| Project/Area Number |
07672386
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biological pharmacy
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| Research Institution | Teikyo University |
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Principal Investigator |
KARASAWA Ken Teikyo University, Faculty of Pharmaceutical Sciences, Lecturer, 薬学部, 講師 (50186029)
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| Project Period (FY) |
1995 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1996: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1995: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Keywords | PAF (Platelet-Activating Factor) / PAF-Acetylhydrolase / Molecular Cloning / PAT |
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| Research Abstract |
1.Molecular cloning of the cDNA of plasma platelet-activating factor-acetylhydrolase Purified guinea pig plasma platelet-activating factor-acetylhydrolase (Mw 63 kDa) was digested with lysylendopeptidase. The 9 resulting peptides were isolated by reverse-phase HPLC,and their amino acid sequences were determined using a gas-phase amino acid sequencer. The deduced oligonucleotide primers were then syntesized on the basis of the petide sequences, and PCR was performed using first-strand cDNA derived from guinea pig liver poly (A)^+ RNA as a template. A specific fragment of about 600 bp was amplified. Four positive clones were screened from 300,000 pfu of a guinea pig liver cDNA library using the PCR fragment as a probe. None of these contained the N-terminal region of the protein, and 5' RACE was performed. The cDNA encoded 436 amino acids (predicted Mw 49 kDa) and contained consensus sequences for an active site of serine-esterases and three putative N-linked glycosylation sites.
2.Gene expression and antibody preparation
The full-length cDNA was ligated in an E.coli expression vector with a His tag. The recombinant protein showed platelet-activating factor-acetylhydrolase activity, and was purified using a Ni^+ column and SDS-PAGE.The purified protein was used to immunize a rabbit, and a mono-specific antibody was obtained. This antibody recognized a 63-kDa protain contained in guinea pig plasma, suggesting that the enzyme was modified by blycosylation. The significance of this post-translational modification remaines to be elucidated.
Cloning of the genomic DNA is performed. Recently, the expression of this enzyme was reported to be regulated by estradiol in the liver and macrophages and by interleukins in mast cells. Analysis of the regulation and function of this enzyme at the molecular level is planned using promoter region analysis and immunochemical methods.
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