Orientation of NAHD-linked ferric chelate (turbo) reductase in plasma membranes from roots ofPlantago lanceolata

Summary Plasma membrane vesicles isolated from roots ofPlantago lanceolata L. revealed approximately 70% right-side-out orientation based on structure-linked latency with H⁺-ATPase as a marker. Incubation with 0.05% Brij 58 caused the formation of sealed insideout vesicles, evidenced by assaying ATP-dependent proton pumping activity with the optical pH probe acridine orange. NADH-linked FeEDTA reductase activity was stimulated by including either Triton X-100 or Brij 58 in the assay medium. The activity of inverted (Brijtreated) vesicles was not further increased by the addition of Triton, suggesting that maximum activity was obtained in inside-out vesicles. Iron deficiency resulted in a ca. 2-fold increase in the specific activity of both ATPase and Fe(III) chelate reductase but did not cause significant alterations with respect to the effect of detergents. It is concluded that in vitro both donor and acceptor sites of NADH-FeEDTA reductase are located on the cytosolic face of the membrane and trans-oriented flow of electrons is not detectable in plasma membrane vesicles. Unlike Fe chelate reduction in vivo, the plasma membrane-bound reductase activity was insensitive towards application of the translation inhibitor cycloheximide prior to isolation of the membranes, implying the involvement of a regulatory enzyme in the electron transport in vivo.Abbreviations BPDS bathophenanthroline disulfonate - BTP 1,3-bis[tris(hydroxymethyl)methylamino]-propane - PM plasma membrane