Distribution and structure of the vacuolar H+ ATPase in endosomes and lysosomes from LLC-PK1 cells.
Distribution and structure of the vacuolar H+ ATPase in endosomes and lysosomes from LLC-PK1 cells.
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LLC-PK1 细胞内体和溶酶体中液泡 H ATP 酶的分布和结构。
DOI：
10.1016/0014-4827(91)90063-z
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发表时间：
1991
影响因子：
3.7
通讯作者：
Gluck,S
中科院分区：
文献类型：
--
作者：
Rodman,JS;Stahl,PD;Gluck,S
在胞吞途径中，质子泵使几个细胞内膜区室酸化。我们研究了LLC-PK 1细胞中液泡H+ ATP酶的分布和富含内体或溶酶体的膜组分中生物合成标记酶的结构。LLC-PK 1细胞被允许内化细胞色素包被的胶体金作为内吞区室的标记物。通过用免疫金或免疫过氧化物酶技术检测的针对液泡泵的单克隆抗体对细胞进行染色，在这些细胞中鉴定出质子泵。H+ ATP酶标记见于类似于内体和溶酶体的结构，但不在高尔基体或质膜上。为了检查这些隔室中H+ ATP酶的结构，我们用[35 S]甲硫氨酸标记LLCPK 1细胞24小时，并使用Percoll梯度获得富含内体或溶酶体的组分。用单克隆抗H + ATP酶抗体从两种组分中免疫沉淀的H+ ATP酶具有70、56和31 kDa的标记多肽。在二维凝胶上，内体和溶酶体组分的H+ ATP酶的比较显示，70-，56-和31-kDa亚基在两个组分中是相似的。结果表明，这些细胞中的液泡H ~+ ATP酶主要分布在内体和溶酶体中，并且在两个区室中的酶的结构相似。
Vacuolar proton pumps acidify several intracellular membrane compartments in the endocytic pathway. We have examined the distribution of the vacuolar H+ATPase in LLC-PK1cells and the structure of the biosynthetically labeled enzyme in membrane fractions enriched for endosomes or lysosomes. LLC-PK1cells were allowed to internalize cytochromec-coated colloidal gold as a marker for endocytic compartments. Proton pumps were identified in these cells by staining the cells with a monoclonal antibody against the vacuolar pump detected with either immunogold or immunoperoxidase techniques. H+ATPase labeling was seen on structures resembling endosomes and lysosomes, but not on Golgi or plasma membrane. To examine the structure of the H+ATPase in these compartments, we labeled LLCPK1cells for 24 h with [35S]methionine and used a Percoll gradient to obtain fractions enriched for endosomes or lysosomes. H+ATPase immunoprecipitated from both fractions with monoclonal anti-H+ATPase antibodies had labeled polypeptides of 70, 56, and 31 kDa. On two-dimensional gels, a comparison of the H+ATPase from the endosomal and lysosomal fractions revealed that the 70-, 56-, and 31-kDa subunits were similar in both fractions. The results show that the vacuolar H+ATPase in these cells is distributed primarily in endosomes and lysosomes and that the structure of the enzyme is similar in both compartments.