Assembly of the peripheral domain of the bovine vacuolar H(+)-adenosine triphosphatase.
Assembly of the peripheral domain of the bovine vacuolar H(+)-adenosine triphosphatase.
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牛液泡 H()-腺苷三磷酸酶外周结构域的组装。
DOI：
10.1002/jcp.1041560106
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发表时间：
1993
影响因子：
5.6
通讯作者：
Forgac,M
中科院分区：
文献类型：
--
作者：
Myers,M;Forgac,M
在牛肾上皮细胞系中研究了液泡质子转位腺苷三磷酸酶（V-ATP酶）的外周部分（V1）的生物合成和组装。细胞的单层培养物用 Tran35S 标记进行代谢放射性标记，随后使用针对牛脑包被的囊泡质子泵产生的单克隆抗体对 V-ATP 酶进行免疫沉淀。从牛肾细胞系免疫沉淀的 V-ATP 酶具有与牛脑包被的囊泡质子泵和从其他肾组织制备的 V-ATP 酶非常相似的亚基组成。对细胞进行放射性标记的次数不断增加，结果表明 V1 或 V-ATP 酶的外周部分在 10-15 分钟内组装完毕；完整的 V1V0 复合体也可在 10-15 分钟内检测到。在免疫沉淀之前将细胞分为细胞质和膜成分，结果表明细胞质中存在大量 V1；在牛脑细胞质中也发现了类似的复合物。脉冲追踪研究表明，该胞质池不是膜结合 V1V0 的必然前体，并且稍后不会与膜 V1 群体交换。当在 15°C 或存在布雷菲德菌素 A 的情况下进行脉冲追踪研究时，没有观察到组装的质量差异。这表明 V1V0 的组装可能在内质网中完成，然后酶分布到整个细胞中，具有未知功能的 V1 的胞质库与完全组装的复合物并行存在。 © 1993 Wiley-Liss, Inc.
The biosynthesis and assembly of the peripheral sector (V1) of the vacuolar protontranslocating adenosine triphosphatase (V‐ATPase) was studied in a bovine kidney epithelial cell line. Monolayer cultures of cells were metabolically radiolabeled with Tran35S‐label and the V‐ATPase subsequently immunoprecipitated using a monoclonal antibody raised against the bovine brain‐coated vesicle proton pump. The V‐ATPase immunoprecipitated from the bovine kidney cell line has a subunit composition very similar to that of the bovine brain‐coated vesicle proton pump and the V‐ATPase prepared from other kidney tissues. Radiolabeling the cells for increasing times showed that the V1or peripheral portion of the V‐ATPase is assembled within 10–15 min; the intact V1V0complex is also detectable within 10–15 min. Fractionation of the cells into cytosolic and membrane components prior to immunoprecipitation revealed that there is a significant pool of V1in the cytosol; a similar complex is also found in bovine brain cytosol. Pulse‐chase studies suggest that this cytosolic pool is not an obligate precursor for membranebound V1V0and does not exchange with the membrane V1population at later times. No qualitative differences in assembly were observed when pulse‐chase studies were performed at 15°C or in the presence of brefeldin A. This suggests that assembly of V1V0is probably completed in the endoplasmic reticulum prior to distribution of the enzyme throughout the cell, with a cytosolic pool of V1of unknown function existing in parallel with the fully assembled complex. © 1993 Wiley‐Liss, Inc.