REDUCED GLUTATHIONE (GSH) TRANSPORT INTO CULTURED TOBACCO CELLS

Uptake of glutathione (GSH) into photoheterotrophic and heterotrophic tobacco cells follows biphasic Michaelis-Menten kinetics in the concentration range 0.01-1.0 mM. K(m) for the high affinity system was 17 +/- 3-mu-M for heterotrophic cells and 18 +/- 1-mu-M for photoheterotrophic cells; K(m) for the low affinity system was 310 +/- 70-mu-M for heterotrophic cells and 780 +/- 70-mu-M for photoheterotrophic cells. Maximum transport velocities for each system were in the same range for heterotrophic and photoheterotrophic cells affinity system: 19-20 nmol GSH min-1 g-1 DW, low affinity system: 167-177 nmol GHS min-1 g-1 DW). GSH uptake was linear for about 2 h and showed maximum uptake rates at pH 5.5-6.5. Metabolic inhibitors reduced GSH transport into the cells significantly, indicating active, energy driven uptake of GSH. gamma-Glutamyl transpeptidase, an enzyme involved in GSH degradation in animal cells, is not likely to participate in GSH uptake by tobacco CellS. L-Cysteine caused competitive inhibition of GSH transport in the high affinity systems of photoheterotrophic and heterotrophic cells (K(I): 55 +/- 6.0-mu-M and 12 +/- 0.6-mu-M cysteine, respectively). Other amino acids did not considerably affect GSH transport. In the presence of sulfate GSH transport into photoheterotrophic cells decreased severely (low affinity system), whereas transport into heterotrophic cells remained unaffected.