CHARACTERIZATION OF THE COATED VESICLE PROTON PUMP

这项研究的目的是确定结构，离子 三磷酸腺苷的转运特性和胞内分布 依赖于包膜囊泡的质子泵。这台泵 S似乎在细胞膜运输中起着至关重要的作用 通过提供配体-受体所需的酸性环境 受体介导的解离和受体再循环 内吞作用。此外，涂层囊泡泵代表了一种新的 一类由三磷酸腺苷驱动的质子泵，可酸化各种 胞内隔膜，包括内体、溶酶体、高尔基体 植物和低等真核生物的液泡。 我们的工作将集中在进一步的结构表征 包覆泡囊质子泵及其关系的确定 到其他细胞内细胞器的质子泵。我们有 包被囊泡(H+)-ATPase的分离成功 将这个泵重新构建成人造脂泡。这个 纯化的酶含有9种相对分子质量的多肽 17000-10万。在标签研究的基础上我们建议 73,000个道尔顿亚基参与了ATP的水解和 17,000个道尔顿亚基构成DCCD可抑制质子的一部分 频道。计划进行进一步的研究来表征这种反应性 这些多肽上的结合位点。亚基化学计量学和接近度 将通过氨基酸分析和共价交联来测量， 分别进行了分析。将对亚基进行糖基化或 外周膜缔合，以及每一种 将通过标记来确定与膜相关的亚基 使用膜封闭和疏水试剂，并通过 蛋白质分解。完整络合物的离子输运性质 分离的17,080个道尔顿亚基也将被调查。 我们还成功地分离出了一系列单抗。 识别天然的洗涤剂增溶(H+)的抗体- ATPase和免疫使该酶以活性形式沉淀。这些 抗体将被用来测试免疫学关系 在不同的细胞内质子泵之间使用 免疫细胞化学技术和免疫沉淀法 来自分离细胞器的交叉反应物种。这些抗体 也将被用来探测该泵在 质膜。

The objectives of this research are to determine the structure, ion transport properties and intracellular distribution of the ATP- dependent proton pump of clathrin-coated vesicles. This pump appears to play s crucial role in intracellular membrane traffic by providing the acidic environment required for ligand-receptor dissociation and receptor recycling following receptor-mediated endocytosis. In addition, the coated vesicle pump represents a new class of ATP-driven proton pumps which acidify a variety of intracellular compartments, including endosomes, lysosomes, Golgi and the vacuoles of plants and lower eukaryotes. Our work will focus on further structural characterization of the coated vesicle proton pump and on determination of its relationship to the proton pumps of other intracellular organelles. We have succeeded in isolation of the coated vesicle (H+)-ATPase and in reconstitution of this pump into artificial lipid vesicles. The purified enzyme contains nine polypeptides of molecular weight 17,000-100,000. On the basis of labeling studies we have suggested that the 73,000 dalton subunit is involved in ATP hydrolysis and the 17,000 dalton subunit forms part of a DCCD-inhibitable proton channel. Further studies are planned to characterize the reactive sites on these polypeptides. Subunit stoichiometry and proximity will be measured by amino acid analysis and covalent crosslinking, respectively. Subunits will be tested for glycosylation or peripheral membrane association, and the disposition of each subunit with respect to the membrane will be determined by labeling with membrane impermeant and hydrophobic reagents and by proteolysis. The ion transport properties of the intact complex and the isolated 17,080 dalton subunit will also be investigated. We have also succeeded in isolation of a series of monoclonal antibodies which recognize the native, detergent-solubilized (H+)- ATPase and immuneprecipitate this enzyme in an active form. These antibodies will be used to test the immunological relationship between the various intracellular proton pumps using both immunocytochemical techniques and by immuneprecipitation of crossreactive species from isolated organelles. These antibodies will also be used to probe for the presence of this pump in the plasma membrane.