Rapid purification of the gastric H+/K(+)-ATPase complex by tomato-lectin affinity chromatography.
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.
Callaghan
J M
JM
Department of Pathology and Immunology, Monash University Medical School, Prahran, Victoria, Australia.
Toh
B H
BH
Simpson
R J
RJ
Baldwin
G S
GS
Gleeson
P A
PA
D000251:Adenosine Triphosphatases / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity / Q000379:methods; D006026:Glycoside Hydrolases; D017506:H(+)-K(+)-Exchanging ATPase; D037102:Lectins; D008566:Membranes / Q000201:enzymology; D008969:Molecular Sequence Data; D013270:Stomach / Q000201:enzymology*; D013552:Swine
D037102:Lectins; D006026:Glycoside Hydrolases; C020668:glycanase; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase