGG GAT 281 Val Gly Phe Pro Pro Val Thr Gly Pro His Leu Val Gly Cys Gly Asp 10 15 20 GTG ATG GAG GGT CAG AAT CTC CAG GGG AGC TTC TTT CGA CTC TTC TAC 329 Val Met Glu Gly Gln Asn Leu Gln Gly Ser Phe Phe Arg Leu Phe Tyr 25 30 35 CCC TGC CAA AAG GCA GAG GAG ACC ATG GAG CAG CCC CTG TGG ATT CCC 377 Pro Cys Gln Lys Ala Glu Glu Thr Met Glu Gln Pro Leu Trp Ile Pro 40 45 50 CGC TAT GAG TAC TGC ACT GGC CTG GCC GAG TAC CTG CAG TTT AAT AAG 425 Arg Tyr Glu Tyr Cys Thr Gly Leu Ala Glu Tyr Leu Gln Phe Asn Lys 55 60 65 70 CGC TGC GGG GGC TTG CTG TTC AAC CTG GCG GTG GGA TCT TGT CGC CTG 473 Arg Cys Gly Gly Leu Leu Phe Asn Leu Ala Val Gly Ser Cys Arg Leu 75 80 85 CCT GTT AGC TGG AAT GGC CCC TTT AAG ACA AAG GAC TCT GGA TAC CCC 521 Pro Val Ser Trp Asn Gly Pro Phe Lys Thr Lys Asp Ser Gly Tyr Pro 90 95 100 TTG ATC ATC TTC TCC CAT GGC CTA GGA GCC TTC AGG ACT TTG TAT TCA 569 Leu Ile Ile Phe Ser His Gly Leu Gly Ala Phe Arg Thr Leu Tyr Ser 105 110 115 GCC TTC TGC ATG GAG CTG GCC TCA CGT GGC TTT GTG GTT GCT GTG CCA 617 Ala Phe Cys Met Glu Leu Ala Ser Arg Gly Phe Val Val Ala Val Pro 120 125 130 GAG CAC AGG GAC CGG TCA GCG GCA ACC ACC TAT TTC TGC AAG CAG GCC 665 Glu His Arg Asp Arg Ser Ala Ala Thr Thr Tyr Phe Cys Lys Gln Ala 135 140 145 150 CCA GAA GAG AAC CAG CCC ACC AAT GAA TCG CTG CAG GAG GAA TGG ATC 713 Pro Glu Glu Asn Gln Pro Thr Asn Glu Ser Leu Gln Glu Glu Trp Ile 155 160 165 CCT TTC CGT CGA GTT GAG GAA GGG GAG AAG GAA TTT CAT GTT CGG AAT 761 Pro Phe Arg Arg Val Glu Glu Gly Glu Lys Glu Phe His Val Arg Asn 170 175 180 CCC CAG GTG CAT CAG CGG GTA AGC GAG TGT TTA CGG GTG TTG AAG ATC 809 Pro Gln Val His Gln Arg Val Ser Glu Cys Leu Arg Val Leu Lys Ile 185 190 195 CTG CAA GAG GTC ACT GCT GGG CAG ACT GTC TTC AAC ATC TTG CCT GGT 857 Leu Gln Glu Val Thr Ala Gly Gln Thr Val Phe Asn Ile Leu Pro Gly 200 205 210 GGC TTG GAT CTG ATG ACT TTG AAG GGC AAC ATT GAC ATG AGC CGT GTG 905 Gly Leu Asp Leu Met Thr Leu Lys Gly Asn Ile Asp Met Ser Arg Val 215 220 225 230 GCT GTG ATG GGA CAT TCA TTT GGA GGG GCC ACA GCT ATT CTG GCT TTG 953 Ala Val Met Gly His Ser Phe Gly Gly Ala Thr Ala Ile Leu Ala Leu 235 240 245 GCC AAG GAG ACC CAA TTT CGG TGT GCG GTG GCT CTG GAT GCT TGG ATG 1001 Ala Lys Glu Thr Gln Phe Arg Cys Ala Val Ala Leu Asp Ala Trp Met 250 255 260 TTT CCT CTG GAA CGT GAC TTT TAC CCC AAG GCC CGA GGA CCT GTG TTC 1049 Phe Pro Leu Glu Arg Asp Phe Tyr Pro Lys Ala Arg Gly Pro Val Phe 265 270 275 TTT ATC AAT ACT GAG