1997 Fiscal Year Final Research Report Summary
ANALYSIS OF ADP-RIBOSYLATION ON MOUSE LYMPHOCYTES USING BIOTINYL NAD
| Project/Area Number |
08680687
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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 |
Functional biochemistry
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| Research Institution | SHIMANE MEDICAL UNIVERSITY |
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Principal Investigator |
TSUCHIYA Mikako SHIMANE MEDICAL UNIV.BIOCHEMISTRY.PROFESSOR, 医学部, 教授 (90188582)
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| Project Period (FY) |
1996 – 1997
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| Keywords | ADP-ribosyltransferase / ADP-ribosylation / biotinyl NAD / lymphocytes |
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| Research Abstract |
Biotinyl NAD was prepared from 5- (N-succinimidyloxycarbonyl) pentyl D-biotinamide and N6 [[(6-aminohexyl) carbamoyl] methyl] NAD,purified by HPLC and confirmed to be a substrate for ADP-ribosylation catalyzed by chicken and mouse ADP-ribosyltransferases. When biotinyl NAD was used as a substrate for detection of specific AD-ribosylation of tuftsin in chicken plasma, the modification of the peptide was not detected by biotin-avidin-peroxydase reaction. After incubated with biotinyl NAD,mouse lymphocytes were stained with FITC-avidin and analyzed by cell sorter. The intensity of the fluorescent labeling of the cells did not decrease by addition of ADP-ribose, NAD or arginine in the labeling reaction or ADP-ribosylarginine hydrolase treatment after the labeling. The cells which do not have the ADP-ribosyltransferase were also stianed to a similar extent. Thus, the labeling does not exactly reflect the ADP-reibosylation occurring on the lymphocytes under the conditions we used for staining and washing of cells, probably because of non-specific and non-covalent binding of biotinyl NAD.To determine the subset of lymphocytes which have ADP-ribosyltransferase of its target protein, more stringent conditions may bed needed for labeling and washing.
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