PH-DEPENDENT PROCESSING OF SECRETOGRANIN-II BY THE ENDOPEPTIDASE PC2 IN ISOLATED IMMATURE SECRETORY GRANULES

have previously characterized the processing of secretogranin II (SgII ) in PC12 cells that were stably transfected with the endopeptidase PC 2. Here we show that processing of SgII can be observed in isolated im mature secretory granules (ISGs) derived from this cell line in a temp erature- and ATP-dependent manner. The stimulatory effect of ATP on pr ocessing can be attributed to the activation of the vacuolar H+-ATPase and a concomitant decrease in intragranular pH. The immature secretor y granule therefore provides an adequate environment for correct proce ssing of SgII by PC2. The rate of SgII processing was strongly depende nt 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 v alues. The extent of processing in ISGs incubated in the presence of A TP at physiological pH was compared with the standard curve, and the i ntragranular pH was determined. From these observations, we propose an intragranular pH of 6.3+/-0.1 for ISGs in a physiological buffer in t he presence of ATP. Hence, the pH of ISGs seems to be similar to the p H of the trans-Golgi network (TGN) and is clearly higher than the pH o f mature secretory granules (pH 5.0-5.5). Interestingly, no processing of SgII could be observed in a membrane fraction that is highly enric hed in TGN under conditions for which processing was readily obtained in isolated ISGs.