Immunopurification of gastric parietal cell tubulovesicles.
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.
Bayle
D
D
INSERM Unité 10, Hôpital Bichat, Paris, France.
Benkouka
F
F
Robert
J C
JC
Peranzi
G
G
Soumarmon
A
A
D000251:Adenosine Triphosphatases / Q000276:immunology; D000818:Animals; D000911:Antibodies, Monoclonal; D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D017506:H(+)-K(+)-Exchanging ATPase; D007425:Intracellular Membranes / Q000201:enzymology; D010295:Parietal Cells, Gastric / Q000201:enzymology*; D011233:Precipitin Tests / Q000379:methods; D011817:Rabbits; D013552:Swine
D000911:Antibodies, Monoclonal; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase