In situ, real time measurement of enzyme activities in the cells

原位、实时测量细胞内的酶活性

基本信息

批准号：
05558092
负责人：
HOSHI Motonori
金额：
$ 8.45万
依托单位：
Tokyo Institute of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-05558092/
关键词：
Protease Enzyme Activity Microinjection 蛍光物質ヒトデ卵成熟酵素活性

项目摘要

A method was investigated for monitoring an activity of protease (s) in cytosol of a single starfish oocyte using succinyl-Phe-Leu-Arg-coumarylamido-4-methanesulfonic acid as the substrate to be injected into the cell. After preincubation of immature oocytes with a proteasome inhibitor, N-carbobenzoxy-L-leucinyl-L-leucinyl-L-norvalinal, the initial hydrolysis of the substrate was remarkably inhibited. The inhibitor blocked 1-methyladenine-triggered cyclin degradation which is known to be mediated by proteasome. Similar results were obtained when Carbobenzoxy-leucyl-leucyl-leucinal was applied to the oocytes. These results suggested that the protease activity measured by this method is mainly attributable to cytoplasmic proteasome. Indeed, calpain inhibitor E-64 had no effect on the hydrolysis of the substrate.The hydrolysis of the substrate was partially inhibited by bestatin, suggesting that the substrate was cleaved by aminopeptidase. Thus, the initial velocity of hydrolysis of the substrate (V0) by proteasome was assayd in a living oocyte after preinjection of bestatin. The values of V0 increased gradually after 1-methyladenine addition and reached to a maximum level at the time corresponding to cyclin degradation. The calculated maximum velocity of a mature oocyte was approximately three times higher than that of an immature oocyte. The Michealis-Menten constant value was also higher in the mature oocytes than in the immature one. These results suggest that proteasome-dependent proteolysis is regulated not only by ubiquitination of substrates as generally believed but also by the proteasome activity by itself.

研究了一种方法，使用琥珀酸 - phe-leu-arg-arg-coumarylamido-4-甲基磺酸作为底物作为底物作为底物作为注射到细胞中的蛋白酶在单个海星卵母细胞中蛋白酶的活性。在未成熟的卵母细胞与蛋白酶体抑制剂预孵育后，N-碳二氧基-L-葡萄糖蛋白L-葡萄糖蛋白基-L-甲基甲基蛋白酶的初始水解受到了明显抑制。抑制剂阻断了1-甲基杜解触发的细胞周期蛋白降解，该降解是由蛋白酶体介导的。当将碳苯二唑 - 达糖基 - 核苷 - 蛋白酶应用于卵母细胞时，获得了相似的结果。这些结果表明，该方法测量的蛋白酶活性主要归因于细胞质蛋白酶体。实际上，钙蛋白酶抑制剂E-64对底物的水解没有影响。底物的水解被Bestatin部分抑制，这表明底物被氨基肽酶切割。因此，蛋白酶体对底物的水解（V0）的初始速度是在预注射bestatin后的活卵母细胞中的测定。 1-甲基趋化后V0的值逐渐增加，并在对应于细胞周期蛋白降解的时间达到最大水平。成熟卵母细胞计算出的最大速度大约是未成熟卵母细胞的最大速度。成熟卵母细胞中的Michealis-Menten恒定值也高于未成熟的卵母细胞。这些结果表明，蛋白酶体依赖性蛋白水解不仅受到普遍认为的底物的泛素化的调节，而且还通过蛋白酶体活性本身来调节。