AAA TTC CAG ACA ATG GAG AGT GTC AAT TTG ATG 1097 Phe Ile Asn Thr Glu Lys Phe Gln Thr Met Glu Ser Val Asn Leu Met 280 285 290 AAG AAG ATA TGT GCC CAG CAT GAA CAG TCT AGG ATC ATA ACC GTT CTT 1145 Lys Lys Ile Cys Ala Gln His Glu Gln Ser Arg Ile Ile Thr Val Leu 295 300 305 310 GGT TCT GTT CAT CGG AGT CAA ACT GAC TTT GCT TTT GTG ACT GGC AAC 1193 Gly Ser Val His Arg Ser Gln Thr Asp Phe Ala Phe Val Thr Gly Asn 315 320 325 TTG ATT GGT AAA TTC TTC TCC ACT GAA ACC CGT GGG AGC CTG GAC CCC 1241 Leu Ile Gly Lys Phe Phe Ser Thr Glu Thr Arg Gly Ser Leu Asp Pro 330 335 340 TAT GAA GGG CAG GAG GTT ATG GTA CGG GCC ATG TTG GCC TTC CTG CAG 1289 Tyr Glu Gly Gln Glu Val Met Val Arg Ala Met Leu Ala Phe Leu Gln 345 350 355 AAG CAC CTC GAC CTG AAA GAA GAC TAT AAT CAA TGG AAC AAC CTT ATT 1337 Lys His Leu Asp Leu Lys Glu Asp Tyr Asn Gln Trp Asn Asn Leu Ile 360 365 370 GAA GGC ATT GGA CCG TCG CTC ACC CCA GGG GCC CCC CAC CAT CTG TCC 1385 Glu Gly Ile Gly Pro Ser Leu Thr Pro Gly Ala Pro His His Leu Ser 375 380 385 390 AGC CTG T AGGCACAACT GGCCATTTGT AAAGTCACTT CAGCCAAGTT TTCATTTGGG 1442 Ser Leu AGCTACCCAA GGGCACCCAT GAGCTCCTAT CAAGAAGTGA TCAACGTGAC CCCTTTTCAC 1502 AGATTGAAAG GTGTAATCAC ACTGCTGCTT GGATAACTGG GTACTTTGAT CTTAGATTTG 1562 ATCTTAAAAT CACTTTGGGA CTGGGATCCC TTGCTGATTG ACAAACAGAC TTTCTGGGAC 1622 CTTGATGGAG TGGGGAACAA GCAGTAGAGT GGGACTGGGG GAGACCCAGG CCCCGGGCTG 1682 AGCACTGTGA GGCCTGGATG TGAAGACTCA GCCCAGCGAA GCTCATTCCC TTACCCCCGG 1742 CCAGTGCTGC TGCTTCAGTG GAAGAGATGA AGCCAAAGGA CAGAATGAAA ATCCCTACCT 1802 TCAGAGACTC TAGCCCAGCC CAACACCATC TCTTCCTACC TCTCAGCCTT CTCCCTCCCC 1862 AGGGCCACTT GTTGAAGTCT GAGCACTTTA TGTAAATTTC TAGGTGTGAG CCGTGATCAC 1922 ATTTTCTATT TATTTCCAAG TCTTCTCATT GTATGGAACA TAGTACTACT TATACTTACA 1982 GTAGTAAGTT ATACTTGTGA GCCCACAGAG TGGCAGACAG CATGGCTCTC ACAGCACAGG 2042 GAGAAAAACT GAGGTACACA GAGGTACCTC AGAAGCTCTG GATGTCTTTG GGGGTTTTGC 2102 TAAGTGTATC TTGATAGGAA ACAACAAAAG CAGGTTGAGA TGGGGAAGAT GACAGAACAA 2162 CAGTGTTAAA TGGCCATTTG CACAGGCCTT TGCCACAACA GAGAAGTAGT TTGGTCAGCT 2222 AAAACTCAGC TGCAGCCTGG ACAGTAGAGC GAGACCCCAT CTTAAAAATA AAGAAGGCTG 2282 GGCGTGGTGG CTCATGCCTG TAATCCCAGC ACTTTGGGAG GCCAAGGCAG GCAGATCACT 2342 TAAGGCCAGG AGTTCAAGAC CACCTGGCCA ACATGGTGAA ACCCCGTCTC TACTAAAAAT 2402 ACAAAAAATT AGCCTGGCGT AATGGCAGGC GCCTATAATC CCAGCTACTC AGGAGGCTGA 2462 AGCAGAAGAA TCACTTGAAC CTAGGAGGCG GAGGTTGCAG TGAGTCAAGA TCGCGCCACT 2522 GCACTCCAGC CTGGGTGACA GAGCAAGACT CTGTCTT 2559

