The reactivation of nitrate reductase from spinach (Spinacia oleracea L.) inactivated by NADH and cyanide, using trivalent manganese either generated by illuminated chloroplasts or as manganipyrophosphate

Abstract

Nitrate reductase of spinach (Spinacia oleracea L.) leaves which had been inactivated in vitro by treatment with NADH and cyanide, was reactivated by incubation with oxidant systems and measured as FMNH2-dependent activity. Reactivation was produced with trivalent manganese compounds represented either by manganipyrophosphate or produced by oxidation of Mn(2+) ions in the presence of illuminated chloroplasts and compared with reactivation obtained with ferricyanide. Reactivation in the chloroplast system was equivalent to that with ferricyanide when orthophosphate was used but was variable and weak in the presence of pyrophosphate, although manganipyrophosphate was formed, freely. Reactivation by manganipyrophosphate in dark reaction conditions was less effective than with ferricyanide but was not inhibited by the addition of pyrophosphate. Reactivation with illuminated unheated chloroplasts was dependent on added manganese and oxidation of manganese in the presence of pyrophosphate was abolished by boiling the chloroplasts. In the presence of orthophosphate however, boiled, illuminated chloroplasts reactivated the enzyme in the absence of added manganese. Reactivation occurred spontaneously in air, more slowly than with the other oxidants, but to a similar extent to that produced by manganipyrophosphate. The results provide a possible model for physiological reactivation mechanisms.