SOLUBILIZATION, PURIFICATION, AND CHARACTERIZATION OF THE PLASMA MEMBRANE ATPASE OF OAT ROOTS

Plasma membrane adenosine triphosphatases have been implicated in being closely involved with ion transport in plant roots. One hypothesis dealing with ion uptake into roots is that there are two ATPases in the plasma membrane responsible for ion transport, one for anion transport and one for cation transport. Solubilization of the plasma membrane would allow for the purification of the ATPase activities associated with it and for the characterization of these activities. Plasma membrane-enriched fractions from oat (Avena sativa L. cult. Goodfield) root homogenates have been solubilized using the neutral detergent octyl-(beta)-D-glucopyranoside in combination with glycerol. Two ATPase activities were purified from the solubilized material using isoelectric focusing procedures in horizontal polyacrylamide slab gels. One ATPase activity was optimally active at pH 6.5, was stimulated by KCl, and had an isoelectric point of about pH 7.8. The other ATPase activity was optimally active at pH 8.5, was stimulated by KHCO(,3), and had an isoelectric point of about pH 4.2. When solubilized mitochondria were tested, however, the ATPase activity migrated to the same position in the isoelectric focusing gels as the pH activity from the plasma membrane-enriched fraction. Orthovanadate inhibited the pH 6.5 ATPase activity but not the pH 8.5 ATPase activity or the mitochondrial ATPase activity. Azide, on the other hand, had no effect on the pH 6.5 ATPase while inhibiting both the pH 8.5 ATPase and the mitochondrial ATPase. It is concluded that the pH 8.5 ATPase activity is due to mitochondrial contamination of the plasma membrane-enriched fraction from oat root homogenates, and that the plasma membrane of oat roots contains only one ATPase