A novel approach to construct a single recombinant baculovirus expressing two protein subunits simultaneously by replacing polyhedrin as well as p10 coding sequences is described. The recombinant baculovirus expressed the α‐ as well as the β‐subunit of the gastric H.K‐ATPase. Sf9 cells infected with this virus exhibited a K+‐ and SCH 28080‐sensitive ATP‐dependent phosphorylation capacity in purified Sf9 membranes similar to Natlve H,K‐ATPase. This activity was not present in control membranes containing only one of the two H,K‐ATPase subunits. We therefore conclude that both subunits are essential for the phosphorylation capacity of H.K‐ATPase.

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The monoclonal antibody 5-B6, directed against the alpha-subunit of pig gastric H+,K(+)-ATPase (Mg(2+)-dependent H(+)-transporting and K(+)-stimulated ATPase), was shown to be a potent inhibitor of the K(+)-ATPase activity, thereby binding to the cytoplasmic side of the alpha-subunit of the enzyme [Van Uem, Peters & De Pont (1990) Biochim. Biophys. Acta 1023, 56-62]. In order to define the epitope for 5-B6 on pig gastric H+,K(+)-ATPase more precisely, the alpha-subunit of the enzyme was subjected to limited proteolysis followed by chemical cleavage. Restricted proteolysis with papain followed by sequence analysis yielded an immunoreactive fragment of 27 kDa beginning at Ser379. This fragment was water-soluble and possessed the fluorescein isothiocyanate-reaction site. Limited tryptic digestion in the presence of K+ gave rise to an immunoreactive 56 kDa fragment beginning at Ile456, thus restricting the location of the epitope from Ile456 to the C-terminal end of the 27 kDa fragment (around residue 620). Further degradation of the 27 kDa fragment by means of formic acid cleavage at Asp-Pro bonds resulted initially in the formation of two non-immunoreactive fragments of 17 kDa and 11 kDa, indicating that the epitope for 5-B6 has to be localized around the chemical cleavage sites Asp507 and/or Asp510. Comparison of the primary structure of the alpha-subunits of gastric H+,K(+)-ATPase and non-immunoreactive rat kidney Na+,K(+)-ATPase shows almost no similarity for the sequence containing these formic acid cleavage sites (Thr504-Leu-Glu-Asp-Pro-Arg-Asp-Pro-Arg512), whereas the adjacent sequences are nearly 100% identical. These findings strongly suggest that the epitope for 5-B6 includes (part of) this sequence.

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