Claims (11)

1. A protein having activities of a human platelet activating factor acetylhydrolase, and represented by an amino acid sequence represented by the following formula (I) or an amino acid sequence having homology therewith:
Met Gly Val Asn Gln Ser Val Gly Phe Pro Pro Val Thr Gly Pro His Leu Val Gly Cys Gly Asp Val Met Glu Gly Gln Asn Leu Gln Gly Ser Phe Phe Arg Leu Phe Tyr Pro Cys Gln Lys Ala Glu Glu Thr Met Glu Gln Pro Leu Trp Ile Pro Arg Tyr Glu Tyr Cys Thr Gly Leu Ala Glu Tyr Leu Gln Phe Asn Lys Arg Cys Gly Gly Leu Leu Phe Asn Leu Ala Val Gly Ser Cys Arg Leu Pro Val Ser Trp Asn Gly Pro Phe Lys Thr Lys Asp Ser Gly Tyr Pro Leu Ile Ile Phe Ser His Gly Leu Gly Ala Phe Arg Thr Leu Tyr Ser Ala Phe Cys Met Glu Leu Ala Ser Arg Gly Phe Val Val Ala Val Pro Glu His Arg Asp Arg Ser Ala Ala Thr Thr Tyr Phe Cys Lys Gln Ala Pro Glu Glu Asn Gln Pro Thr Asn Glu Ser Leu Gln Glu Glu Trp Ile Pro Phe Arg Arg Val Glu Glu Gly Glu Lys Glu Phe His Val Arg Asn Pro Gln Val His Gln Arg Val Ser Glu Cys Leu Arg Val Leu Lys Ile Leu Gln Glu Val Thr Ala Gly Gln Thr Val Phe Asn Ile Leu Pro Gly Gly Leu Asp Leu Met Thr Leu Lys Gly Asn Ile Asp Met Ser Arg Val Ala Val Met Gly His Ser Phe Gly Gly Ala Thr Ala Ile Leu Ala Leu Ala Lys Glu Thr Gln Phe Arg Cys Ala Val Ala Leu Asp Ala Trp Met Phe Pro Leu Glu Arg Asp Phe Tyr Pro Lys Ala Arg Gly Pro Val Phe Phe Ile Asn Thr Glu Lys Phe Gln Thr Met Glu Ser Val Asn Leu Met Lys Lys Ile Cys Ala Gln His Glu Gln Ser Arg Ile Ile Thr Val Leu Gly Ser Val His Arg Ser Gln Thr Asp Phe Ala Phe Val Thr Gly Asn Leu Ile Gly Lys Phe Phe Ser Thr Glu Thr Arg Gly Ser Leu Asp Pro Tyr Glu Gly Gln Glu Val Met Val Arg Ala Met Leu Ala Phe Leu Gln Lys His Leu Asp Leu Lys Glu Asp Tyr Asn Gln Trp Asn Asn Leu Ile Glu Gly Ile Gly Pro Ser Leu Thr Pro Gly Ala Pro His His Leu Ser Ser Leu
2. A DNA encoding said protein of claim 1.
3. An expression vector having said DNA of claim 2.
4. Recombinant host cells transformed by said expression vector of claim 3.
5. A process for the production of a protein having activities of a human platelet activating factor acetylhydrolase, which comprises culturing said recombinant host cells of claim 4 and collecting said protein from the resulting cultured matter.
