Analysis of gastric acid secretary mechanism by identifying proton pomp beta subunit binding protein

我们制备的多肽由约30个氨基酸组成，存在于质子泵H+K+ATPaseβ亚基N端的胞内部分。将这些多肽应用于亲和小球，制备该柱是为了鉴定与这些多肽结合的未知蛋白质。将大鼠胃粘膜裂解液上柱，提取未知蛋白质，然后进行电泳法。电泳法测定分离蛋白的氨基酸序列。用免疫组织化学方法研究了该蛋白在胃粘膜壁细胞中的定位。制备同位素标记的GST-细胞内β亚基部分融合蛋白作为表达克隆的探针。已有研究表明，无论有无胃酸分泌刺激，都存在调节质子泵功能的蛋白质。利用亲和柱系统和GST融合蛋白进行表达克隆，我们可以分离出与质子泵H+K+ATPaseβ亚基结合的候选蛋白，并阐明结合蛋白与β亚基的相互作用。

We made peptides constituted about 30 amino acids, which exist intracellular portion of N end of proton pump H+K+ATPase beta subunit. Applying this peptides to the affinylbeads, the column was prepared for the purpose of identification of unknown proteins that bind to those peptides. Rat gastric mucosa lysate was applied to the column and the extraction of unknown proteins was done, followed by electrophoresis. Amino acid sequencing of the isolated protein was done after electrophoresis. The localization of this protein in the parietal cells in gastric mucosa was studied by immunohistochemical method. Isotope labeled GST-intracellular beta subunit portion fusion protein as the probe for expression cloning was prepared. It has been suggested that there are proteins which regulate proton pump function with or without the stimuli of gastric acid secretion. Using the affinity column system and expression cloning conducting by GST fusion protein, we could isolate the candidate protein that binds to beta submit of proton pump H+K+ATPase and clarify the interaction between the binding proteins and beta subunit.