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

研究了一种以琥珀酰- ph - leu - arg -香豆胺-4-甲磺酸为底物注入海星卵母细胞，监测胞浆中蛋白酶活性的方法。用蛋白酶体抑制剂n -carbobenzoxy- l- leucinyl- l- leucinyl- l- normvalinal对未成熟卵母细胞进行预孵育后，底物的初始水解被显著抑制。该抑制剂阻断了由蛋白酶体介导的1-甲基腺嘌呤引发的细胞周期蛋白降解。将Carbobenzoxy-leucyl-leucyl-leucinal应用于卵母细胞时也获得了类似的结果。这些结果表明，用这种方法测定的蛋白酶活性主要归因于细胞质蛋白酶体。事实上，calpain抑制剂E-64对底物的水解没有影响。贝司他汀部分抑制了底物的水解，表明底物是被氨基肽酶裂解的。因此，在预注射贝斯特汀后，在活卵母细胞中测定蛋白酶体水解底物的初始速度（V0）。加入1-甲基腺苷后，V0值逐渐升高，在周期蛋白降解对应的时间达到最大值。计算出成熟卵母细胞的最大速度约为未成熟卵母细胞的三倍。成熟卵母细胞的Michealis-Menten常数也高于未成熟卵母细胞。这些结果表明，蛋白酶体依赖的蛋白质水解不仅受底物泛素化的调节，而且还受蛋白酶体自身活性的调节。

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.