6. An antibody against said protein of claim 1.
7. A DNA encoding a protein having activities of a bovine platelet activating factor acetylhydrolase, and represented by an amino acid sequence represented by the following formula (III) or an amino acid sequence having homology therewith:
Met Gly Val Asn Gln Ser Val Ser Phe Pro Pro Val Thr Gly Pro His Leu Val Gly Cys Gly Asp Val Met Glu Gly Gln Ser Leu Gln Gly Ser Phe Phe Arg Leu Phe Tyr Pro Cys Gln Glu Ala Glu Glu Thr Ser Glu Gln Pro Leu Trp Ile Pro Arg Tyr Glu Tyr Cys Ala Gly Leu Ala Glu Tyr Leu Lys Phe Asn Lys Arg Trp Gly Gly Leu Leu Phe Asn Leu Gly Val Gly Ser Cys Arg Leu Pro Val Ser Trp Asn Gly Pro Phe Lys Thr Lys Asp Ser Gly Tyr Pro Leu Ile Ile Phe Ser His Gly Met Gly Ala Phe Arg Thr Val Tyr Ser Ala Phe Cys Met Glu Leu Ala Ser Arg Gly Phe Val Val Ala Val Pro Glu His Arg Asp Gly Ser Ala Ala Ala Thr Cys Phe Cys Lys Gln Thr Pro Glu Glu Asn Gln Pro Asp Asn Glu Ala Leu Lys Glu Glu Trp Ile Pro His Arg Gln Ile Glu Glu Gly Glu Lys Glu Phe Tyr Val Arg Asn Tyr Gln Val His Gln Arg Val Ser Glu Cys Val Arg Val Leu Lys Ile Leu Gln Glu Val Thr Ala Gly Gln Ala Val Leu Asn Ile Leu Pro Gly Gly Leu Asp Leu Met Thr Leu Lys Gly Gly Ile Asp Val Ser Arg Val Ala Val Met Gly His Ser Phe Gly Gly Ala Thr Ala Ile Leu Ala Leu Ala Lys Glu Met Gln Phe Arg Cys Ala Val Ala Leu Asp Ala Trp Met Phe Pro Leu Glu His Asp Phe Tyr Pro Thr Ala Arg Gly Pro Ile Phe Phe Ile Asn Ala Glu Lys Phe Gln Thr Val Glu Thr Val Asn Leu Met Lys Lys Ile Cys Asp Gln His His Gln Ser Arg Ile Ile Thr Val Leu Gly Ser Val His Arg Ser Leu Thr Asp Phe Val Phe Val Ala Gly Asn Trp Ile Ser Lys Phe Phe Ser Ser His Thr Arg Gly Ser Leu Asp Pro Tyr Glu Gly Gln Glu Thr Val Val Arg Ala Met Leu Ala Phe Leu Gln Lys His Leu Asp Leu Lys Glu Asp Tyr Asp Gln Trp Asn Asn Phe Ile Glu Gly Ile Gly Pro Ser Leu Thr Pro Gly Ala Pro His His Leu Ser Ser Leu
8. An expression vector having said DNA of claim 7.
9. Recombinant eucaryotic host cells transformed by said expression vector of claim 8.
10. A process for the production of a protein having activities of a bovine platelet activating factor acetylhydrolase, which comprises culturing said recombinant eucaryotic host cells of claim 9 and collecting said protein from the resulting cultured matter.
