Rapid purification of the gastric H+/K+-ATPase complex by tomato-lectin affinity chromatography

Abstract

We have previously shown that tomato lectin binds specifically to the 60-90 kDa membrane glycoprotein of parietal cell tubulovesicles, the β-subunit of the gastric H+/K+-ATPase (proton pump) [Callaghan, Toh, Pettitt, Humphris and Gleeson (1990) J. Cell Sci. 95, 563-576; Toh, Gleeson, Simpson, Moritz, Callaghan, Goldkorn, Jones, Martinelli, Mu, Humphris, Pettitt, Mori, Masuda, Sobieszczuk, Weinstock, Mantamadiotis and 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. 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 subunit. The two components were identified as the 95 kDa α-subunit and the 60-90 kDa β-subunit of the gastric H+/K+-ATPase by immunoreactivity with monospecific antibodies and by tryptic peptide sequences of the tomato-lectin-bound material. The β-subunit was present in approximately equimolar amounts to the catalytic α-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.