RAPID PURIFICATION OF THE GASTRIC H+/K+-ATPASE COMPLEX BY TOMATO-LECTIN AFFINITY-CHROMATOGRAPHY
- Author(s)
- CALLAGHAN, JM; Toh, BH; Simpson, RJ; Baldwin, GS; Gleeson, PA;
- Journal Title
- BIOCHEMICAL JOURNAL
- Publication Type
- Journal Article
- Abstract
- 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, Moritz, 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.
- Publisher
- PORTLAND PRESS
- Keywords
- AMINO-ACID-SEQUENCE; BETA-SUBUNIT; AUTOIMMUNE GASTRITIS; ADENOSINE-TRIPHOSPHATASE; PURIFIED MICROSOMES; PERNICIOUS-ANEMIA; MUCOSAL MEMBRANES; FUNDIC MUCOSA; CDNA CLONING; K+-ATPASE
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Creation Date: 1992-04-01 12:00:00