STRUCTURE AND FUNCTION OF THE VACUOLAR ATPASE

液泡ATPase是一种存在于内膜中的大型复合酶 真核细胞系统。它的作用是使内部酸化 几个细胞器，如溶酶体和分泌囊泡，并产生 通过细胞器驱动运输的电化学梯度 薄膜。我们已经对液泡膜中的ATPase进行了表征 并分离出了编码三种主要的 亚单位。该酶似乎至少有六个额外的亚基。 本方案的目标是：(1)分离其他多肽 与ATPase活性配对，得到部分氨基酸序列， 构建寡核苷酸探针，分离相应基因。 (2)用遗传学来确定必需基因的数量 合成液泡型ATPase。(3)检测齐聚物 用抗体作图的电子显微镜观察ATPase的结构 几种多肽的定位。(4)检查染色体 外显子/内含子的结构、丰度和大小 液泡ATPase基因的转录本。 对这种酶的研究很重要，因为它的核心作用是 在许多细胞器中发挥作用。配体和配体的解偶联 内小体中的受体，分泌中神经递质的包装 小泡，以及溶酶体中酶的水解性都依赖于 对液泡膜ATPase活性的影响。此外，一种液泡型 ATPase在破骨细胞的质膜上已有报道，其中 它在骨重建和质膜上起着重要作用。 在一些肾细胞中，它在尿酸中起作用。

The vacuolar ATPase is a large complex enzyme found in the endomembrane system of eucaryotic cells. It functions to acidify the interior of several organelles, e.g., lysosomes and secretory vesicles, and generates an electrochemical gradient to drive transport across the organellar membrane. We have characterized the ATPase in vacuolar membranes from Neurospora and have isolated the genes that encode three of the major subunits. The enzyme appears to have at least six additional subunits. The goals of this proposal are: (1) To isolate the other polypeptides which copurify with ATPase activity, obtain partial amino acid sequence, construct oligonucleotide probes, and isolate the corresponding genes. (2) To use genetics to determine the number of genes essential for synthesizing the vacuolar ATPase. (3) To examine the oligomeric structure of the ATPase by electron microscopy, using antibodies to map the location of several polypeptides. (4) To examine the chromosomal location, exon/intron structure, and the abundance and size of transcripts of vacuolar ATPase genes. Investigation of this enzyme is important because of the central role it plays in the function of many organelles. The uncoupling of ligands and receptors in endosomes, the packaging of neurotransmitters in secretory vesicles, and the hydrolytic action of enzymes in lysosomes all depend on the activity of the vacuolar ATPase. Furthermore, a vacuolar-type ATPase has been reported on the plasma membranes of osteoclasts, where it plays an important role in bone remodeling, and on the plasma membrane of some renal cells, where it plays a role in urinary acidification.