11. An antibody against a protein having activities of a bovine platelet activating factor acetylhydrolase, and represented by an amino acid sequence represented by the following formula (III) or an amino acid sequence having homology therewith:
Met Gly Val Asn Gln Ser Val Ser Phe Pro Pro Val Thr Gly Pro His Leu Val Gly Cys Gly Asp Val Met Glu Gly Gln Ser Leu Gln Gly Ser Phe Phe Arg Leu Phe Tyr Pro Cys Gln Glu Ala Glu Glu Thr Ser Glu Gln Pro Leu Trp Ile Pro Arg Tyr Glu Tyr Cys Ala Gly Leu Ala Glu Tyr Leu Lys Phe Asn Lys Arg Trp Gly Gly Leu Leu Phe Asn Leu Gly Val Gly Ser Cys Arg Leu Pro Val Ser Trp Asn Gly Pro Phe Lys Thr Lys Asp Ser Gly Tyr Pro Leu Ile Ile Phe Ser His Gly Met Gly Ala Phe Arg Thr Val Tyr Ser Ala Phe Cys Met Glu Leu Ala Ser Arg Gly Phe Val Val Ala Val Pro Glu His Arg Asp Gly Ser Ala Ala Ala Thr Cys Phe Cys Lys Gln Thr Pro Glu Glu Asn Gln Pro Asp Asn Glu Ala Leu Lys Glu Glu Trp Ile Pro His Arg Gln Ile Glu Glu Gly Glu Lys Glu Phe Tyr Val Arg Asn Tyr Gln Val His Gln Arg Val Ser Glu Cys Val Arg Val Leu Lys Ile Leu Gln Glu Val Thr Ala Gly Gln Ala Val Leu Asn Ile Lue Pro Gly Gly Leu Asp Leu Met Thr Leu Lys Gly Gly Ile Asp Val Ser Arg Val Ala Val Met Gly His Ser Phe Gly Gly Ala Thr Ala Ile Leu Ala Leu Ala Lys Glu Met Gln Phe Arg Cys Ala Val Ala Leu Asp Ala Trp Met Phe Pro Leu Glu His Asp Phe Tyr Pro Thr Ala Arg Gly Pro Ile Phe Phe Ile Asn Ala Glu Lys Phe Gln Thr Val Glu Thr Val Asn Leu Met Lys Lys Ile Cys Asp Gln His His Gln Ser Arg Ile Ile Thr Val Leu Gly Ser Val His Arg Ser Leu Thr Asp Phe Val Phe Val Ala Gly Asn Trp Ile Ser Lys Phe Phe Ser Ser His Thr Arg Gly Ser Leu Asp Pro Tyr Glu Gly Gln Glu Thr Val Val Arg Ala Met Leu Ala Phe Leu Gln Lys His Leu Asp Leu Lys Glu Asp Tyr Asp Gln Trp Asn Asn Phe Ile Glu Gly Ile Gly Pro Ser Leu Thr Pro Gly Ala Pro His His Leu Ser Ser Leu
US09/961,253 1996-06-28 2001-09-25 Platelet activating factor acetylhydrolase, and gene thereof Abandoned US20030040093A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US09/961,253 US20030040093A1 (en) 1996-06-28 2001-09-25 Platelet activating factor acetylhydrolase, and gene thereof

