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Immunopurification of gastric parietal cell tubulovesicles
D Bayle1, F Benkouka, J C Robert
1INSERM Unité 10, Hôpital Bichat, Paris, France.
Abstract:
1. The tubulovesicles of hog and rabbit gastric parietal cells were immunopurified from microsomes using monoclonal antibodies against the (H+, K+)-ATPase. 2. The best yields of immunoprecipitation were obtained with an ATPase/mAb molar ratio of 0.3: the immunoprecipitate contained 79 and 90% of the hog and rabbit microsomal PNPPase activity respectively and K(+)-stimulated ATPase specific activity was 221 +/- 29 mumoles Pi per hr and per mg of membrane protein. 3. The immunoprecipitate contained vesicles that were 85% cytoplasmic-side out, like tubulovesicles in vivo, demonstrating that the epitopes were cytoplasmic. 4. The alpha-beta protomer of (H+, K+)-ATPase accounted for 80 +/- 12% of the immunopurified proteins. 5. The major other proteins ran at 80, 75, 69, 57, 47, 44, 39, 34 and 32 kDa on the SDS-PAGE. 6. Comparative analysis between sucrose-gradient purified fractions and immunopurified tubulovesicles demonstrated that carbonic anhydrase and actin were contaminants and that the 53 kDa and presumably the 50 kDa bands of the gradient fraction were alpha and beta subunits of F1 ATPase.
Insights
Researchers immunopurified gastric parietal cell tubulovesicles using antibodies against the H+/K+-ATPase. This method successfully isolated these key proton pump-containing vesicles for further study.
Area of Science:
- Biochemistry
- Cell Biology
- Physiology
Background:
- Gastric acid secretion relies on the H+/K+-ATPase proton pump located in parietal cell tubulovesicles.
- Understanding the precise composition and localization of these vesicles is crucial for comprehending gastric physiology.
Purpose of the Study:
- To develop and validate an immunopurification method for isolating tubulovesicles from hog and rabbit gastric parietal cells.
- To characterize the protein content and orientation of the immunopurified vesicles.
Main Methods:
- Immunoprecipitation using monoclonal antibodies against the H+/K+-ATPase.
- Enzyme activity assays (PNPPase and K+-stimulated ATPase).
- SDS-PAGE analysis to identify protein components.
Main Results:
- Optimal immunoprecipitation yields were achieved at a specific ATPase/mAb molar ratio.
- Immunopurified vesicles showed high K+-stimulated ATPase specific activity and were predominantly cytoplasmic-side out.
- The alpha-beta protomer of H+/K+-ATPase constituted a significant portion of the immunopurified proteins.
- Contaminants like carbonic anhydrase and actin were identified and excluded.
Conclusions:
- Immunopurification is an effective method for isolating functional tubulovesicles enriched in H+/K+-ATPase.
- The cytoplasmic orientation of epitopes was confirmed, validating the method's specificity.
- This technique provides a purified source of tubulovesicles for detailed biochemical and structural analysis.
