SCHULTHEIS POST-DOC/TECHNICIAN SUPPORT

SCHULTHEIS 博士后/技术员支持

• 批准号: 8168281
• 负责人: PATRICK John SCHULTHEIS
• 金额: $ 6.45万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8168281
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Affinity Chromatography; Antibodies; Biological; Biological Assay; Ca(2+) Mg(2+)-ATPase; Ca(2+)-Transporting ATPase; Cations; Cell Line; Cell membrane; Cells; Computer Retrieval of Information on Scientific Projects Database; Defect; Detergents; Development; Drug or chemical Tissue Distribution; Endoplasmic Reticulum; Eukaryota; Funding; Glycoproteins; Grant; H(+)-K(+)-Exchanging ATPase; Heavy Metals; Histidine; In Situ Hybridization; Institution; Ion Transport; Ions; Location; Measures; Membrane; Messenger RNA; Mus; Na(+)-K(+)-Exchanging ATPase; Nickel; Physiological; Postdoctoral Fellow; Process; Protein Isoforms; Proteins; Radioisotopes; Research; Research Personnel; Resources; Source; Specificity; Tissues; United States National Institutes of Health; Vesicle; Western Blotting; Yeasts; cell type; immunocytochemistry; protein degradation; response; uptake

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. P-type ATPases, which transport ions across cell membranes, have been grouped into five subfamilies. The P1-, P2-, and P3-ATPases have been well characterized and include heavy metal ATPases, Na,K-ATPases, H,K-ATPases, SERCAs and other Ca2+-ATPases, and bacterial Mg2+-ATPases. The P4-ATPases are found only in eukaryotes, and have been implicated in the transport of aminophospholipids. Although they are present in all eukaryotes, little is known about the P5-ATPases, other than the observations that loss of Cod1p, one of the two yeast P5-ATPases, leads to defects in glycoprotein processing, endoplasmic reticulum associated protein degradation (ERAD), and constitutive activation of the unfolded protein response (UPR). Thus, our objective is to obtain basic information regarding the distribution and ion specificity of the mammalian P5-ATPases. In preliminary studies we have identified 5 mammalian P5-ATPases, termed Atp13a1-Atp13a5, determined their sequences and mRNA tissue distribution, and begun development of isoform-specific antibodies and expression constructs. In Aim 1 we will develop isoform-specific antibodies and in situ hybridization probes, and determine the membrane location and cell-type distribution of mouse Atp13a1-Atp13a5 using a combination of Western blot analysis of tissues and subcellular membrane fractions, immunocytochemistry, and in situ hybridization. In Aim 2 the ion specificity of the murine P5-ATPases will be determined. Membrane fractions from cell lines in which histidine-tagged versions of Atp13a1-Atp13a5 are over-expressed will be prepared and subjected to detergent solubilization. The His-tagged transporters will then be purified by nickel affinity chromatography and ion specificities will be determined by measuring cation-dependent ATP hydrolysis using sensitive assays of ATPase activity and phosphoenzyme formation. Ion uptake in whole cells or isolated membrane vesicles will also be analyzed using the appropriate radioisotope to confirm ion specificity. These studies will provide basic information that will be critical for more detailed mechanistic studies of the cell biological and physiological functions of this most poorly understood subfamily of mammalian P-type ATPases.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此，可以在其他清晰的条目中表示。列出的机构是 对于中心，这不一定是调查员的机构。 P型ATPases将离子跨细胞膜运输，已分为五个亚家族。 P1，P2-和P3-ATP酶的表征很好，包括重金属ATPases，Na，K-ATPases，H，K-ATPases，SERCA，SERCAS，其他Ca2+-ATPases以及细菌MG2+-ATPases。 P4-ATP酶仅在真核生物中发现，并且与氨基磷脂的运输有关。尽管它们存在于所有真核生物中，但对P5-ATP酶的了解鲜为人知，除了观察到COD1P的丢失（两种酵母P5-ATP酶之一）导致糖蛋白加工，内质网的缺陷，内质网的缺陷，相关蛋白质蛋白质蛋白去散发（ERAD） ，以及展开的蛋白质反应（UPR）的本构激活。因此，我们的目标是获取有关哺乳动物P5-ATP酶的分布和离子特异性的基本信息。在初步研究中，我们已经确定了5个称为ATP13A1-ATP13A5的哺乳动物P5-ATP酶，确定了它们的序列和mRNA组织分布，并开始开发同工型特异性抗体和表达构建体。在AIM 1中，我们将开发同工型特异性抗体和原位杂交探针，并确定小鼠ATP13A1-ATP13A5的膜位置和细胞类型的分布，结合了组织和亚细胞膜分裂，免疫细胞学的蛋白质印迹分析的组合杂交。在AIM 2中，将确定鼠P5-ATP酶的离子特异性。从细胞系中，将制备过表达ATP13A1-ATP13A5的细胞系的膜级分，并进行洗涤剂溶解。然后，将HIS标记的转运蛋白通过镍亲和色谱纯化，离子特异性将通过使用ATPase活性和磷酸酶形成的敏感测定法测量阳离子依赖性ATP水解来确定。全细胞或分离的膜囊泡中的离子摄取也将使用适当的放射性同位素进行分析以确认离子特异性。这些研究将提供基本信息，对于对哺乳动物P型ATPases的亚科细胞生物学和生理功能的更详细的机械研究至关重要。