2000 Fiscal Year Final Research Report Summary
The role of glycohydrates in the function of plasma-type PAF-acetylhydrolase.
| Project/Area Number |
11672186
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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, Pharmaceutical Science, Lecturer, 薬学部, 講師 (50186029)
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| Project Period (FY) |
1999 – 2000
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| Keywords | platelet-activating factor / PAF / gene transfer / glycosylation / PAF-acetylhydrolase |
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| Research Abstract |
We prepared the mutants in which possible N-glycosylation sites (N76, N200 and N324) of guinea pig plasma-type PAF-acetylhydrolase cannot be glycosylated by displacement of each Asn residue with Gln residue according to the method of Kunkel. DNA nucleotide sequences of these mutants DNAs and wild-type DNA were determined, expression vectors were constructed, and COS7 cells were transfected with resulting DNA.
The mutant proteins showed smaller molecular weights than wild-type protein in the western blotting, suggesting that this enzyme was modulated by glycosylation after translation. In addition, wild-type enzyme showed smaller molecular weight in the cells treated with tunicamycin, an inhibitor of N-glycosylation, compared with control cells. The molecular weights expressed in the cells treated with tunicamycin corresponded to those of mutant protein, N76Q/N200Q/N324Q.The enzyme activity of the mutant proteins were altered, suggesting that glycohydrates linked to this enzyme was involved in the regulation of the enzyme activity. Although the mechanism of modulation of enzyme activity remains to be determined at present, there exists possibility that sugar chains could affect the protein structure. Therefore, further study such as crystal structure analysis will be necessary to elucidate the mechanism of modulation of enzyme activity in the future.
Plasma-type PAF acetylhydrolases have been hitherto purified from human plasma besides guinea pig plasma, and human enzyme has been reported to be a glycoprtein as well as guinea-pig enzyme. On the other hand, it is reported that sugar chain affects the activity of plasma-type PAF acetylhydrolase secreted from HepG2, human liver derived cell-line. It is necessary to study the effect of sugar chain on the enzyme activity in detail from now on.
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Research Products
(10 results)
