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

Abstract

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 light. GR was extremely labile, and the concentrations of GSH and GSSG progressively diminished in chloroplasts prepared without ascorbate. The NADPH level did not significantly change during photooxidative treatment. The addition of 10 mM ascorbate to the incubation medium prevented the decrease of GSH and GSSG and strongly protected GR activity. However, ascorbate had no effect on NADPH-dependent inhibition of the chloroplastic GR purified from wheat leaves. We studied the effect of NADPH, temperature, pH and GSSG on the purified enzyme. The inhibition by NADPH was greatly dependent on temperature and pH. NADPH inhibited GR by around 93% up to pH 7.5, but within a range 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 several pyridin nucleotide dehydrogenases. Furthermore, the NADPH-dependent inhibition was almost completely prevented by GSSG, but not by GSH.