Discrimination between temperature- and brefeldin A-sensitive steps in the sulfation, phosphorylation, and cleavage of progastrin and its derivatives.
Discrimination between temperature- and brefeldin A-sensitive steps in the sulfation, phosphorylation, and cleavage of progastrin and its derivatives.
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前胃泌素及其衍生物硫酸化、磷酸化和裂解中温度敏感步骤和布雷菲德菌素 A 敏感步骤的区别。
DOI：
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发表时间：
1994
影响因子：
4.8
通讯作者：
G. Dockray
中科院分区：
文献类型：
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作者：
A. Varró;J. Henry;C. Vaillant;G. Dockray
促酸激素胃泌素的成熟包括前体裂解、酪氨酸硫酸化、丝氨酸磷酸化和COOH末端酰胺化。我们已经使用布雷菲德菌素A和在22摄氏度下孵育来确定这些修饰发生的位置和时间。在22摄氏度下孵育的胃泌素细胞的免疫金研究显示肿胀的高尔基池，其终端区域与前胃泌素免疫反应性的积累。在22 ℃时，[3 H]酪氨酸和[35 S]硫酸盐被掺入前胃泌素中，但Arg 94-Arg 95切割和Ser 96磷酸化被抑制。当在22 ℃下脉冲标记120分钟后在37 ℃下追踪时，[35 S]前胃泌素在Arg 94-Arg 95处裂解，t1/2约为10分钟，而[3 H]前胃泌素约为20分钟。大约60%的COOH-末端切割片段被磷酸化，但很少或没有[32 P]磷酸盐掺入前胃泌素。在追逐过程中加入布雷菲德菌素A基本上抑制了[3 H]前胃泌素的裂解，但不抑制[35 S]前胃泌素的裂解。然而，当脉冲标记限于20分钟，在22摄氏度，在随后的追逐中，在37摄氏度的布雷菲德菌素A的存在完全抑制裂解的[35 S]前胃泌素。数据表明，前胃泌素硫酸化发生在trans-Golgi网络中，从该网络退出涉及首先通过布雷菲德菌素A敏感步骤，然后通过温度敏感步骤。Arg 94-Arg 95的裂解和Ser磷酸化密切相关，发生在温度敏感步骤的远端，然后在分泌颗粒中发生酰胺化。已知成熟的分泌颗粒不磷酸化前胃泌素衍生肽，因此磷酸化似乎与肽从反式高尔基体递送到胃泌素细胞中的未成熟分泌颗粒一致，并且可以为肽从反式高尔基体递送到胃泌素细胞中的未成熟分泌颗粒提供标记。
Maturation of the acid-stimulating hormone gastrin involves precursor cleavage, tyrosine sulfation, serine phosphorylation, and COOH-terminal amidation. We have used brefeldin A and incubation at 22 degrees C to determine where and when these modifications occur. Immunogold studies of gastrin cells incubated at 22 degrees C revealed swollen Golgi cisternae, the terminal regions of which were associated with an accumulation of progastrin immunoreactivity. At 22 degrees C, [3H]tyrosine and [35S]sulfate were incorporated into progastrin, but Arg94-Arg95 cleavage, and Ser96 phosphorylation, were inhibited. When pulse labeling at 22 degrees C for 120 min was followed by a chase at 37 degrees C, [35S]progastrin was cleaved at Arg94-Arg95 with a t1/2 of about 10 min, compared with about 20 min for [3H]progastrin. Approximately 60% of the COOH-terminal cleavage fragment was phosphorylated, but there was little or no incorporation of [32P]phosphate into progastrin. Addition of brefeldin A during the chase substantially inhibited cleavage of [3H]progastrin, but not [35S]progastrin. However, when pulse labeling was limited to 20 min at 22 degrees C, the presence of brefeldin A in a subsequent chase at 37 degrees C completely inhibited cleavage of [35S]progastrin. The data indicate that progastrin sulfation occurs in the trans-Golgi network, exit from which involves passage through first a brefeldin A-sensitive and then a temperature-sensitive step. Cleavage at Arg94-Arg95 and Ser phosphorylation are closely linked, occur distal to the temperature-sensitive step, and are followed by amidation in secretory granules. It is known that mature secretory granules do not phosphorylate progastrin-derived peptides, and so phosphorylation appears to coincides with, and may provide a marker for, delivery of peptide from trans-Golgi work to immature secretory granules in gastrin cells.