TOPOLOGICAL ORGANIZATION OF GASTRIC H,K ATPASE

胃 H,K ATP酶的拓扑结构

• 批准号: 6308866
• 负责人: JOHN G FORTE
• 金额: $ 0.99万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2002-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6308866
• 关键词: biomedical resource; proteins; spectrometry; stomach; structural biology

The H,K-ATPase abd Na,K-ATPase are heterodimeric P-type ATPases consisting of an a-subunit that traverses the membrane several times with most of its mass cytoplasmically disposed and a b-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 a-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 a-subunit from Met1-Arg92, Ser165-Arg280,Val351-Lys785,Ala838--Lys851 and Phe997-Tyr1035. Thus, both the N- and C-terminus of the a-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 a-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.

H,K-ATP 酶和 Na,K-ATP 酶是异二聚体 P 型 ATP 酶 由多次穿过膜的α亚基组成 其大部分质量分布在细胞质中，并且有一个 b 亚基 其大部分质量在腔内仅穿过膜一次 处置。 关于拓扑和结构存在一些争论 a-亚基跨膜片段的具体数量，从 7-12。 以前的方法涉及蛋白水解，然后是费力的 使用 Edman 测序进行跨膜肽鉴定或 区域特异性抗体。 由于肽的数量较多， 定义拓扑是一个复杂的问题。 这里我们利用矩阵 辅助激光解吸电离质谱 (MALDI-MS) 识别胃 H,K-ATP 酶的细胞质导向区域。 从兔体内分离出富含 H,K-ATP 酶的胞质侧出囊泡 胃被胰蛋白酶消化并释放肽并通过以下方法进行分析 MALDI-MS 获得细胞质肽的质量。 胰蛋白酶 还通过 RP-HPLC 分离肽，并对级分进行 MALDI-MS 和 PSD 分析。 使用这种方法我们能够 识别 a 亚基中的细胞质定向区域 Met1-Arg92、Ser165-Arg280、Val351-Lys785、Ala838--Lys851 和 Phe997-Tyr1035。 因此，a-亚基的 N 端和 C 端 被确认为细胞质，而 Asn226 和 Asn731 不是 糖基化。 我们目前对胰蛋白酶的观察结果是一致的 与a-亚基的10次跨膜片段假说。 胰凝乳蛋白酶分析似乎进一步定义了拓扑结构 H,K-ATP酶的950-1016区域。 完整分析 胰蛋白酶和胰凝乳蛋白酶释放的肽，以及标记 将进行膜侧试剂以获得拓扑 H,K-ATP酶模型。