Temperature dependence and mercury inhibition of tonoplast-type H +-ATPase

Abstract

The effects of changing temperature on ATP hydrolysis and proton pumping associated with the H +-ATPase of tonoplast membrane vesicles isolated from the maize root microsomal fraction were determined. In the range 5 to 45 °C, the maximal initial rate of ATP hydrolysis obeyed a simple Arrhenius model and the activation energy determined was ~14 kcal/mol. On the other hand, the initial proton pumping rate showed a bell-shaped temperature dependence, with maximum activity around 25 °C. Lineweaver-Burke analysis of the activities showed that the K m of ATP hydrolysis, unlike that of proton pumping, was relatively insensitive to temperature changes. Detailed kinetic analysis of the proton pumping process showed that the increase in membrane leakage to protons during the pumping stage constituted a major reason for the decreased transport. Nitrate-sensitive ATPase activities of the tonoplast vesicles were found to be inhibited by the presence of micromolar concentrations of Hg 2+. The proton pumping process was more sensitive to the presence of Hg 2+. Double-reciprocal analysis of kinetic data indicated that Hg 2+ was a noncompetitive inhibitor of proton pumping but was an uncompetitive inhibitor of ATP hydrolysis. Further kinetic analysis of Hg 2+ effects revealed that the lower proton transport did not result from enhanced membrane leakage but rather from reduced coupling between H + pumping and ATP hydrolysis.