Characterization of ATP-hydrolyzing and ATP-driven proton-translocating activities associated with the peribacteroid membrane from root nodules of Lupinus luteus L

In order to detect and characterize PBM Ht-ATPase from root nodules of Lupinus luteus L., ATP-hydrolyzing and ATP-dependent proton translocating activities associated with this membrane were studied using isolated symbiosomes, vesicle PBM preparations and intact plant tissues. It has been found that ATP-hydrolyzing activity of isolated PBMs is sufficiently high (about 100-150 mu moles of P-i/mg protein.h) in the selected pH range (4.5-8.5) and is characterized by the absence of any pronounced pH optimum, little substrate specificity, the absence of selectivity in relation to Mg2+ and Ca2+ as stimulators of ATP-hydrolysis and even more sensitivity to Ca2+. Mg2+-dependent ATP-hydrolyzing activity was moderately decreased in the presence of some known inhibitors of H+-ATPases, such as vanadate, DCCD and nitrate. On the other hand, Ca2+-dependent ATP-hydrolysis was significantly inhibited by sodium fluoride. ATP-hydrolyzing activity on the PBM of Lapinus luteus L. with many similar properties was also detected cytochemically in electron microscope studies of intact plant tissues. These findings led us to conclude elude that at least one more Ca2+/Mg2+-dependent ATP-hydrolase, in addition to H+-ATPase, is associated with the PBM. This enzyme is likely responsible for the observed background that masks ATP-hydrolyzing activity of the PBM H+-ATPase. ATP-dependent electrogenic proton transport across the PBM was detected in isolated symbiosomes and vesicle PBM preparations using oxonol YI and acridine orange as indicators of Delta psi and Delta pH, respectively. Transport activity of the PBM H+-ATPase was blocked by the above mentioned ATPase inhibitors, except nitrate, which acted only as permeant anion. These results indicate that the PBM of Lupinus luteus L. contains only one H+-translocating ATPase, which belongs to a P-type H+-ATPase.