TOPOLOGICAL ORGANIZATION OF GASTRIC H,K ATPASE

H，K-ATPase和Na，K-ATPase是异源二聚体P型ATPase 由一个a亚基组成的，它穿过细胞膜几次 其大部分质量分布在细胞质中，并且其b亚基 只穿过一次膜，大部分质量都在管腔内 处理掉了。关于拓扑学和 A-亚基跨膜片段的特定数量，从 7-12。以前的方法包括蛋白质分解，然后是费力的 Edman测序法鉴定跨膜多肽 区域特异性抗体。由于多肽的数量很多， 定义拓扑是一个复杂的问题。在这里，我们使用矩阵 辅助激光解吸电离质谱仪 确定胃H，K-ATPase面向细胞质的区域。 兔H，K-ATPase富含胞质倒置囊泡的分离 胃被胰酶消化并释放多肽，并通过 用MALDI-MS获得大量的细胞质多肽。胰酶 用反相高效液相色谱分离多肽，并对分离出的多肽进行分离。 MALDI-MS和PSD分析。使用这种方法，我们能够 鉴定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-末端和C-末端 Asn226和Asn731不是细胞质 糖基化。我们目前对胰酶的观察结果是一致的 与a-亚基的10个跨膜片段假说一致。 用胰凝乳酶的分析似乎进一步定义了 H，K-ATPase的950-1016区。完整的分析 胰酶和胰凝乳酶释放的多肽，以及标记 膜侧试剂将被执行以获得拓扑结构 H，K-ATPase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.