TOPOLOGICAL ORGANIZATION OF GASTRIC H,K ATPASE

H，K-ATP酶和Na，K-ATP酶均为异二聚体P型ATP酶 由a亚单位组成， 其大部分物质位于胞质中， 仅穿过膜一次，其大部分物质在内腔中 处置。 关于拓扑结构有一些争论， 特定数量的a亚基跨膜片段，从 7-12. 以前的方法涉及蛋白质水解，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。 由于大量的肽， 定义拓扑是一个复杂的问题。 在这里，我们利用矩阵 辅助激光解吸电离质谱（MALDI-MS） 鉴定胃H，K-ATP酶的细胞质定向区域。 从家兔体内分离的富含H，K-ATP酶的胞质外侧囊泡 胃被胰蛋白酶消化并释放肽， MALDI-MS以获得细胞质肽的质量。 胰蛋白 还通过RP-HPLC分离肽，并将级分进行纯化。 MALDI-MS和PSD分析。 使用这种方法，我们能够 鉴定a亚基中的胞质定向区域， Met 1-Arg 92、Ser 165-Arg 280、Val 351-Lys 785、Ala 838-Lys 851和 Phe997-Tyr1035。 因此，α-亚基的N-和C-末端均 Asn 226和Asn 731均为胞浆型， 糖基化 我们目前对胰蛋白酶的观察结果与 与α-亚基的10个跨膜片段假设一致。 胰凝乳蛋白酶分析似乎进一步定义了 H，K-ATPase的950-1016区。 全面分析 胰蛋白酶和胰凝乳蛋白酶释放的肽，以及用 将进行膜侧试剂，以达到拓扑 H，K-ATPase的模型。

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.