Activation of the proteasomes of sand dollar eggs at fertilization depends on the intracellular pH rise.
Activation of the proteasomes of sand dollar eggs at fertilization depends on the intracellular pH rise.
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受精时沙钱卵蛋白酶体的激活取决于细胞内 pH 值的上升。
DOI：
10.1006/dbio.1999.9239
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Steinhardt,RA
中科院分区：
文献类型：
--
作者：
Chiba,K;Alderton,JM;Hoshi,M;Steinhardt,RA
使用不透膜的荧光底物琥珀酰-苯丙氨酸-亮氨酸-精氨酸-香豆酰氨基-4-甲磺酸，在活沙钱卵中测量了受精时细胞内蛋白酶体的激活机制。当将底物显微注射到未受精卵中时，底物水解的初始速度（V0）较低。受精后5至10分钟测量的V0是受精前水平的5至9倍，并且在整个第一个细胞周期中保持较高水平。底物的水解被 clasto-lactacystin β-内酯（一种蛋白酶体的特异性抑制剂）抑制。体外证据表明，钙可能参与蛋白酶体活性的调节，以抑制与 PA 28 与 20S 蛋白酶体结合相关的肽酶活性的增加，或刺激 PA 700-蛋白酶体复合物的活性。由于受精后细胞内游离 Ca2+ 浓度 ([Ca2+]i) 和细胞内 pH (pHi) 均增加，因此在 [Ca2+]i 和 pHi 在活化过程中独立变化的条件下测量蛋白酶体底物的水解。当未受精卵暴露于 pH 9 海水中的 15 mM 氯化铵中，其 pHi 升高时，V0 增加到与受精后测量的水平相当。相比之下，无钠海水中钙离子载体诱导的[Ca2+]升高而不改变pHi，对未受精卵的V0没有影响。此外，当未受精卵显微注射调节 pHi 的缓冲液时，V0 以 pH 依赖性方式增加。这些结果表明，受精时的 pH 升高是蛋白酶体激活的必要先决条件，蛋白酶体是细胞周期调节的重要组成部分。
The mechanism of the activation of intracellular proteasomes at fertilization was measured in living sand dollar eggs using the membrane-impermeant fluorogenic substrate, succinyl-Phe-Leu-Arg-coumarylamido-4-methanesulfonic acid. When the substrate was microinjected into unfertilized eggs, the initial velocity of hydrolysis of the substrate (V0) was low.V0measured 5 to 10 min after fertilization was five to nine times the prefertilization level and remained high throughout the first cell cycle. Hydrolysis of the substrate was inhibited byclasto-lactacystin β-lactone, a specific inhibitor of the proteasome. There has beenin vitroevidence that calcium may be involved in regulation of proteasome activity to either inhibit the increase in peptidase activity associated with PA 28 binding to the 20S proteasome or stimulate activity of the PA 700–proteasome complex. Since both intracellular free Ca2+concentration ([Ca2+]i) and intracellular pH (pHi) increase after fertilization, hydrolysis of the proteasome substrate was measured under conditions in which [Ca2+]iand pHiwere varied independently during activation. When the pHiof unfertilized eggs was elevated by exposure to 15 mM ammonium chloride in pH 9 seawater,V0increased to a level comparable to that measured after fertilization. In contrast, [Ca2+]ielevation without pHichange, induced by calcium ionophore in sodium-free seawater, had no effect onV0in the unfertilized egg. Moreover, when unfertilized eggs were microinjected with buffers modulating pHi,V0increased in a pH-dependent manner. These results indicate that the pHirise at fertilization is the necessary prerequisite for activation of the proteasome, an essential component in the regulation of the cell cycle.
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