STRUCTURAL ELUCIDATION OF GASTRIC H+, K+ ATPASE

胃H+，K+-ATPase是一种阳离子逆向转运蛋白，包括 M1=94,000个亚基的a和b亚基，m1=60-80,000， 分别为(1)。克隆的大鼠a亚基由1033个氨基酸组成。 酸(相对分子质量=114,012)和5个潜在的糖基化位点(2)， 已被提议不以其糖基化为基础 疑似位于细胞膜的细胞质一侧， 不能与凝集素Sepharose柱结合，以及非特异性 高碘酸化后丹磺酰肼对α-亚基的染色 凝胶条带的处理(3)。我们最近证实， A-亚基绝对不是糖基化的基础 电印迹条带单糖分析(4)。克隆人 大鼠b亚基由294个氨基酸组成(相对分子质量为33,689)，其中7个 潜在的N-糖基化位点(5)。兔b亚基染色 与小麦胚芽结合的十二烷基硫酸钠凝胶上碳水化合物呈阳性 凝集素和蓖麻毒素A琼脂糖柱，改变了流动性 用PNGase处理，发现含有己糖和己糖胺 (3)。我们发现兔的b-亚基含有一定比例的半乳糖 和GlcNAc到人类，这表明存在乳糖胺型 低聚糖(4)。此外，对蛋白质上GalN的检测 在去除所有N-连接的寡糖后，表明存在 O-连接结构。B亚基被发现缺乏 唾液酸，唾液酸基转移酶活性以来的一个值得注意的发现 在胃肠道组织中具有区域特异性(6)。我们建议 扩展这些对该膜结构的初步研究 具有以下特定目的的糖蛋白。(1)确定 B-亚基糖型的精确相对分子质量 基质辅助激光解吸MS(2)定位 用LC/电喷雾MS分析糖基化(3) 阐明低聚糖的结构并最终定义 在每个肽轨迹上进行合奏。由于糖基化作用，b亚基 以涂片形式迁移，经十二烷基硫酸钠-PAGE分析，估计分子量为 60-80 kDa。精确测定个体的分子量 糖型可以估计糖基化的数量。 站点存在，并提供“全球”测量以指导 针对具体目标的额外结构研究的战略2和 3.糖基化的7个潜在N-连接位点的数目 或者，某些蛋白质是否被可变的糖基化还不得而知。 将使用LC/电喷雾MS来确定哪些N-连接位点是 糖基化并定位O-糖基化基因座。我们的第三个 其具体目的是阐明低聚糖的结构。上一首 使用高效液相图谱方法的研究表明， 酶促释放的b亚基上的寡糖不会 与任何常见的乳糖胺或高甘露糖共洗脱 低聚糖。使用HPLC的单寡糖将是 制备了它们，并用MALD-MS测定了它们的分子量。 结构将使用化学和化学相结合的方法来推断 酶促裂解和适当的质谱分析 可在中心购买。1.Forte，J.G.和Lee，H.C.(1977) 胃肠病学，73,921-2。2.Shull，G.E.和Lingrel，J.B.(1986) J.Biol.化学。261,16788-16791。3.冈本，C.T.，卡皮罗，J.M. 斯莫尔卡，A.和福特，J.G.(1990)Biochim。生物群落。Acta 1037， 360-372。4.WeitzHandler，M.，Kadlecik，D.，Avdalovic，N.，Forte， J.，Chow，D.和Townsen，R.R.接受J.Biol。化学。(2月/3月) 首页--期刊主要分类--期刊细介绍--期刊题录与文摘--期刊详细文摘内容 J.G.和Levenson，R.(1990)J.Biol.化学。265,19878-19884。6. J·J·泰杰斯·罗斯·J·韦恩斯坦·J·保尔森(1988)J. 化学。263、6302-6309。

The gastric H+,K+-ATPase is a cation antiporter which consists of a and b subunits with M1 = 94,000 subunits with M1 = 60-80,000, respectively (1). The cloned rat a subunit consists of 1033 amino acids (MW = 114,012) and 5 potential glycosylation sites (2), which have been proposed not to be glycosylated on the basis of their suspected location on the cytoplasmic side of the plasma membrane, the inability to bind to lectin Sepharose columns, and the non-specific staining of the a-subunit with dansyl hydrazine after periodate treatment of gel bands (3). We have recently confirmed that the a-subunit is definitively not glycosylated on the basis of monosaccharide analysis of the electro-blotted band (4). The cloned rat b subunit consists of 294 amino acids (MW = 33,689) with 7 potential N-glycosylation sites (5). The rabbit b subunit stained positively for carbohydrate on SDS gels, bound to wheat germ agglutinin and Ricin A Sepharose columns, changed mobility after treatment with PNGase and was found to contain hexose and hexosamines (3). We found that the b-subunit from rabbit contained a ratio of Gal and GlcNAc to Man which suggested the presence of lactosamine-type oligosaccharides (4). Further, the detection of GalN on the protein after removal of all N-linked oligosaccharides indicated the presence of O-linked structures. The b subunit was found to be devoid of sialic acids, a noteworthy finding since sialyl transferase activity is regio-specific in gastrointestinal tissues (6). We propose to extend these initial studies on the structure of this membrane glycoprotein with the following specific aims. (1) Determine the precise molecular weight of the glycoforms of the b-subunit using matrix-assisted laser desorption MS. (2) Locate the sites of glycosylation through analyses using LC/electrospray MS. (3) Elucidate the oligosaccharide structures and eventually define the ensemble at each eptide locus. Due to glycosylation, the b subunit migrates as a smear by SDS PAGE with an estimated molecular weight of 60-80 kDa. Precise molecular weight determination of the individual glycoforms may enable an estimation of the number of glycosylation sites which are present and provide "global" measurements to direct strategies for the additional structural studies in specific aims2 and 3. The number of the 7 potential N-linked sites which are glysoylated or whether somesites are variably glycosylated is not known. LC/electrospray MS will be used to ascertain which N-linked sites are glycosylated and to locate the O-glycosylated loci. Our third specific aim is to elucidate the oligosaccharide structures. Previous studies, using an HPLC mapping approach, indicated that the enzymatically-released oligosaccharides on the b subunit do not co-elute with any of the commonly found lactosamine or high-mannose oligosaccharides. Using HPLCsingular oligosaccharides will be prepared and their molecular weight determined using MALD-MS. Structures will be deduced using a combination of chemical and enzymatic cleavages and appropriate mass spectrometric analyses available at the Center. 1. Forte, J.G. and Lee, H.C. (1977) Gastroenterology, 73, 921-2. 2. Shull, G.E. and Lingrel, J.B. (1986) J. Biol. Chem. 261, 16788-16791. 3. Okamoto, C.T., Karpilow, J.M., Smolka, A. and Forte, J.G. (1990) Biochim. Biophys. Acta 1037, 360-372. 4. Weitzhandler, M., Kadlecik, D., Avdalovic, N., Forte, J., Chow, D. and Townsend, R.R. accepted J. Biol. Chem. (Feb/Mar) 5. Canfield, V.A., Okamoto, C.T., Chow, D., Dortman, J., Gros, P., Forte, J.G. and Levenson, R. (1990) J. Biol. Chem. 265, 19878-19884. 6. Taatjes, D.J., Roth, J., Weinstein, J., Paulson, J.C. (1988) J. Biol. Chem. 263, 6302-6309.