Site-directed antibodies as topographical probes of the gastric H,K-ATPase alpha-subunit.
Site-directed antibodies as topographical probes of the gastric H,K-ATPase alpha-subunit.
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定点抗体作为胃 H,K-ATP 酶 α 亚基的拓扑探针。
DOI：
10.1016/0005-2736(92)90116-4
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
Swiger,KM
中科院分区：
文献类型：
--
作者：
Smolka,A;Swiger,KM
胃酸由 ATP 驱动的 H+ 和 K+ 交换剂（H,K-ATP 酶）分泌，这是壁细胞的一种完整的顶膜蛋白。尽管酶的一级结构已知，但其高级结构尚不确定。为了获得天然微粒体 H,K-ATP 酶的拓扑探针，将猪胃 H,K-ATP 酶 α 亚基的 17 个氨基末端（N 肽）和 16 个羧基末端（C 肽）残基对应的合成肽与匙孔血蓝蛋白 (KLH) 偶联。用肽-KLH 缀合物对兔子进行免疫，并通过酶联免疫吸附测定 (ELISA)、免疫印迹和免疫细胞化学测试其血清的特异性。所有血清均与吸附到微量滴定孔上的合成肽、肽-BSA 缀合物和微粒体 H,K-ATP 酶显示出高 ELISA 反应性（某些效价 > 1:104）。通过 SDS-PAGE 解析的 H,K-ATP 酶免疫印迹显示 N 肽和 C 肽抗体均与单个 94 kDa 条带发生反应。所有血清对猪胃粘膜切片中的壁细胞进行选择性染色。免疫前血清在所有测定中均给出阴性或弱信号。在竞争 ELISA 中，通过添加微粒体 H,K-ATP 酶，将 N 肽抗体（而非 C 肽抗体）从相应结合的合成肽中取代。其中一种N肽抗体抑制H,K-ATP酶活性超过50%；当 ATP 或 K+ 与酶结合时，该抗体的结合会减少。这些结果表明细胞质导向的 α 亚基 N 末端可能在构象上参与 H,K-ATP 酶催化循环，并表明针对合成 H,K-ATP 酶肽的抗体是天然微粒体 H,K-ATP 酶拓扑的潜在有用探针。
Gastric acid is secreted by an ATP-driven H+and K+exchanger (H,K-ATPase), an integral apical membrane protein of parictal cells. Although the primary structure of the enzyme is known, its higher order structure is uncertain. In order to acquire topographical probes of native, microsomal H,K-ATPase, synthetic peptides corresponding to the 17 amino-terminal (N-peptide) and 16 car☐yl-terminal (C-peptide) residues of pig gastric H,K-ATPase α-subunit were coupled to keyhole limpet hemocyanin (KLH). Rabbits were immunized with peptide-KLH conjugates and their sera were tested for specificity by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and immunocytochemistry. All sera showed high ELISA reactivities with synthetic peptides, peptide-BSA conjugates, and microsomal H,K-ATPase adsorbed to microtiter wells (some titers > 1:104). Immunoblots of H,K-ATPase resolved by SDS-PAGE showed both N-peptide and C-peptide antibodies reacting with a single 94 kDa band. All sera selectively stained parictal cells in pig gastric mucosal sections. Preimmune sera gave negative or weak signals in all assays. In competition ELISAs, N-peptide antibodies, but not C-peptide antibodies, were displaced from the corresponding bound synthetic peptides by added microsomal H,K-ATPase. One of the N-peptide antibodies inhibited H,K-ATPase activity by more than 50%; binding of this antibody was decreased when ATP or K+were bound to the enzyme. These results indicate a cytoplasmically-oriented α-subunit N-terminus which may participate conformationally in the H,K-ATPase catalytic cycle, and suggest that antibodies against synthetic H,K-ATPase peptides are potentially useful probes of native microsomal H,K-ATPase topography.