Development of inositol 1,4,5-trisphosphate assay kit

肌醇1,4,5-三磷酸测定试剂盒的研制

• 批准号: 61870017
• 负责人: TAKENAWA Tadaomi
• 金额: $ 5.06万
• 依托单位: Tokyo Metropolitan Institute of Gerontology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research
• 财政年份: 1986
• 资助国家: 日本
• 起止时间: 1986 至 1988
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-61870017/
• 关键词: Inositol 1,4,5-trisphosphate; イノシトール1,4,5ートリスホスフェイト; ホスファチジルイノシトール4,5ービスホスフェイト; セカンドメッセンジャー; イノシトール1.45-トリホスフェイト; モノクローナル抗体

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.

在肌醇磷脂介导的信号转导系统中，信号是通过磷脂酰肌醇4，5-二磷酸（PIP_2）分解为肌醇1，4，5-三磷酸（IP_3）和甘油二酯（DG）而产生的。因此，决定反应强度的最重要因素之一似乎是PIP_2（产生脂质的信号）、IP_3和DG（第二信使）。本研究试图建立一种新的方法，利用抗PIP_2抗体或抗IP_3抗体，定量检测细胞内的PIP_2和IP_3。制备了抗PIP_2和抗IP_3单克隆抗体。抗PIP_2抗体对PIP_2有高度特异性，与PIP或PI无交叉反应。另一方面，由于抗IP_3抗体与1，3，4，5-IP_3或1，3，4，5-IP_4的交叉反应程度低10倍，因此，我们建立了一种薄层免疫染色检测微量IP_2的方法。从细胞中提取PIP_2（1 × 10^6）并上样至TLC板后，展开板。用抗体检测PIP_2。在此方法中，测定了痕量（20 pmol 1 - 1 nmol）的PIP_2，表明灵敏度比通常测定无机磷酸盐的方法高1,000倍。

项目成果

Matsuoka; Fukami; Nakanishi; Kawai; Takenawa: "Mitogenesis in Response to PDGF and Bombesisn Abolished by Microinjection of Antibody to PIP_2" Science. 239. 640-643 (1988)

松冈；

Uno;Fukami;Kato;Takenawa;Ishikawa: Nature. 333. 188-190 (1988)

宇野；深见；加藤；竹绳；石川：自然。

M.Sato;S.Ando;K.Mithui;Y.Homma;T.Takenawa: Biochem.Biophys.Res.Commun.137. 23-28 (1986)

M.Sato；S.Ando；K.Mithui；Y.Homma；T.Takenawa：Biochem.Biophys.Res.Commun.137。

Matsuoka,K.;Fukami,K.;Nakanishi;Kawai;Takenawa: Science. 239. 640-643 (1988)

松冈，K.；深见，K.；中西；河合；竹绳：科学。

M.Kato;T.Tkenawa: J.Biol.Chem.

M.Kato；T.Tkenawa：J.Biol.Chem。