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
JP8188369A JPH1017600A (en) 1996-06-28 1996-06-28 Blood platelet activating factor acetyl hydrolase and its gene
JP8-188369 1996-06-28
US08/886,152 US5880273A (en) 1996-06-28 1997-06-30 Platelet activating factor acetylhydrolase, and gene thereof
US09/196,222 US6323017B1 (en) 1996-06-28 1998-11-20 Platelet activating factor acetylhydrolase, and gene thereof
US09/961,253 US20030040093A1 (en) 1996-06-28 2001-09-25 Platelet activating factor acetylhydrolase, and gene thereof

Related Parent Applications (2)

Application Number Title Priority Date Filing Date
US08/886,152 Division US5880273A (en) 1996-06-28 1997-06-30 Platelet activating factor acetylhydrolase, and gene thereof
US09/196,222 Division US6323017B1 (en) 1996-06-28 1998-11-20 Platelet activating factor acetylhydrolase, and gene thereof

Publications (1)

Publication Number Publication Date
US20030040093A1 true US20030040093A1 (en) 2003-02-27

Family

ID=16222415

Family Applications (3)

Application Number Title Priority Date Filing Date
US08/886,152 Expired - Fee Related US5880273A (en) 1996-06-28 1997-06-30 Platelet activating factor acetylhydrolase, and gene thereof
US09/196,222 Expired - Fee Related US6323017B1 (en) 1996-06-28 1998-11-20 Platelet activating factor acetylhydrolase, and gene thereof
US09/961,253 Abandoned US20030040093A1 (en) 1996-06-28 2001-09-25 Platelet activating factor acetylhydrolase, and gene thereof

Family Applications Before (2)

Application Number Title Priority Date Filing Date
US08/886,152 Expired - Fee Related US5880273A (en) 1996-06-28 1997-06-30 Platelet activating factor acetylhydrolase, and gene thereof
US09/196,222 Expired - Fee Related US6323017B1 (en) 1996-06-28 1998-11-20 Platelet activating factor acetylhydrolase, and gene thereof

Country Status (6)

Country Link
US (3) US5880273A (en)
EP (1) EP0816504B1 (en)
JP (1) JPH1017600A (en)
AT (1) ATE315092T1 (en)
DE (1) DE69735039D1 (en)
PT (1) PT816504E (en)

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0658205B1 (en) 1993-06-25 2000-03-15 Smithkline Beecham Plc Lipoprotein associated phospholipase a2, inhibitors thereof and use of the same in diagnosis and therapy
EP0948605A1 (en) * 1997-08-13 1999-10-13 Icos Corporation Truncated platelet-activating factor acetylhydrolase
EP1124970A1 (en) * 1998-10-28 2001-08-22 Smithkline Beecham Plc Process of purification of low density lipoprotein associated phospholipase a2 using immobilized metal affinity chromatography
KR19990084203A (en) * 1999-09-28 1999-12-06 이승렬 Method and apparatus of controlling the receiving calls for mobile telephone units
EP1318154B1 (en) * 2001-12-07 2004-06-16 SIRS-Lab GmbH Compounds for determination of the activity of phospholipase A2
US20050249828A1 (en) * 2002-04-15 2005-11-10 James Logie Canola protein isolate compositions
US6890831B2 (en) * 2002-06-03 2005-05-10 Sanyo Electric Co., Ltd. Method of fabricating semiconductor device
EP1633864B1 (en) 2003-05-28 2010-04-07 Glaxo Group Limited High throughput assay of lp-pla2 activity
US20150017671A1 (en) 2004-04-16 2015-01-15 Yaping Shou Methods for detecting lp-pla2 activity and inhibition of lp-pla2 activity
EP1735457A4 (en) 2004-04-16 2007-08-29 Glaxo Group Ltd METHODS FOR DETECTING Lp-PLA2 ACTIVITY AND INHIBITION OF Lp-PLA2 ACTIVITY
US20140283157A1 (en) 2013-03-15 2014-09-18 Diadexus, Inc. Lipoprotein-associated phospholipase a2 antibody compositions and methods of use

