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

研究了使用琥珀酰-Phe-Leu-Arg-香豆酰氨基-4-甲磺酸作为待注射到细胞中的底物来监测单个海星卵母细胞的胞质溶胶中的蛋白酶活性的方法。用蛋白酶体抑制剂N-苄氧羰基-L-亮氨酰-L-亮氨酰-L-正缬氨酸预孵育未成熟卵母细胞后，底物的初始水解被显著抑制。该抑制剂阻断1-甲基腺嘌呤触发的细胞周期蛋白降解，这是已知的蛋白酶体介导的。当Carbobenzoxy-leucyl-leucyl-leucinal应用于卵母细胞时，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胞质蛋白酶体。事实上，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，表明底物被氨肽酶切割。因此，在预注射bestatin后的活卵母细胞中测定了蛋白酶体水解底物的初速度（V0）。加入1-甲基腺嘌呤后V0值逐渐增加，并在细胞周期蛋白降解时达到最大值。计算出的成熟卵母细胞的最大速度约为未成熟卵母细胞的3倍。成熟卵母细胞的米氏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调节不仅由底物的泛素化，一般认为，但也由蛋白酶体本身的活性。

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.