Asano S, Tabuchi Y, Takeguchi N
Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University.
J Biochem. 1989 Dec;106(6):1074-9. doi: 10.1093/oxfordjournals.jbchem.a122967.
A monoclonal antibody (designated as HK4001) was prepared against hog gastric H+,K(+)-ATPase. It dose-dependently inhibited the H+,K(+)-ATPase activity, formation of the K(+)-sensitive phosphoenzyme, and proton uptake into gastric vesicles. The H+,K(+)-ATPase activity was completely inhibited by addition of the antibody at a molar ratio of 1:2 (antibody/catalytic subunit) at pH 7.8. The maximal inhibition decreased with decrease in pH of the medium (7.8 greater than 7.4 greater than 6.2). The Fab fragment obtained by digestion of the antibody with papain was also inhibitory. The antibody did not inhibit the K(+)-dependent p-nitrophenylphosphatase or the labeling of the enzyme with fluorescein isothiocyanate. It inhibited gastric H+,K(+)-ATPase from rabbits and rats, but did not cross-react with related cation-transport ATPases (Na+,K(+)-ATPase or Ca2(+)-ATPase) or H(+)-ATPase in the multivesicular body. From these and related findings, the antibody was suggested to recognize a highly specific site on the cytosolic surface of H+,K(+)-ATPase. The conformation of the epitope was conserved after treatment with Triton X-100, but not sodium dodecyl sulfate. In addition, judging from the stoichiometry of inactivation of H+,K(+)-ATPase by this antibody, the functional unit of H+,K(+)-ATPase was suggested to be a dimer or a tetramer (not a trimer) of the catalytic unit.
制备了一种针对猪胃H⁺,K⁺-ATP酶的单克隆抗体（命名为HK4001）。它呈剂量依赖性地抑制H⁺,K⁺-ATP酶活性、K⁺敏感磷酸酶的形成以及质子摄入胃小泡。在pH 7.8时，以1:2（抗体/催化亚基）的摩尔比加入该抗体可完全抑制H⁺,K⁺-ATP酶活性。最大抑制作用随培养基pH值降低而减小（7.8＞7.4＞6.2）。用木瓜蛋白酶消化该抗体得到的Fab片段也具有抑制作用。该抗体不抑制K⁺依赖性对硝基苯磷酸酶或用异硫氰酸荧光素对该酶的标记。它抑制兔和大鼠的胃H⁺,K⁺-ATP酶，但不与相关的阳离子转运ATP酶（Na⁺,K⁺-ATP酶或Ca²⁺-ATP酶）或多泡体中的H⁺-ATP酶发生交叉反应。根据这些及相关发现，提示该抗体识别H⁺,K⁺-ATP酶胞质表面的一个高度特异性位点。用Triton X-100处理后表位的构象得以保留，但用十二烷基硫酸钠处理后则不然。此外，根据该抗体使H⁺,K⁺-ATP酶失活的化学计量关系，提示H⁺,K⁺-ATP酶的功能单位是催化单位的二聚体或四聚体（而非三聚体）。
