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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(Kcccived 2 June128 Augusl 1995) -ESR 95 0889/6 The putative phospholipase C inhibitor, U73122, transiently increascs the cytosolic free Ca' ' concentration in rabbit pancreatic acinar cells by stimulating the release of CaZt froin intracellular stores [Willems, Van de Put, Engbersen, Bosch, Van Hoof & De Pont (1994) Pfliigers Arch. 427, 233-2431. In order to elucidate the exact mechanism of action or U73122 we studied its cffects on both Ca' ' -stimulated Mg"-dependent ATPase activity and Ca2 ' -sti tnulated ATP-dependent Ca2+ uptake in rat liver microsotnes. In addition, we studied its effects on Ca' ' release from steady-state loaded microsoincs. The effects of U73122 were compared with those or thimerosal, describcd in the literature as inhibiting CaZ+-ATPases and scnsitizing inositol 1,4,5-trisphosphate-operated Ca2+ release channels, and thapsigargin, a specific inhibitor of sarcnplasmic and endoplasmic reticuluni C$+-ATPases. Both U73122 (lC5,, = 9 pM) and thimerosal (1C5() = 11 pM) dose-dependently inhibited Ca''-stimulated Mg' ' -dependent ATPase activity, without significantly affecting Mg"-stimulated ATPasc activity. Similarly, both U73122 (IC<,, = 9 pM) and thimerosal (IC5,, = 14 pM) dose-dependently inhibited ATP-dependent Ca2+ uptake. At concentrations beyond 20 pM, U73122 stimulatcd Ca'+ release from stcady-stale loaded microsomcs at a rate considerably higher than obtained with a maximally inhibitory concentration of thapsigargin ('I pM). This observation, which was not reached with equally inhibitory concentrations of thimerosal, demonstrates that higher U73 122 concentrations cause an additional increase of the passive Ca2+ lcak. The data presented demonstratc that U73122 stimulates the release of actively stored Ca2+ primarily through inhibition of the internal Ca2+ pump.Keywords: U73122 ; thimerosal ; Ca2+-ATPase; endoplasmic reticulum ; hepatocytes. In order to explorc the involverncnt of phospholipase C in the mechanism of action of Ca2+-mobilizing stimuli, recent studies havc used the aininosteroid U73 122 [ S -101. lJ731 22 was originally described as a potent inhibitor of platelet aggregation induced by a variety of agoni ts 151. The inhibitory action of U73122 was paralleled by inhib ion of the agonist-stimulated

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