Spearman T N, Hurley K P, Olivas R, Ulrich R G, Butcher F R
J Cell Biol. 1984 Oct;99(4 Pt 1):1354-63. doi: 10.1083/jcb.99.4.1354.
Rat parotid minces were labeled with [32P]Pi, stimulated with isoproterenol, homogenized in sucrose, and fractionated on continuous sucrose density gradients. We analyzed the resulting fractions by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiograms were made from the gels. Comparison of fractions from control and isoproterenol-stimulated minces revealed seven phosphoproteins that were affected by isoproterenol. The subcellular location of these proteins was determined by comparing their distribution in the sucrose gradients with that of a number of enzymes that are characteristic of specific organelles. Isoproterenol decreased the phosphorylation of two cytoplasmic proteins (Mr 16,000 and 18,000) and increased the phosphorylation of a third (Mr 14,000). The phosphorylation of two endoplasmic reticulum proteins was increased by isoproterenol (Mr 20,500 and 22,500), as was an Mr 31,000 protein which was probably the S6 ribosomal protein. The phosphorylation of a secretory granule protein (Mr 24,000) was decreased by isoproterenol. We then developed a purification scheme for parotid secretory granules. By using this method, we demonstrated that the phosphorylation of the Mr 24,000 was also decreased by carbamylcholine. Granules purified by this method also contained a small number of other phosphoproteins whose phosphorylation was increased only by isoproterenol. Secretory granule-associated stimulus-affected phosphoproteins were found in the particulate fraction when the granules were hypotonically lysed, and were not extracted from the particulate fraction by washing with 0.6 M KCl.
将大鼠腮腺切碎组织用[32P]磷酸进行标记，用异丙肾上腺素刺激，在蔗糖中匀浆，然后在连续蔗糖密度梯度上进行分级分离。我们通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳分析所得级分，并从凝胶制作放射自显影片。比较对照和异丙肾上腺素刺激的切碎组织的级分，发现有7种磷蛋白受异丙肾上腺素影响。通过将它们在蔗糖梯度中的分布与一些特定细胞器特有的酶的分布进行比较，确定了这些蛋白质的亚细胞定位。异丙肾上腺素降低了两种细胞质蛋白（分子量分别为16,000和18,000）的磷酸化水平，并增加了第三种（分子量为14,000）的磷酸化水平。异丙肾上腺素增加了两种内质网蛋白（分子量分别为20,500和22,500）的磷酸化水平，还增加了一种分子量为31,000的蛋白（可能是S6核糖体蛋白）的磷酸化水平。异丙肾上腺素降低了一种分泌颗粒蛋白（分子量为24,000）的磷酸化水平。然后我们制定了腮腺分泌颗粒的纯化方案。通过使用这种方法，我们证明卡巴胆碱也降低了分子量为24,000的蛋白的磷酸化水平。用这种方法纯化的颗粒还含有少量其他磷蛋白，其磷酸化仅由异丙肾上腺素增加。当颗粒被低渗裂解时，在颗粒部分发现了与分泌颗粒相关的受刺激影响的磷蛋白，并且用0.6 M氯化钾洗涤颗粒部分不能将其从颗粒部分中提取出来。
