TOPOLOGICAL ORGANIZATION OF GASTRIC H,K ATPASE

胃 H,K ATP酶的拓扑结构

• 批准号: 8169725
• 负责人: JOHN G FORTE
• 金额: $ 0.88万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-12 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8169725
• 关键词: ATP phosphohydrolase; Antibodies; Complex; Computer Retrieval of Information on Scientific Projects Database; Funding; Grant; H(+)-K(+)-Exchanging ATPase; Institution; Label; Membrane; Modeling; Na(+)-K(+)-Exchanging ATPase; Oryctolagus cuniculus; Peptides; Proteolysis; Reagent; Research; Research Personnel; Resources; Side; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stomach; Time; Trypsin; United States National Institutes of Health; Vesicle; chymotrypsin

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. The H,K-ATPase abd Na,K-ATPase are heterodimeric P-type ATPases consisting of an alpha-subunit that traverses the membrane several times with most of its mass cytoplasmically disposed and a beta-subunit that traverses the membrane just once with most of its mass lumenally disposed. There has been some argument regarding the topology and specific number of alpha-subunit transmembrane segments, varying from 7-12. Previous approaches involve proteolysis followed by laborious transmembrane peptide identification using Edman sequencing or regio-specific antibodies. Due to the large number of peptides, defining topology is a complex problem. Here we utilize Matrix Assisted Laser Desorption Ionization mass spectrometry (MALDI-MS) to identify cytoplasmically oriented regions of the gastric H,K-ATPase. H,K-ATPase-enriched cytoplasmic-side-out vesicles isolated from rabbit stomach were trypsinized and released peptides and analyzed by MALDI-MS to obtain the masses of cytoplasmic peptides. Tryptic peptides were also separated by RP-HPLC and the fractions subjected to MALDI-MS and PSD analysis. Using this approach we were ble to identify cytoplasmically oriented regions in the alpha-subunit from Met1-Arg92, Ser165-Arg280,Val351-Lys785,Ala838--Lys851 and Phe997-Tyr1035. Thus, both the N- and C-terminus of the alpha-subunit were confirmed to be cytoplasmic and Asn226 and Asn731 were not glycosylated. Our current observations with trypsin are consistent with the 10 transmembrane segment hypothesis of the alpha-subunit. Analysis with chymotrypsin appears to further defines the topology in the 950-1016 region of the H,K-ATPase. Complete analysis of the tryptic and chymotryptic released peptides, as well as labeling with membrane-sided reagents will be performed to arrive at a topological model of the H,K-ATPase.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 H,K-ATP 酶和 Na,K-ATP 酶是异二聚体 P 型 ATP 酶，由 α 亚基和 β 亚基组成，α 亚基多次穿过膜，其大部分质量分布在细胞质中，而 β 亚基仅穿过膜一次，大部分质量分布在细胞质中。其质量的管腔分布。关于 α 亚基跨膜片段的拓扑结构和具体数量（从 7 到 12 个不等）存在一些争论。以前的方法涉及蛋白水解，然后使用埃德曼测序或区域特异性抗体进行费力的跨膜肽鉴定。由于肽数量众多，定义拓扑是一个复杂的问题。在这里，我们利用基质辅助激光解吸电离质谱 (MALDI-MS) 来识别胃 H,K-ATP 酶的细胞质导向区域。从兔胃中分离出富含H,K-ATP酶的胞质侧出囊泡，用胰蛋白酶处理并释放肽，并通过MALDI-MS进行分析以获得胞质肽的质量。还通过 RP-HPLC 分离胰蛋白酶肽，并对级分进行 MALDI-MS 和 PSD 分析。使用这种方法，我们能够从 Met1-Arg92、Ser165-Arg280、Val351-Lys785、Ala838-Lys851 和 Phe997-Tyr1035 的 α 亚基中识别细胞质定向区域。因此，证实α亚基的N端和C端均在细胞质中，并且Asn226和Asn731未糖基化。我们目前对胰蛋白酶的观察与α亚基的10次跨膜片段假说一致。胰凝乳蛋白酶分析似乎进一步定义了 H,K-ATP 酶 950-1016 区域的拓扑结构。对胰蛋白酶和胰凝乳蛋白酶释放的肽进行完整分析，并用膜侧试剂进行标记，以获得 H,K-ATP 酶的拓扑模型。