The Vacuolar ATPase: Sulfite Stabilization and the Mechanism of Nitrate Inactivation

Using vacuolar membranes from Neurospora crassa, we observed that sulfite prevented the loss of vacuolar ATPase activity that otherwise occurred during 36 h at room temperature. Sulfite neither activated nor changed the kinetic behavior of the enzyme. Further, in the presence of sulfite, the vacuolar ATPase was not inhibited by nitrate.We tested the hypothesis that sulfite acts as a reducing agent to stabilize the enzyme, while nitrate acts as an oxidizing agent, inhibiting the enzyme by promoting the formation of disulfide bonds. All reducing agents tested, dithionite, selenite, thiophosphate, dithiothreitol and glutathione, prevented the loss of ATPase activity. On the other hand, all oxidizing agents tested, bromate, iodate, arsenite, perchlorate, and hydrogen peroxide, were potent inhibitors of ATPase activity. The inhibitory effect of the oxidizing agents was specific for the vacuolar ATPase. The mitochondrial ATPase, assayed under identical conditions, was not inhibited by any of the oxidizing agents. Analysis of proteins with two-dimensional gel electrophoresis indicated that nitrate can promote the formation of disufide bonds between proteins in the vacuolar membrane. These data suggest a mechanism to explain why nitrate specifically inhibits vacuolar ATPases, and they support the proposal by Feng and Forgac (Feng, Y., and Forgac, M.(1994) J. Biol. Chem. 269, 13244-13230) that oxidation and reduction of critical cysteine residues may regulate the activity of vacuolar ATPases in vivo. Using vacuolar membranes from Neurospora crassa, we observed that sulfite prevented the loss of vacuolar ATPase activity that otherwise occurred during 36 h at room temperature. Sulfite neither activated nor changed the kinetic behavior of the enzyme. Further, in the presence of sulfite, the vacuolar ATPase was not inhibited by nitrate. We tested the hypothesis that sulfite acts as a reducing agent to stabilize the enzyme, while nitrate acts as an oxidizing agent, inhibiting the enzyme by promoting the formation of disulfide bonds. All reducing agents tested, dithionite, selenite, thiophosphate, dithiothreitol and glutathione, prevented the loss of ATPase activity. On the other hand, all oxidizing agents tested, bromate, iodate, arsenite, perchlorate, and hydrogen peroxide, were potent inhibitors of ATPase activity. The inhibitory effect of the oxidizing agents was specific for the vacuolar ATPase. The mitochondrial ATPase, assayed under identical conditions, was not inhibited by any of the oxidizing agents. Analysis of proteins with two-dimensional gel electrophoresis indicated that nitrate can promote the formation of disufide bonds between proteins in the vacuolar membrane. These data suggest a mechanism to explain why nitrate specifically inhibits vacuolar ATPases, and they support the proposal by Feng and Forgac (Feng, Y., and Forgac, M.(1994) J. Biol. Chem. 269, 13244-13230) that oxidation and reduction of critical cysteine residues may regulate the activity of vacuolar ATPases in vivo.