TOPOLOGICAL ORGANIZATION OF GASTRIC H,K ATPASE

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH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 中心，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亚基穿过膜数次，其大部分质量位于胞质中，β亚基仅穿过膜一次，其大部分质量位于内腔中。关于α-亚基跨膜片段的拓扑结构和具体数目存在一些争论，从7-12不等。以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。由于大量的肽，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），以确定细胞质定向区域的胃H，K-ATP酶。从兔胃分离的富含H，K-ATP酶的胞质侧外囊泡经胰蛋白酶消化并释放肽，并通过MALDI-MS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。利用这种方法，我们能够确定α-亚基中的细胞质定向区域为Met 1-Arg 92，Ser 165-Arg 280，Val 351-Lys 785，Ala 838-Lys 851和Phe 997-Tyr 1035。因此，确认α-亚基的N-和C-末端均为胞质，Asn 226和Asn 731未糖基化。我们目前对胰蛋白酶的观察结果与α亚基的10个跨膜片段假说一致。胰凝乳蛋白酶分析似乎进一步确定了H，K-ATP酶950-1016区域的拓扑结构。将对胰蛋白酶和胰凝乳蛋白酶释放的肽进行完整分析，以及用膜侧试剂进行标记，以获得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.