Solubilization and purification of the ATPase from the tonoplast of Hevea

Abstract

The tonoplast‐bound ATPase of Hevea brasiliensis (caoutchouc tree) was solubilized with dichloromethan and purified 100‐fold with two ammonium sulfate precipitation steps and a G‐200 gel filtration step. The resulting ATPase activity eluted according to a molecular mass of approximately 200 kDa and chromatographed at an isoelectric pH of 5.3. Subunits of molecular mass 100 kDa, 68 kDa, 24 kDa and 12 kDa appeared after treatment with 1% sodium dodecyl sulfate or spontaneously during storage of the solubilized ATPase. Dodecyl sulfate/polyacrylamide gel electrophoresis yielded four polypeptides of molecular mass 54 kDa, 66 kDa, 23 kDa and 13 kDa. From protein determination by ultraviolet absorption and Coomassie stain it appears that the 54‐kDa and the 66‐kDa polypeptides exist in multiple copies. No close resemblance to the membrane‐bound ATPase of mitochondria, plastids, plasmalemma, chromaffin granules and synaptic vesicles is seen. No antibody crossreaction to F₁ of bacteria is observed. Therefore it is concluded that the vacuolar ATPase represents a novel type of ATPase. Many properties of the tonoplast‐bound ATPase such as pH‐dependence, substrate specificity, ion‐dependence and inhibitor sensitivity did not change when the enzyme had been solubilized and purified. The phosphatase activity was lost during the purification procedure. The stimulation of ATP‐hydrolysis in tonoplast vesicles by uncouplers and ionophores was absent in the solubilized ATPase, and also the stimulation by chloride was significantly reduced. Anion channel blockers, such as triphenyltin and 4,4′‐diisothiocyano‐2,2′‐disulfonic acid stilbene, which are strong inhibitors of membrane‐bound ATPase, fully or partly lost their inhibiting effect after solubilization of the ATPase. These results are interpreted to indicate that ionophores do not directly affect the ATPase molecule, whereas chloride might have a small direct effect on the ATPase besides its effect as a permeating anion.