Further Characterization on the Transport Property of Plasmalemma NADH Oxidation System in Isolated Corn Root Protoplasts

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Abstract
Recent experiments show that exogenous NADH increases the O2 consumption and uptake of inorganic ions into isolated corn root protoplasts. A mild treatment of protoplasts with trypsin released most of the NADH oxidation system from the plasmalemma (Lin 1982 Plant Physiol 70: 326-328). Further studies on this system showed that exogenous NADH (1.5 mM) tripled the proton efflux from the protoplasts thus generating a greater electrochemical proton gradient across the plasmalemma. Trypsin also released ubiquinone (11.95 nanomoles/mg protein) but not flavin or cytochrome from the system. Kinetic analyses showed that 1.5 mM NADH quadrupled Vmax of the mechanism I (saturable) component of K+ uptake, while Km was not affected. Diethylstibestrol and vanadate inhibited basal (ATPase-mediated) K+ influx and H+ efflux, while NADH-stimulated K+ uptake was not or only slightly inhibited. .rho.-Chloromercuribenzene-sulfonic acid, N,N''-dicyclohexylcarbodiimide, ethidium bromide, and oligomycin inhibited both ATPase- and NADH-mediated H+ and K+ fluxes. A combination of 10 mM fusicoccin and 1.5 mM NADH gave an 11-fold increase of K+ influx and a more than 3-fold increase of H+ efflux. A plasmalemma ATPase is apparently not involved in the NADH-mediated ion transport mechanism. NADH oxidase is a -SH containing enzyme (protein) and the protein channel is an important element in this transport system. Fusicoccin synergistically stimulates the effects of NADH on K+ uptake.