Jones C M, Toh B H, Pettitt J M, Martinelli T M, Humphris D C, Callaghan J M, Goldkorn I, Mu F T, Gleeson P A
Department of Pathology and Immunology, Monash University Medical School, Melbourne, Australia.
Eur J Biochem. 1991 Apr 10;197(1):49-59. doi: 10.1111/j.1432-1033.1991.tb15881.x.
The gastric H+/K(+)-transporting adenosine triphosphatase (H+/K+ ATPase) (proton pump) consists of a catalytic alpha-subunit and a recently proposed 60-90-kDa glycoprotein beta-subunit. Using dog gastric membranes as the antigen, we have produced two murine monoclonal antibodies, 4F11 (IgG1) and 3A6 (IgA), which are specific for the 60-90-kDa glycoprotein. The monoclonal antibodies (1) specifically stained the cytoplasm of unfixed and formalin-fixed dog gastric parietal cells; (2) specifically reacted by ELISA with gastric tubulovesicular membranes; (3) recognised epitopes located on the luminal face of parietal cell tubulovesicular membranes, the site of the proton pump, by immunogold electron microscopy; (4) immunoblotted a 60-90-kDa molecule from tubulovesicular membranes and a 35-kDa component from peptide N-glycosidase-F-treated membrane extracts; (5) immunoblotted the 60-90-kDa parietal cell autoantigen associated with autoimmune gastritis and pernicious anemia, purified by chromatography on parietal cell autoantibody- or tomato-lectin-Sepharose 4B affinity columns, and the 35-kDa protein core of this autoantigen; this autoantigen has amino acid sequence similarity to the beta-subunit of the related Na+/K(+)-transporting adenosine triphosphatase (Na+/K+ ATPase) [Toh et al. (1990) Proc. Natl Acad. Sci. 87, 6418-6422]; (6) co-precipitated a molecule of 95 kDa with the 60-90-kDa molecule from 125I-labelled detergent extracts of dog tubulovesicular membranes; and (7) co-purified the catalytic alpha-subunit of the H+/K+ ATPase with the 60-90-kDa molecule by immunoaffinity chromatography of tubulovesicular membrane extracts on a monoclonal antibody 3A6-Sepharose 4B column, indicating a physical association between the two molecules. These results provide further evidence that the 60-90-kDa glycoprotein is the beta-subunit of the gastric H+/K+ ATPase. We conclude that the monoclonal antibodies specifically recognise luminal epitopes on the 35-kDa core protein of the 60-90-kDa beta-subunit of the gastric proton pump, a major target molecule in autoimmune gastritis and pernicious anaemia. These monoclonal antibodies will be valuable probes to study the structure and function of this associated beta-subunit, as well as the ontogeny of the gastric proton pump.
胃H⁺/K⁺转运ATP酶（质子泵）由一个催化性α亚基和一个最近提出的60 - 90kDa糖蛋白β亚基组成。我们以犬胃膜作为抗原，制备了两种鼠单克隆抗体，4F11（IgG1）和3A6（IgA），它们对60 - 90kDa糖蛋白具有特异性。这些单克隆抗体：（1）能特异性地对未固定和经福尔马林固定的犬胃壁细胞的细胞质进行染色；（2）通过酶联免疫吸附测定法与胃微管泡膜发生特异性反应；（3）通过免疫金电子显微镜识别位于壁细胞微管泡膜腔面（质子泵所在部位）的表位；（4）对微管泡膜中的一个60 - 90kDa分子以及经肽N - 糖苷酶 - F处理的膜提取物中的一个３５kDa成分进行免疫印迹分析；（5）对通过壁细胞自身抗体或番茄凝集素 - Sepharose 4B亲和柱层析纯化的与自身免疫性胃炎和恶性贫血相关的60 - 90kDa壁细胞自身抗原以及该自身抗原的35kDa蛋白核心进行免疫印迹分析；这种自身抗原与相关的Na⁺/K⁺转运ATP酶（Na⁺/K⁺ATP酶）的β亚基具有氨基酸序列相似性 [Toh等人（1990年）《美国国家科学院院刊》87，6418 - 6422]；（6）从犬微管泡膜的¹²⁵I标记去污剂提取物中与60 - 90kDa分子共沉淀出一个95kDa的分子；（7）通过在单克隆抗体3A6 - Sepharose 4B柱上对微管泡膜提取物进行免疫亲和层析，将H⁺/K⁺ATP酶的催化性α亚基与60 - 90kDa分子共纯化，表明这两个分子之间存在物理关联。这些结果进一步证明60 - 90kDa糖蛋白是胃H⁺/K⁺ATP酶的β亚基。我们得出结论，这些单克隆抗体特异性识别胃质子泵60 - 90kDaβ亚基的35kDa核心蛋白上的腔表位，该核心蛋白是自身免疫性胃炎和恶性贫血中的一个主要靶分子。这些单克隆抗体将成为研究这种相关β亚基的结构和功能以及胃质子泵个体发生的有价值的探针。
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