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. 2000 Jan 15;345(Pt 2):239–245.

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

J J Lacapère 1, J C Robert 1, A Thomas-Soumarmon 1
PMCID: PMC1220752  PMID: 10620500

Abstract

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.

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Selected References

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  1. Bayle D., Benkouka F., Robert J. C., Peranzi G., Soumarmon A. Immunopurification of gastric parietal cell tubulovesicles. Comp Biochem Physiol B. 1992 Apr;101(4):519–525. doi: 10.1016/0305-0491(92)90331-k. [DOI] [PubMed] [Google Scholar]
  2. Benkouka F., Péranzi G., Robert J. C., Lewin M. J., Soumarmon A. A monoclonal antibody which inhibits H+/K(+)-ATPase activity but not chloride conductance. Biochim Biophys Acta. 1989 Dec 28;987(2):205–211. doi: 10.1016/0005-2736(89)90546-4. [DOI] [PubMed] [Google Scholar]
  3. Callaghan J. M., Toh B. H., Simpson R. J., Baldwin G. S., Gleeson P. A. Rapid purification of the gastric H+/K(+)-ATPase complex by tomato-lectin affinity chromatography. Biochem J. 1992 Apr 1;283(Pt 1):63–68. doi: 10.1042/bj2830063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Coll R. J., Murphy A. J. Purification of the CaATPase of sarcoplasmic reticulum by affinity chromatography. J Biol Chem. 1984 Nov 25;259(22):14249–14254. [PubMed] [Google Scholar]
  5. Cornelius F., Skou J. C. Reconstitution of (Na+ + K+)-ATPase into phospholipid vesicles with full recovery of its specific activity. Biochim Biophys Acta. 1984 May 30;772(3):357–373. doi: 10.1016/0005-2736(84)90153-6. [DOI] [PubMed] [Google Scholar]
  6. Hebert H., Xian Y., Hacksell I., Mårdh S. Two-dimensional crystals of membrane-bound gastric H,K-ATPase. FEBS Lett. 1992 Mar 9;299(2):159–162. doi: 10.1016/0014-5793(92)80237-b. [DOI] [PubMed] [Google Scholar]
  7. Knol J., Sjollema K., Poolman B. Detergent-mediated reconstitution of membrane proteins. Biochemistry. 1998 Nov 17;37(46):16410–16415. doi: 10.1021/bi981596u. [DOI] [PubMed] [Google Scholar]
  8. Kragh-Hansen U., le Maire M., Møller J. V. The mechanism of detergent solubilization of liposomes and protein-containing membranes. Biophys J. 1998 Dec;75(6):2932–2946. doi: 10.1016/S0006-3495(98)77735-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kragh-Hansen U., le Maire M., Nöel J. P., Gulik-Krzywicki T., Møller J. V. Transitional steps in the solubilization of protein-containing membranes and liposomes by nonionic detergent. Biochemistry. 1993 Feb 16;32(6):1648–1656. doi: 10.1021/bi00057a032. [DOI] [PubMed] [Google Scholar]
  10. Lacapère J. J., Stokes D. L., Olofsson A., Rigaud J. L. Two-dimensional crystallization of Ca-ATPase by detergent removal. Biophys J. 1998 Sep;75(3):1319–1329. doi: 10.1016/S0006-3495(98)74050-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lambert O., Levy D., Ranck J. L., Leblanc G., Rigaud J. L. A new "gel-like" phase in dodecyl maltoside-lipid mixtures: implications in solubilization and reconstitution studies. Biophys J. 1998 Feb;74(2 Pt 1):918–930. doi: 10.1016/S0006-3495(98)74015-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Le Maire M., Moller J. V., Tanford C. Retention of enzyme activity by detergent-solubilized sarcoplasmic Ca2+ -ATPase. Biochemistry. 1976 Jun 1;15(11):2336–2342. doi: 10.1021/bi00656a014. [DOI] [PubMed] [Google Scholar]
