Wheat chloroplastic glutathione reductase activity is regulated by the combined effect of pH, NADPH and GSSG

To study the mechanism of regulation of chloroplastic glutathione reductase (GR) under photooxidative conditions, GR activity, and the levels of NADPH , GSH and GSSG were measured in wheat chloroplasts under photooxidative lig ht. GR was extremely labile, and the concentrations of GSH and GSSG progres sively diminished in chloroplasts prepared without ascorbate, The NADPH lev el did not significantly change during photooxidative treatment. The additi on of 10 mM ascorbate to the incubation medium prevented the decrease of GS H and GSSG and strongly protected GR activity. However, ascorbate had no ef fect on NADPH-dependent inhibition of the chloroplastic GR purified from wh eat leaves. We studied the effect of NADPH, temperature, pH and GSSG on the purified enzyme. The inhibition by NADPH was greatly dependent on temperat ure and DH, NADPH inhibited GR by around 93% up to pH 7.5, but within a ran ge of 8.0 to 9.5 the inhibition was only marginal. The pH dependence of the NADPH inhibitory effect could be due, at least in part, to different rates in the generation of NADPH-X, a derivative of NADPH which inactivates seve ral pyridin nucleotide dehydrogenases. Furthermore, the NADPH-dependent inh ibition was almost completely prevented by GSSG, but not by GSH.