Changes in nitrate reductase protein during loss and gain of nitrate reductase activity in Chlorella vulgaris

Abstract

Nitrate reductase (NADH) (NADH: nitrate oxidoreductase, EC 1.6.6.1) in Chlorella vulgaris exists, in vivo, in at least two forms. One is active, and the other, a reversibly inactived form, accumulates when nitrate assimilation is inhibited. This inactive form converts, in vivo, to the active form under other culture conditions which promote nitrate metabolism. This inactive form can also be activated in vitro by oxidation with reagents such as ferricyanide. The only inactive form which is characterized is a cyanide complex of the reduced enzyme that also can be formed in vitro. The possibility of other inactive forms has been suggested, but none has been reported. In the present study, active and ferricyanide-activatable nitrate reductase activities were determined in crude extracts from cells grown under very different conditions. Nitrate reductase protein in these extracts was determined by rocket immunoelectrophoresis. Conditions which lower the level of active and activatable nitrate reductase activites concomitantly lower nitrate reductase protein. Similarly, when total activities increase, there is a corresponding increase in nitrate reductase protein. Since all changes in nitrate reductase activity may be accounted for by corresponding changes in nitrate reductase protein, the cyanide complex of nitrate reductase is the only major inactive form of nitrate reductase in Chlorella.