The role of plasma membrane-bound activities in nitrate transport into sealed plasma membrane vesicles from Cucumis sativus L. roots

Abstract

Nitrate uptake was examined on sealed plasma membrane vesicles isolated from cucumber (Cucumis sativus L.) roots by two phase system. Latency and lack of trypsin inactivation of H+-ATPase proved the right-side out orientation of plasma membrane vesicles. NADH-loaded plasma membrane vesicles reduced the external ferricyanide. Triton X-100 stimulation of the ferricyanide reduction by vesicles loaded with NADH indicated that oxidoreductase, which catalyzes this reduction is a transmembrane protein, with reactive sites for NADH and ferricyanide on inner and outer sides of membrane, respectively. Nitrate was transported into sealed, right-side out plasma membrane vesicles with imposed proton gradient. The nitrate uptake increased when plasmalemma vesicles were preloaded with ATP. In the presence of vanadate or dicyclohexylcarbodiimide NO 3 − transport into ATP-loaded vesicles was inhibited. Stimulation of nitrate transport was also observed in experiments with NADHATP-loaded vesicles but only when ferricyanide was added externally. Addition of quinacrine, an effective inhibitor of plasmalemma oxidoreductase in cucumber roots, to the external solution decreased nitrate uptake by vesicles with NADH and ATP inside. Results presented here are consistent with the hypothesis that proton-nitrate symport is involved in active nitrate transport in plant cells. Moreover, the uptake of nitrate by plasma membrane vesicles is related not only to the plasma membrane ATPase but also to the oxidoreductase activity.