1988 Fiscal Year Final Research Report Summary
Development of inositol 1,4,5-trisphosphate assay kit
| Project/Area Number |
61870017
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| Research Category |
Grant-in-Aid for Developmental Scientific Research
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| Allocation Type | Single-year Grants |
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| Research Field |
Pathological medical chemistry
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| Research Institution | Tokyo Metropolitan Institute of Gerontology |
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Principal Investigator |
TAKENAWA Tadaomi Department of Pharmacology, Tokyo Metropolitan Institute of Gerontology, 薬理学科, 部長 (40101315)
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| Co-Investigator(Kenkyū-buntansha) |
FUKAMI Kiyoko Department of Pharmacology, Tokyo Metropolitan Institute of Gerontology, 薬理学部, 助手 (40181242)
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| Project Period (FY) |
1986 – 1988
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| Keywords | Inositol 1,4,5-trisphosphate |
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| Research Abstract |
In inositol phospholipid-mediated signal transduction system, signals are caused through the breakdown of phosphatidyl inositol 4,5-bisphosphate (PIP_2) to inositol 1,4,5-trisphosphate (IP_3) and diacylglycerol (DG). Therefore, one of the most important factor to determine the strength of responses seems to be PIP_2 ( a signal producing lipid), IP_3 and DG (2nd messenger). But these materials are contained too low to measure in cells.
We tried to develop a new method to quantify PIP_2 and IP_3 using anti-PIP_2 antibody or anti-IP_3 antibody. Monoclonal anti-PIP_2 antibody and anti-IP_3 antibody were developed. anti-PIP_2 antibody showed a high specificity for PIP_2 and did not cross react with PIP ot PI. On the other hand, anti-IP_3 antibody was found to be fairly low specificity for IP_3, since the antibody cross reacted with 1,3,4-IP_3 or 1,3,4,5-IP_4 with a 10-fold lesser extent.
Therefore, we extablished a method to measure a trace amount of PIP_2 by TLC immunostaining. After PIP_2 was extracted from cells (1 x 10^6) and applied on TLC plate, the plate was developed. PIP_2 was detected with the antibody. In this method, a trace amount (20 pmo1 - 1 nmol) of PIP_2 was determined, suggesting 1,000 times higher sensitivity than usual method for measuring inorganic phosphate.
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