Chiba K, Sato E, Hoshi M
Department of Life Sciences, Tokyo Institute of Technology, Yokohama.
J Biochem. 1997 Aug;122(2):286-93. doi: 10.1093/oxfordjournals.jbchem.a021751.
A method was investigated for monitoring the activity of protease(s) in cytosol of a single starfish oocyte using succinyl-Phe-Leu-Arg-coumarylamido-4-methanesulfonic acid as the substrate, which was 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. However, calpain inhibitor E-64 did not inhibit the hydrolysis of the substrate. These results suggested that the protease activity measured by this method is mainly attributable to cytoplasmic proteasome. 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 assayed in a living oocyte after preinjection of bestatin. The values of V0 increased gradually after 1-methyladenine addition and reached the maximum level at the time corresponding to cyclin degradation. The calculated maximum velocity of hydrolysis by a mature oocyte was approximately three times higher than that by an immature oocyte. The Michaelis-Menten constant value was also higher in mature than immature oocytes. These results suggest that proteasome-dependent proteolysis is regulated not only by ubiquitination of substrates, as is generally believed, but also by the proteasome activity itself.
研究了一种使用琥珀酰 - 苯丙氨酸 - 亮氨酸 - 精氨酸 - 香豆素酰胺基 - 4 - 甲磺酸作为底物监测单个海星卵母细胞胞质溶胶中蛋白酶活性的方法，该底物被注入细胞中。在用蛋白酶体抑制剂N - 苄氧羰基 - L - 亮氨酰 - L - 亮氨酰 - L - 正缬氨酸对未成熟卵母细胞进行预孵育后，底物的初始水解受到显著抑制。该抑制剂阻断了1 - 甲基腺嘌呤触发的细胞周期蛋白降解，已知这是由蛋白酶体介导的。然而，钙蛋白酶抑制剂E - 64并未抑制底物的水解。这些结果表明，通过该方法测得的蛋白酶活性主要归因于细胞质蛋白酶体。底物的水解受到抑氨肽酶的部分抑制，表明底物是由氨肽酶切割的。因此，在预注射抑氨肽酶后，在活卵母细胞中测定了蛋白酶体对底物的初始水解速度（V0）。添加1 - 甲基腺嘌呤后，V0值逐渐增加，并在与细胞周期蛋白降解相对应的时间达到最高水平。成熟卵母细胞计算出的最大水解速度约为未成熟卵母细胞的三倍。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，蛋白酶体依赖性蛋白水解不仅如通常所认为的那样受底物泛素化调节，还受蛋白酶体活性本身的调节。
