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 detergents, a rapid loss of ATPase activity is generally observed. In this article, SDS/PAGE of octa(ethylene glycol)dodecyl monoether (C(12)E(8))- and n-dodecyl beta-d-maltoside-solubilized microsomes and their purifications by affinity chromatography on Reactive Red column reveal that inactivation is due to two main effects. (i) Solubilization activates an aspartic protease that cleaves 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-maltoside is the detergent, the soluble H(+), K(+)-ATPase is highly active (2.5 micromol/mg per h at pH 6.0 and 5 degrees C) as long as ATP is added. When C(12)E(8) is used, the detergent induces an inactivation due to delipidation, since addition of lipids restores activity. The two subunits of the H(+), K(+)-ATPase are present in equimolar ratio in the n-dodecyl beta-d-maltoside-purified complex. Moreover, two main types of complex (330 and 660 kDa) were resolved in non-denaturing gels and should be the dimeric (alphabeta)(2) and tetrameric (alphabeta)(4) heterodimers respectively. In conclusion, purification of active, stable, soluble complexes of H(+),K(+)-ATPase with few lipids (a lipid/protein ratio of 0.25, w/w) has been achieved. This material should be useful for further structural studies.
Lacapère
J J
JJ
Section de Recherche, UMR-168 CNRS, LCR-8 CEA, Institut Curie, 11 rue P. et M. Curie, 75231 Paris, France. lacapere@curie.fr
Robert
J C
JC
Thomas-Soumarmon
A
A
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors; D002846:Chromatography, Affinity; D003902:Detergents; D004795:Enzyme Stability; D005026:Ethylene Glycols / Q000494:pharmacology; D005753:Gastric Mucosa / Q000201:enzymology*; D005960:Glucosides / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology; D010436:Pepstatins / Q000494:pharmacology; D011480:Protease Inhibitors; D000254:Sodium-Potassium-Exchanging ATPase / Q000302:isolation & purification*; D012995:Solubility; D013552:Swine; D013696:Temperature
D003902:Detergents; D005026:Ethylene Glycols; D005960:Glucosides; D010436:Pepstatins; D011480:Protease Inhibitors; C014195:Streptomyces pepsin inhibitor; C040358:dodecyl maltoside; D000255:Adenosine Triphosphate; D016282:Aspartic Acid Endopeptidases; D000254:Sodium-Potassium-Exchanging ATPase; C031375:pepstatin