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3563091B2 (en) * 1993-08-03 2004-09-08 第一サントリーファーマ株式会社 Oxidized phospholipid degrading enzymes and their genes
DE69434609T2 (en) * 1993-10-06 2006-09-21 Icos Corp., Bothell Acethylhydrolase of the platelet activating factor
EP0853673A1 (en) * 1995-09-29 1998-07-22 Smithkline Beecham Plc COMPOUND HAVING SEQUENCE HOMOLOGY WITH LIPOPROTEIN ASSOCIATED PHOSPHOLIPASE A2 (Lp-PLA2)/PAF ACETYL HYDROLASE

Also Published As

Publication number Publication date
PT816504E (en) 2006-05-31
EP0816504A2 (en) 1998-01-07
EP0816504B1 (en) 2006-01-04
JPH1017600A (en) 1998-01-20
US5880273A (en) 1999-03-09
EP0816504A3 (en) 1999-01-07
ATE315092T1 (en) 2006-02-15
DE69735039D1 (en) 2006-03-30
US6323017B1 (en) 2001-11-27

Similar Documents

Publication Publication Date Title
US5702936A (en) Cyclic GMP-binding, cyclic GMP-specific phosphodiesterase materials and methods
US5354677A (en) Intracellular phospholipase A2 enzyme
US5338678A (en) Expression of DNA sequences encoding a thermally stable cytosine deaminase from saccharomyces
US5532152A (en) Platelet-activating factor acetylhydrolase
CA2153254C (en) Cloning of enterokinase and method of use
KR920007666B1 (en) Modified fibrinolytic agents
RU2093519C1 (en) Method of enzymatic preparing fibroblast basic growth factor b fgf (10-155) and pharmaceutical composition based on thereof
EP0402108B1 (en) Thermally stable cytosine deaminase
US6323017B1 (en) Platelet activating factor acetylhydrolase, and gene thereof
US6074864A (en) Cloned DNA for synthesizing unique glucocerebrosidase
JP3472587B2 (en) Bone-related carboxypeptidase-like protein and method for producing the same
CA1332814C (en) Method for producing human pancreatic phospholipase a _
WO1995009913A9 (en) Human monocyte/macrophage derived metalloproteinase inhibitor
WO1996023869A1 (en) RECOMBINANT α-GALACTOSIDASE ENZYME
JPH10201487A (en) Fin-1 nucleic acid and protein sequence in mammal and its use
US6015685A (en) Ancrod proteins, their preparation and use
WO1995009913A1 (en) Human monocyte/macrophage derived metalloproteinase inhibitor
JP3533699B2 (en) Prophenol oxidase and phenol oxidase from silkworm
EP0303997B1 (en) Acyl-peptide hydrolase and methods of production and use
JPH074238B2 (en) Process for producing peptide C-terminal amidating enzyme
JPH09168390A (en) Human vacuolar atp-ase (14-kda subunit)gene
EP0870038A1 (en) INTERLEUKIN-1$g(b) CONVERTING ENZYME LIKE CYSTEINE PROTEASE
JPH03183485A (en) Production of human nerve growth factor with yeast
HK1037882A (en) Cyclic gmp-binding, cyclic gmp-specific phosphodiesterase materials and methods
JPH0686675A (en) Murine dehydropeptidase-i

Legal Events

Date Code Title Description
AS Assignment

Owner name: LEICA MIKROSYSTEME AKTIENGESELLSCHAFT, AUSTRIA

Free format text: CHANGE OF NAME;ASSIGNOR:LEICA AG;REEL/FRAME:009864/0032

Effective date: 19990114

AS Assignment

Owner name: DAIICHI SUNTORY PHARMA CO., LTD., JAPAN

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:SUNTORY LIMITED;REEL/FRAME:013864/0028

Effective date: 20030306

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION