TOPOLOGICAL ORGANIZATION OF GASTRIC H,K ATPASE

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，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亚基穿过膜多次，其大部分质量在细胞质上分布，β亚基只穿过膜一次，其大部分质量在细胞质上分布。关于α -亚基跨膜段的拓扑结构和具体数目，有一些争论，从7-12不等。以前的方法包括蛋白水解，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。由于肽的数量众多，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（MALDI-MS）来鉴定胃H， k - atp酶的细胞质导向区域。从兔胃中分离的富含H， k - atp酶的细胞质侧边囊泡被胰蛋白酶化并释放多肽，用MALDI-MS分析得到细胞质多肽的质量。用反相高效液相色谱（RP-HPLC）分离色氨酸肽，并进行MALDI-MS和PSD分析。使用这种方法，我们能够在Met1-Arg92， Ser165-Arg280,Val351-Lys785，Ala838- Lys851和Phe997-Tyr1035的α -亚基中鉴定细胞质定向区域。因此，α -亚基的N端和c端都被证实是细胞质的，Asn226和Asn731没有糖基化。我们目前对胰蛋白酶的观察结果与α -亚基的10个跨膜段假说是一致的。用胰凝乳酶进行分析似乎进一步确定了H， k - atp酶950-1016区域的拓扑结构。对胰蛋白酶和凝乳胰蛋白酶释放肽的完整分析，以及用膜侧试剂进行标记，将得到H， k - atp酶的拓扑模型。

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.