Callaghan J M, Toh B H, Simpson R J, Baldwin G S, Gleeson P A
Department of Pathology and Immunology, Monash University Medical School, Prahran, Victoria, Australia.
Biochem J. 1992 Apr 1;283 ( Pt 1)(Pt 1):63-8. doi: 10.1042/bj2830063.
We have previously shown that tomato lectin binds specifically to the 60-90 kDa membrane glycoprotein of parietal cell tubulovesicles, the beta-subunit of the gastric H+/K(+)-ATPase (proton pump) [Callaghan, Toh, Pettitt, Humphris & Gleeson (1990) J. Cell Sci. 95, 563-576; Toh, Gleeson, Simpson, Mortiz, Callaghan, Goldkorn, Jones, Martinelli, Mu, Humphris, Pettitt, Mori, Masuda, Sobieszczuk, Weinstock, Mantamadiotis & Baldwin (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6418-6422]. Here we have exploited this interaction for the development of a rapid single-step chromatography procedure for the purification of an active pig gastric proton pump complex. Initially, H+/K(+)-ATPase-enriched membranes, prepared from pig gastric microsomes by density-gradient centrifugation, were extracted in 1% Triton X-100 and passed through a 1 ml tomato lectin-Sepharose 4B column. The bound material, eluted with 20 mM-chitotriose, showed a major band with an apparent molecular mass of 95 kDa, and a faint broad band of 60-90 kDa, by SDS/PAGE. N-Glycanase treatment of the bound material resulted in the appearance of a 35 kDa band, the size of the protein core of the 60-90 kDa glycoprotein beta-subunit. The two components were identified as the 95 kDa alpha-subunit and the 60-90 kDa beta-subunit of the gastric H+/K(+)-ATPase, by immunoreactivity with monospecific antibodies, and by tryptic peptide sequences of the tomato-lectin-bound material. The beta-subunit was present in approximately equimolar amounts to the catalytic alpha-subunit. Whereas the gastric H+/K(+)-ATPase was not active after solubilization in 1% Triton X-100, solubilization of density-gradient-purified membranes in the non-ionic detergent, C12E8, followed by chromatography of the extract on tomato lectin-Sepharose 4B, resulted in the purification of the gastric H+/K(+)-ATPase complex which exhibited K(+)-dependent phosphatase activity. This is the first report of a rapid purification of a partially active solubilized gastric H+/K(+)-ATPase complex.
我们之前已经表明，番茄凝集素能特异性结合壁细胞微管泡的60 - 90 kDa膜糖蛋白，即胃H⁺/K⁺-ATP酶（质子泵）的β亚基[卡拉汉、托、佩蒂特、汉弗里斯和格利森（1990年）《细胞科学杂志》95卷，563 - 576页；托、格利森、辛普森、莫里茨、卡拉汉、戈德 Korn、琼斯、马蒂内利、穆、汉弗里斯、佩蒂特、森、增田、索别斯祖克、温斯托克、曼塔马迪奥蒂斯和鲍德温（1990年）《美国国家科学院院刊》87卷，6418 - 6422页]。在此，我们利用这种相互作用开发了一种快速单步色谱法，用于纯化活性猪胃质子泵复合物。最初，通过密度梯度离心从猪胃微粒体中制备富含H⁺/K⁺-ATP酶的膜，将其在1% Triton X - 100中提取，然后通过1 ml番茄凝集素 - 琼脂糖4B柱。用20 mM - 壳三糖洗脱结合的物质，通过SDS/PAGE显示出一条表观分子量为95 kDa的主要条带和一条60 - 90 kDa的微弱宽带。对结合物质进行N - 糖苷酶处理后出现了一条35 kDa的条带，这是60 - 90 kDa糖蛋白β亚基的蛋白质核心大小。通过与单特异性抗体的免疫反应以及番茄凝集素结合物质的胰蛋白酶肽序列，确定这两个组分分别为胃H⁺/K⁺-ATP酶的95 kDaα亚基和60 - 90 kDaβ亚基。β亚基与催化性α亚基的含量大致相等。虽然胃H⁺/K⁺-ATP酶在1% Triton X - 100中溶解后无活性，但将密度梯度纯化的膜在非离子去污剂C12E8中溶解，然后将提取物在番茄凝集素 - 琼脂糖4B上进行色谱分离，得到了具有K⁺依赖性磷酸酶活性的胃H⁺/K⁺-ATP酶复合物的纯化产物。这是关于快速纯化部分活性溶解的胃H⁺/K⁺-ATP酶复合物的首次报道。
