Arvan P, Castle J D
J Cell Biol. 1986 Oct;103(4):1257-67. doi: 10.1083/jcb.103.4.1257.
Secretion granules have been isolated from the parotid glands of rats that have been chronically stimulated with the beta-adrenergic agonist, isoproterenol. These granules are of interest because they package a quantitatively different set of secretory proteins in comparison with granules from the normal gland. Polypeptides enriched in proline, glycine, and glutamine, which are known to have pI's greater than 10, replace alpha-amylase (pI's = 6.8) as the principal content species. The internal pH of granules from the treated rats ranges from 7.8 in a potassium sulfate medium to 6.9 in a choline chloride medium. The increased pH over that of normal parotid granules (approximately 6.8) appears to reflect the change in composition of the secretory content. Whereas normal mature parotid granules have practically negligible levels of H+ pumping ATPase activity (Arvan, P., G. Rudnick, and J. D. Castle, 1985, J. Biol. Chem., 260, 14945-14952) the isolated granules from isoproterenol-treated rats undergo a time-dependent internal acidification (approximately 0.2 pH unit) that requires the presence of ATP and is abolished by an H+ ionophore. Additionally, an inside-positive granule transmembrane potential develops after ATP addition that depends upon ATP hydrolysis. Two independent methods have been used that exclude the possibility that contaminating organelles are the source of the H+-ATPase activity. Together these data provide clear evidence for the presence of an H+ pump in the membranes of parotid granules from chronically stimulated rats. However, despite the presence of H+-pump activity, fluorescence microscopy with the weak base, acridine orange, reveals that the intragranular pH in live cells is greater than that of the cytoplasm.
已从用β-肾上腺素能激动剂异丙肾上腺素长期刺激的大鼠腮腺中分离出分泌颗粒。这些颗粒之所以令人感兴趣，是因为与正常腺体的颗粒相比，它们包装了一组数量上不同的分泌蛋白。富含脯氨酸、甘氨酸和谷氨酰胺的多肽（已知其pI大于10）取代了α-淀粉酶（pI = 6.8）成为主要的含量成分。处理过的大鼠的颗粒内部pH值在硫酸钾培养基中为7.8，在氯化胆碱培养基中为6.9。与正常腮腺颗粒（约6.8）相比，pH值升高似乎反映了分泌内容物组成的变化。正常成熟的腮腺颗粒的H + 泵ATP酶活性实际上可以忽略不计（Arvan，P.，G. Rudnick和J. D. Castle，1985，《生物化学杂志》，260，14945 - 14952），而异丙肾上腺素处理过的大鼠分离出的颗粒会经历时间依赖性的内部酸化（约0.2个pH单位），这需要ATP的存在并且会被H + 离子载体消除。此外，添加ATP后会产生一个内膜为正电位的颗粒跨膜电位，这取决于ATP的水解。已经使用了两种独立的方法来排除污染细胞器是H + -ATP酶活性来源的可能性。这些数据共同为长期刺激的大鼠腮腺颗粒膜中存在H + 泵提供了明确的证据。然而，尽管存在H + 泵活性，但用弱碱吖啶橙进行荧光显微镜观察发现，活细胞内颗粒的pH值高于细胞质的pH值。
