pH-dependent processing of secretogranin II by the endopeptidase PC2 in isolated immature secretory granules.
pH-dependent processing of secretogranin II by the endopeptidase PC2 in isolated immature secretory granules.
复制标题
在分离的未成熟分泌颗粒中，肽链内切酶 PC2 对分泌粒素 II 进行 pH 依赖性加工。
作者：
S. Urbé;A. Dittié;S. Tooze
我们以前已经在PC12细胞中描述了分泌颗粒素II(SGII)的加工过程，这些细胞稳定地转染了内肽酶PC2。在这里，我们表明，在分离的未成熟分泌颗粒(ISGs)中可以观察到SgII的处理，这种处理依赖于温度和ATP。ATP对加工的刺激作用可以归因于液泡H(+)-ATPase的激活和伴随的颗粒内pH的下降。因此，未成熟的分泌颗粒为PC2正确处理SGII提供了足够的环境。SgII的加工速度强烈依赖于颗粒内的pH，提示SgII的加工可以作为颗粒内部的pH指示剂。在一定pH值范围内平衡的ISGs中，使用SGII处理制备标准曲线。将在生理pH下存在的ATP孵育的ISGs的加工程度与标准曲线进行比较，并测定了颗粒内的pH。根据这些观察，我们建议在存在ATP的生理缓冲液中，ISGs的颗粒内pH为6.3+/-0.1。因此，ISGs的pH似乎类似于反高尔基网络(TGN)的pH，但明显高于成熟分泌颗粒的pH(pH 5.0-5.5)。有趣的是，在分离的ISGs中容易获得加工的条件下，在高度富含TGN的膜部分中不能观察到SgII的加工。
We have previously characterized the processing of secretogranin II (SgII) in PC12 cells that were stably transfected with the endopeptidase PC2. Here we show that processing of SgII can be observed in isolated immature secretory granules (ISGs) derived from this cell line in a temperature- and ATP-dependent manner. The stimulatory effect of ATP on processing can be attributed to the activation of the vacuolar H(+)-ATPase and a concomitant decrease in intragranular pH. The immature secretory granule therefore provides an adequate environment for correct processing of SgII by PC2. The rate of SgII processing was strongly dependent on the intragranular pH, suggesting that processing of SgII can be used as a pH indicator for the granule interior. A standard curve was prepared using SgII processing in ISGs equilibrated at a range of pH values. The extent of processing in ISGs incubated in the presence of ATP at physiological pH was compared with the standard curve, and the intragranular pH was determined. From these observations, we propose an intragranular pH of 6.3 +/- 0.1 for ISGs in a physiological buffer in the presence of ATP. Hence, the pH of ISGs seems to be similar to the pH of the trans-Golgi network (TGN) and is clearly higher than the pH of mature secretory granules (pH 5.0-5.5). Interestingly, no processing of SgII could be observed in a membrane fraction that is highly enriched in TGN under conditions for which processing was readily obtained in isolated ISGs.