  13. Lund S., Orlowski S., de Foresta B., Champeil P., le Maire M., Møller J. V. Detergent structure and associated lipid as determinants in the stabilization of solubilized Ca2+-ATPase from sarcoplasmic reticulum. J Biol Chem. 1989 Mar 25;264(9):4907–4915. [PubMed] [Google Scholar]
  14. Møller J. V., Juul B., le Maire M. Structural organization, ion transport, and energy transduction of P-type ATPases. Biochim Biophys Acta. 1996 May 6;1286(1):1–51. doi: 10.1016/0304-4157(95)00017-8. [DOI] [PubMed] [Google Scholar]
  15. Møller J. V., le Maire M. Detergent binding as a measure of hydrophobic surface area of integral membrane proteins. J Biol Chem. 1993 Sep 5;268(25):18659–18672. [PubMed] [Google Scholar]
  16. Nandi J., Zhou M. A., Ray T. K. Purification and partial characterization of the (H+,K+)-transporting adenosinetriphosphatase from fundic mucosa. Biochemistry. 1987 Jul 14;26(14):4264–4272. doi: 10.1021/bi00388a013. [DOI] [PubMed] [Google Scholar]
  17. Okamoto C. T., Karpilow J. M., Smolka A., Forte J. G. Isolation and characterization of gastric microsomal glycoproteins. Evidence for a glycosylated beta-subunit of the H+/K(+)-ATPase. Biochim Biophys Acta. 1990 Mar 1;1037(3):360–372. doi: 10.1016/0167-4838(90)90038-h. [DOI] [PubMed] [Google Scholar]
  18. Pettitt J. M., Humphris D. C., Barrett S. P., Toh B. H., van Driel I. R., Gleeson P. A. Fast freeze-fixation/freeze-substitution reveals the secretory membranes of the gastric parietal cell as a network of helically coiled tubule. A new model for parietal cell transformation. J Cell Sci. 1995 Mar;108(Pt 3):1127–1141. doi: 10.1242/jcs.108.3.1127. [DOI] [PubMed] [Google Scholar]
  19. Rabon E. C., Bassilian S., Jakobsen L. J. Glutaraldehyde crosslinking analysis of the C12E8 solubilized H,K-ATPase. Biochim Biophys Acta. 1990 Jul 6;1039(3):277–289. doi: 10.1016/0167-4838(90)90260-m. [DOI] [PubMed] [Google Scholar]
  20. Rabon E. C., Gunther R. D., Bassilian S., Kempner E. S. Radiation inactivation analysis of oligomeric structure of the H,K-ATPase. J Biol Chem. 1988 Nov 5;263(31):16189–16194. [PubMed] [Google Scholar]
  21. Rabon E., Gunther R. D., Soumarmon A., Bassilian S., Lewin M., Sachs G. Solubilization and reconstitution of the gastric H,K-ATPase. J Biol Chem. 1985 Aug 25;260(18):10200–10207. [PubMed] [Google Scholar]
  22. Robert J. C., Benkouka F., Bayle D., Hervatin F., Soumarmon A. H+/K(+)-ATPase contents of human, rabbit, hog and rat gastric mucosa. Biochim Biophys Acta. 1990 May 9;1024(1):167–172. doi: 10.1016/0005-2736(90)90220-i. [DOI] [PubMed] [Google Scholar]
  23. Saccomani G., Shah G., Spenney J. G., Sachs G. Characterization of gastric mucosal membranes. VIII. The localization of peptides by iodination and phosphorylation. J Biol Chem. 1975 Jun 25;250(12):4802–4809. [PubMed] [Google Scholar]
  24. Schägger H., von Jagow G. Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem. 1991 Dec;199(2):223–231. doi: 10.1016/0003-2697(91)90094-a. [DOI] [PubMed] [Google Scholar]
  25. Shin J. M., Sachs G. Dimerization of the gastric H+, K(+)-ATPase. J Biol Chem. 1996 Jan 26;271(4):1904–1908. doi: 10.1074/jbc.271.4.1904. [DOI] [PubMed] [Google Scholar]
