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.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 H，K-ATPase和Na，K-ATPase是异二聚体的P型ATPase，由一个α-亚基和一个β-亚基组成，α-亚基穿过膜多次，其大部分质量以胞质形式分布，而β-亚基仅穿过膜一次，其大部分质量以管腔形式分布。关于阿尔法亚单位跨膜片段的拓扑结构和具体数量一直存在一些争论，从7-12个不等。以前的方法包括蛋白分解，然后使用Edman测序或区域特异性抗体进行繁琐的跨膜多肽鉴定。由于多肽的数量巨大，定义拓扑结构是一个复杂的问题。在这里，我们利用基质辅助激光解吸电离质谱仪(MALDI-MS)来识别胃H，K-ATPase的细胞质定向区域。从兔胃分离的H，K-ATPase胞质倒置囊泡经胰酶消化后释放多肽，经MALDI-MS分析得到大量胞质多肽。用反相高效液相色谱分离胰蛋白酶多肽，并用MALDI-MS和PSD进行分析。利用这种方法，我们能够从MET1-Arg92、Ser165-Arg280、Val351-Lys785、Ala838-Lys851和Phe997-Tyr1035的α亚基中鉴定出面向细胞质的区域。因此，α-亚基的N-末端和C-末端都是细胞质的，Asn226和Asn731没有糖基化。我们目前对胰酶的观察结果与阿尔法亚单位跨膜片段10的假说是一致的。用胰凝乳酶分析似乎进一步定义了H，K-ATPase 950-1016区域的拓扑。对胰酶和胰凝乳酶释放的多肽进行完整的分析，以及膜侧试剂的标记，以得到H，K-ATPase的拓扑模型。