Efficient solubilization and purification of the gastric H+,K+-ATPase for functional and structural studies

When gastric H+,K+-ATPase-containing microsomes are solubilized by detergen ts, a rapid loss of ATPase activity is generally observed. In this article, SDS/PAGE of octa(ethylene glycol)dodecyl monoether (C12E8)- and n-dodecyl beta-D-maltoside-solubilized microsomes and their purifications by affinity chromatography on Reactive Red column reveal that inactivation is due to t wo main effects. (i) Solubilization activates an aspartic protease that cle aves down the alpha-subunit of the H+,K+-ATPase. Addition of pepstatin A at slightly acidic pH and at low temperature prevents the proteolysis. (ii) A too-harsh delipidation inactivates the ATPase. When n-dodecyl-beta-D-malto side is the detergent, the soluble H+,K+-ATPase is highly active (2.5 mu mo l/mg per h at pH 6.0 and 5 degrees C) as long as ATP is added. When C12E8 i s used, the detergent induces an inactivation due to delipidation, since ad dition of lipids restores activity. The two subunits of the H+,K+-ATPase ar e present in equimolar ratio in the n-dodecyl beta-D-maltoside-purified com plex. Moreover, two main types of complex (330 and 660 kDa) were resolved i n nondenaturing gels and should be the dimeric (alpha beta)(2) and tetramer ic (alpha beta)(4) heterodimers respectively. In conclusion, purification o f active, stable, soluble complexes of H+,K+-ATPase with few lipids (a lipi d/protein ratio of 0.25, w/w) has been achieved. This material should be us eful for further structural studies.