  26. Shin J. M., Sachs G. Identification of a region of the H,K-ATPase alpha subunit associated with the beta subunit. J Biol Chem. 1994 Mar 25;269(12):8642–8646. [PubMed] [Google Scholar]
  27. Soumarmon A., Abastado M., Bonfils S., Lewin M. J. C1-transport in gastric micorsomes. An ATP-dependent influx sensitive to membrane potential and to protein kinase inhibitor. J Biol Chem. 1980 Dec 25;255(24):11682–11687. [PubMed] [Google Scholar]
  28. Soumarmon A., Grelac F., Lewin M. J. Solubilization of active (H+ + K+)-ATPase from gastric membrane. Biochim Biophys Acta. 1983 Aug 10;732(3):579–585. doi: 10.1016/0005-2736(83)90234-1. [DOI] [PubMed] [Google Scholar]
  29. Soumarmon A., Robert J. C., Lewin M. J. Depolymerization of solubilized gastric (H+ + K+)-ATPase by n-octylglucoside or cholate. Biochim Biophys Acta. 1986 Aug 7;860(1):109–117. doi: 10.1016/0005-2736(86)90504-3. [DOI] [PubMed] [Google Scholar]
  30. Tai M. M., Im W. B., Davis J. P., Blakeman D. P., Zurcher-Neely H. A., Heinrikson R. L. Evidence for the presence of a carbohydrate moiety in fluorescein isothiocyanate labeled fragments of rat gastric (H+-K+)-ATPase. Biochemistry. 1989 Apr 18;28(8):3183–3187. doi: 10.1021/bi00434a011. [DOI] [PubMed] [Google Scholar]
  31. Takaya J., Omori K., Taketani S., Kobayashi Y., Tashiro Y. Solubilization, purification, and characterization of (H+, K+)ATPase from hog gastric microsomes. J Biochem. 1987 Oct;102(4):903–911. doi: 10.1093/oxfordjournals.jbchem.a122131. [DOI] [PubMed] [Google Scholar]
  32. Tyagarajan K., Chow D. C., Smolka A., Forte J. G. Structural interactions between alpha- and beta-subunits of the gastric H,K-ATPase. Biochim Biophys Acta. 1995 May 24;1236(1):105–113. doi: 10.1016/0005-2736(95)00044-4. [DOI] [PubMed] [Google Scholar]
  33. Usumoto R., Okada Y., Tsuji T. Effect of proton pump inhibitor, omeprazole, on expression of rat parietal cell specific carbohydrate antigen, type 2 chain N-acetyllactosamine. Dig Dis Sci. 1994 Jan;39(1):169–176. doi: 10.1007/BF02090078. [DOI] [PubMed] [Google Scholar]
  34. Vinson P. K., Talmon Y., Walter A. Vesicle-micelle transition of phosphatidylcholine and octyl glucoside elucidated by cryo-transmission electron microscopy. Biophys J. 1989 Oct;56(4):669–681. doi: 10.1016/S0006-3495(89)82714-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Walter A., Vinson P. K., Kaplun A., Talmon Y. Intermediate structures in the cholate-phosphatidylcholine vesicle-micelle transition. Biophys J. 1991 Dec;60(6):1315–1325. doi: 10.1016/S0006-3495(91)82169-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Xian Y., Hebert H. Three-dimensional structure of the porcine gastric H,K-ATPase from negatively stained crystals. J Struct Biol. 1997 Apr;118(3):169–177. doi: 10.1006/jsbi.1997.3847. [DOI] [PubMed] [Google Scholar]
  37. Young H. S., Rigaud J. L., Lacapère J. J., Reddy L. G., Stokes D. L. How to make tubular crystals by reconstitution of detergent-solubilized Ca2(+)-ATPase. Biophys J. 1997 Jun;72(6):2545–2558. doi: 10.1016/S0006-3495(97)78898-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. van Driel I. R., Callaghan J. M. Proton and potassium transport by H+/K(+)-ATPases. Clin Exp Pharmacol Physiol. 1995 Dec;22(12):952–960. doi: 10.1111/j.1440-1681.1995.tb02332.x. [DOI] [PubMed] [Google Scholar]

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