TOPOLOGICAL ORGANIZATION OF GASTRIC H,K ATPASE

胃 H,K ATP酶的拓扑结构

• 批准号: 7724159
• 负责人: JOHN G FORTE
• 金额: $ 0.19万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7724159
• 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; Numbers; 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代表。列出的机构是 对于中心，这不一定是调查员的机构。 H，K-ATPase ABD Na，K-ATPase是杂二聚体P型ATPases，由α-单位组成，该Alpha-subunit多次遍历膜，大部分质量细胞骨均具有大部分质量细胞质量，并且beta-subunit仅遍历其大部分质量，其大部分质量均经过质量。关于拓扑和特定数量的alpha-subunit跨膜段的论点，范围从7-12不等。先前的方法涉及蛋白水解，然后使用Edman测序或区域特异性抗体进行费力的跨膜肽鉴定。由于肽的数量大量，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法（MALDI-MS）来鉴定胃H，K-ATPase的细胞质取向区域。将富含K-ATPase的细胞质侧囊泡从兔胃中分离并释放肽，并通过MALDI-MS分析以获得细胞质肽的质量。胰蛋白酶的肽也通过RP-HPLC分离，并经过MALDI-MS和PSD分析。使用这种方法，我们可以从Met1-Arg92，Ser165-Arg280，Val351-Lys785，Ala838- lys851和Phe997-Tyr1035中鉴定出来自Met1-Arg92，Ser165-Arg280，Ser165-Arg280，Ser165-Arg280，Ser165-Arg280的胞质方向区域。因此，α-亚基的N-和C末端都被证实为细胞质，ASN226和ASN731均未糖基化。我们目前使用胰蛋白酶的观察结果与α-亚基的10个跨膜段假设一致。用甲胆素分析似乎进一步定义了H，K-ATPase的950-1016区域中的拓扑。将对胰蛋白酶和胰蛋白酶释放的肽以及用膜侧试剂进行标记的完整分析，以达到H，K-ATPase的拓扑模型。