Regulation of nitrite reductase and its relationship to the regulation of nitrate reductase in cultured tobacco cells

Abstract

1. 1. The assay for nitrite reductase (EC 1.6.6.4) activity of Ramirez et al. (Biochim. Biophys. Acta, 140 (1966) 58), utilizing methyl viologen reduced by sodium hydrosulfite as electron donor, was optimized for the enzyme from the XD line of cultured tobacco cells. The enzyme reduced nitrite to ammonia under the assay conditions. 2. 2. Nitrite reductase activity was low in cells which had been starved for nitrogen, or which had been grown on the nitrogen in urea or casein hydrolysate. 3. 3. Both nitrate reductase (NADH: nitrate oxidoreductase, EC 1.6.6.1) and nitrite reductase activities developed in cells exposed to either nitrate or nitrite. Nitrate reductase developed more rapidly in response to nitrite than nitrate. 4. 4. Tungstate, a specific inhibitor of development of nitrate reductase activity, and hence of nitrite synthesis, did not inhibit development of nitrite reductase activity in response to nitrate. This is taken as evidence that nitrate does not have to be reduced to nitrite to cause an increase in nitrite reductase activity. 5. 5. Casein hydolysate enhanced the development of both enzyme activities in response to nitrite, but inhibited development of both activities in response to nitrate. 6. 6. Nitrite reductase is more stable and its decay kinetics are more complex in vivo, compared to nitrate reductase activity. After removal of nitrate from the medium, nitrite reductase at first decayed with a half-life of 28 h, but later with a half-life of 124 h, while nitrate reductase decayed with a half-life of approximately 6 h.