Proton transport ATP dependent driven by the plasma membrane ATPase from Arabidopsis thaliana leaves: biochemical characterization

Abstract

The plasma membrane from Arabidopsis thaliana leaves was purified by phase partitioning and the use of enzyme markers showed that this fraction was highly enriched in plasma membrane. This fraction was almost devoid of phosphohydrolase activities originating from endomembranes (tonoplastes, mitochondria, non specific phosphatases and golgi apparatus). The H⁺ATPase in this fraction was Mg²⁺ dependent and stimulated by K⁺ and valinomycin. It was almost unsensitive to nitrate (tonoplaste ATPase inhibitor) but sensitive to vanadate (plasma membrane ATPase inhibitor) and other known ATPase inhibitors, especially the omeprazol inhibited both ATPase activity and plant growth. This activity was specific for ATP with a Kmapp of 392 μM and had a pH optimum around 6.7. On the other hand, 1 μM of lysophosphatidylcholine stimulated the H⁺ transport activity into the purified plasma membrane vesicles. Higher concentration of this detergent (30 μM) was inhibitory.