BIOGENESIS OF SECRETORY GRANULES IN PAROTID ACINAR CELLS

在之前的研究(1997)中，我们证明了ADP核糖化因子(Arf)存在于大鼠腮腺腺泡细胞的胞浆部分，并且Arf以GTP依赖的方式移位到分泌颗粒中。到目前为止，已有6个哺乳动物Arf基因(Arf1-Arf6)被报道。在牛肾上腺嗜铬细胞中，Arf6与分泌颗粒有关。现在，我们成功地证明了以下两点，以阐明Arf蛋白在分泌颗粒中的作用。这些结果发表在《生物化学和生物物理档案》357、147-154(1998)1上。我们利用针对Arf1、Arf3、Arf5和Arf6的抗体鉴定了易位到分泌颗粒的Arf亚型。结果：1.与Arf6定位于牛肾上腺嗜铬细胞分泌颗粒相反，大鼠腮腺腺泡细胞颗粒膜组分中未检测到Arf6。Arf1转位至分泌颗粒的免疫电子显微镜观察为直接证实Arf1已转位至完整的分泌颗粒，移位实验后分离分泌颗粒，用抗Arf抗体进行免疫电子显微镜观察。成熟的分泌颗粒表面可见金粒，直径约1微米。

In the previous study (1997), we demonstrated that ADP-ribosylation factor (Arf) is present in the cytosolic fraction of rat parotid acinar cells and Arf translocates to the secretory granules in a GTp-dependent manner. So far, six mammalian Arf genes (Arf1-Arf6) have been reported. In bovine adrenal chromaffin cells, Arf6 is associated to the secretory granules. Now we have succeeded in demonstrating the following two points to elucidate the role of Arf proteins on the secretory granules. These results were published in Archives of Biochemistry and Biophysics 357, 147-154 (1998).1. Identification of the Arf isotype as Arfi1that translocates to the secretory granulesWe identified the Arf isotype that translocates to the secretory granules by using antibodiesspecific for Arf1, Arf3, Arf5, and Arf6. In contrast to the Arf6 localization to the secretory granules in bovine adrenal chromaffin cells, Arf6 was not detected in the granule membrane fraction of rat parotid acinar cells.2. Demonstration of the Arfi translocation to the secretory granules by immunoelectron microscopyTo confirm directly that Arf1 is translocated to the intact secretory granules, the secretorygranules were isolated after the translocation experiment and used for the immunoelectron microscopy with anti-Arf antibody. Gold particles were detected around the surface of the mature secretory granules, about 1 mum in diameter.