The gastric H+/K+ ‐transporting adenosine triphosphatase (H+/K+ ATPase) (proton pump) consists of a catalytic α‐subunit and a recently proposed 60–90‐kDa glycoprotein β‐subunit. Using dog gastric membranes as the antigen, we have produced two murine monoclonal antibodies, 4F11 (IgG1) and 3A6 (IgA), which are specific for the 60–90‐kDa glycoprotein. The monoclonal antibodies (1) specifically stained the cytoplasm of unfixed and formalin‐fixed dog gastric parietal cells; (2) specifically reacted by ELISA with gastric tubulovesicular membranes; (3) recognised epitopes located on the luminal face of parietal cell tubulovesicular membranes, the site of the proton pump, by immunogold electron microscopy; (4) immunoblotted a 60–90‐kDa molecule from tubulovesicular membranes and a 35‐kDa component from peptide N‐glycosidase‐F‐treated membrane extracts; (5) immunoblotted the 60–90‐kDa parietal cell autoantibody‐ or tomato‐lectin–Sepharose 4B affinity columns, and the 35‐kDa protein core of this autoantigen; this autoantigen has amino acid sequence similarity to the β‐subunit of the related NH+/K+ ‐transporting adenosine triphosphatase (Na+/K+ ATPase) [Toh et al. (1990) Proc. Natl Acad. Sci. 87, 6418–6422]; (6) co‐precipitated a molecule of 95kDa with the 60–90‐kDa molecule from 125I‐labelled detergent extracts of dog tubulovesicular membranes; and (7) co‐purified the catalytic α‐subunit of the H+/K+ ATPase with the 60–90‐kDa molecule by immunoaffinity chromatography of tubulovesicular membrane extracts on a monoclonal antibody 3A6‐Sepharose 4B column, indicating a physical association between the two molecules. These results provide further evidence that the 60–90‐kDa glycoprotein is the β‐subunit of the gastric H+/K+ ATPase. We conclude that the monoclonal antibodies specifically recognise luminal epitopes on the 35‐kDa core protein of the 60–90‐kDa β‐subunit of the gastric proton pump, a major target molecule in autoimmune gastritis and pernicious anaemia. These monoclonal antibodies will be valuable probes to study the structure and function of this associated β‐subunit, as well as the ontogeny of the gastric proton pump.
|Number of pages||11|
|Journal||European Journal of Biochemistry|
|Publication status||Published - 1 Jan 1991|