Development of an Automated Dual Type of Micro Stopped-Flow/Flow-Injection Apparatus Applicable for Detection of Minute Alteration of Inorganic Phosphate or Phosphoric Group in Protein Level

一种适用于检测蛋白质水平中无机磷酸盐或磷基微小变化的自动化双型微停流/流动注射装置的研制

• 批准号: 01870077
• 负责人: TANIGUCHI Shigehiko
• 金额: $ 2.18万
• 依托单位: Okayama University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B).
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01870077/
• 关键词: Hard tissues; Phosphorylation; Dephosphorylation; Micro-autoanalyzer; Stopped-flow method; Flow-injection analysis; 骨・硬組織

By combination of two sets of stopped-flow/flow-injection apparatus (single SF-FIA apparatus), an automated dual SF-FIA apparatus was constructed for serial measurements of minute alteration of inorganic phosphate (Pi) or phosphoric groups in protein level related to calcification of hard tissues or phosphorylation/dephosphorylation cycle of growth control system in normal/cancer cells. The dual SF-FIA apparatus was further installed by an intelligent auto sampler in order to carry out some complex pre-treatments of sample. Normally, the quatitative change of phosphorylation/dephosphorylation in protein level by phosphatase or kinase, respectively, can be detected spectrophotometrically through a direct method to free Pi using Malachite Green/molybdate reagent or an indirect method measuring NADH stoichiometrically reflecting ATP/ADP change in consecutive enzyme assay system. Furthermore, the dual SF-FIA apparatus could be applied as a rapid (msec order) stopping tool required for ^<32>P-labeling method by using filtration with milipore membrane. With 29.4 ul for each of sample and assay solution per one cycle measuring, half-hundred of serial measurements by using the dual SF-FIA apparatus are possible for the total volume of 3.0 ml for each of sample and assay solution. The time interval among the serial measurements with the dual SF-FIA apparatus can be set up within an arbitrary range longer than 4 sec.

通过两套停止流动/流动注射装置（单台SF-FIA装置）的组合，构建了一台自动双SF-FIA装置，用于连续测量与正常/癌细胞硬组织钙化或生长控制系统磷酸化/去磷酸化周期相关的蛋白质水平中无机磷酸盐（Pi）或磷酸基的微小变化。在双SF-FIA装置的基础上，安装智能自动进样器，对样品进行一些复杂的前处理。通常情况下，分别通过磷酸酶或激酶磷酸化/去磷酸化在蛋白水平上的定量变化，可以通过用孔雀石绿/钼酸盐试剂直接释放Pi的方法或在连续酶分析系统中间接测量NADH化学计量学反映ATP/ADP变化的方法来分光光度法检测。此外，双SF-FIA装置可用于微孔膜过滤的^<32> p标记方法所需的快速（毫秒级）停止工具。每个样品和分析溶液每一个周期测量29.4 ul，使用双SF-FIA仪器进行50次连续测量，每个样品和分析溶液的总容积为3.0 ml。双SF-FIA装置串行测量的时间间隔可以设置在超过4秒的任意范围内。

项目成果

Kojiro Takahashi: "A kinetic study of phosphorylation/dephosphorylation in protein level by an dual type of micro stoppedーflowーinjecition apparatus." Anal.Biochem.

Kojiro Takahashi：“通过双类型微停流注射装置进行蛋白质水平磷酸化/去磷酸化的动力学研究。”

K. Takahashi, K. Yasuda, T. Kuroishi and S. Taniguchi: "A kinetic study of phosphorylation/dephosphorylation in protein level by a dual type of micro stopped-flow/flow-injection apparatus." Anal. Biochem.

K. Takahashi、K. Yasuda、T. Kuroishi 和 S. Taniguchi：“通过双类型微型停流/流动注射装置进行蛋白质水平磷酸化/去磷酸化的动力学研究。”

Kojiro Takahashi: "A Kinetic study of phosphorylation/dephosphorylation in protein level by an dual type of micro stoppedーflow/flowーinjecition apparatus." Anal.Biochem.

Kojiro Takahashi：“通过双类型微型停流/流动注射装置进行蛋白质水平磷酸化/去磷酸化的动力学研究。”