Two Ca²⁺-dependent ATPases in rat liver plasma membrane. The previously purified (Ca²⁺-Mg²⁺)-ATPase is not a Ca²⁺-pump but an ecto-ATPase

We have shown that the rat liver plasma membrane has at least two (Ca²⁺-Mg²⁺)-ATPases. One of them has the properties of a plasma membrane Ca²⁺-pump (Lin, S.-H. (1985) J. Biol. Chem. 260, 7850-7856); the other one, which we have purified (Lin, S.-H., and Fain, J.N. (1984) J. Biol. Chem. 259, 3016-3020) and characterized (Lin, S.-H. (1985) J. Biol. Chem. 260, 10976-10980) has no established function. In this study we present evidence that the purified (Ca²⁺-Mg²⁺)-ATPase is a plasma membrane ecto-ATPase. In hepatocytes in primary culture, we can detect Ca²⁺-ATPase and Mg²⁺-ATPase activities by addition of ATP to the intact cells. The external localization of the active site of the ATPase was confirmed by the observation that the Ca²⁺-ATPase and Mg²⁺-ATPase activities were the same for intact cells, saponin-treated cells, and cell homogenates. Less than 14% of total intracellular lactate dehydrogenase, a cytosolic enzyme, was released during a 30-min incubation of the hepatocytes with 2 mM ATP. This indicates that the hepatocytes maintained cytoplasmic membrane integrity during the 30-min incubation with ATP, and the Ca²⁺-ATPase and Mg²⁺-ATPase activity measured in the intact cell preparation was due to cell surface ATPase activity. The possibility that the ecto-Ca²⁺-ATPase and Mg²⁺-ATPase may be the same protein as the previously purified (Ca²⁺-Mg²⁺)-ATPase was tested by comparing the properties of the ecto-ATPase with those of (Ca²⁺-Mg²⁺)-ATPase. Both the ecto-ATPase and the (Ca²⁺-Mg²⁺)-ATPase have broad nucleotide-hydrolyzing activity, i.e. they both hydrolyze ATP, GTP, UTP, CTP, ADP, and GDP to a similar extent. The effect of Ca²⁺ and Mg²⁺ on the ecto-ADPase activity is not additive indicating that both Ca²⁺- and Mg²⁺-ATPase activities are part of the same enzyme. The ecto-ATPase activity, like the (Ca²⁺-Mg²⁺)-ATPase, is not sensitive to oligomycin, vanadate, N-ethylmaleimide and p-chloromercuribenzoate; and both the ecto-ATPase and purified (Ca²⁺-Mg²⁺)-ATPase activities are insensitive to protease treatments. These properties indicate that the previously purified (Ca²⁺-Mg²⁺)-ATPase is an ecto-ATPase and may function in regulating the effect of ATP and ADP on hepatocyte Ca²⁺ mobilization (Charest, R., Blackmore, P.F., and Exton, J.H. (1985) J. Biol. Chem. 260, 15789-15794).