Synthesis and Turnover of Nitrate Reductase Induced by Nitrate in Cultured Tobacco Cells

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Synthesis and turnover of nitrate reductase molecules and soluble proteins of tobacco XD cells were studied by labeling pre-existing proteins with 14C-arginine to make them radioactive and with 99 atom % 15N to increase their buoyant density. The cells were then transferred to medium in which new proteins were synthesized from 14N and were radioactively labeled with 3H-arginine. The breakdown of 15N-labeled proteins was followed after transfer of cells to 14N medium by means of the 14C label. The influence of the pre-existing pool of 15N-amino acids on the density of newly synthesized proteins was followed by means of the 3H label. At various times protein was extracted and subjected to isopycnic equilibrium centrifugation in CsCl. Buoyant densities, relative to an added standard, catalase, were determined for nitrate reductase activity, 14C-labeled protein, and 3H-labeled protein. The studies were carried out (a) during induction of nitrate reductase activity, (b) after full induction, when the level of nitrate reductase activity remains constant, and (c) after a shift from inducing to noninducing conditions, when nitrate reductase activity decays. The buoyant density data show that nitrate reductase activity induced by nitrate is an activity of a protein synthesized de novo after addition of inducer. However, even when the enzyme level was constant, the buoyant density of nitrate reductase decreased from that of 15N-nitrate reductase towards that of 14N-nitrate reductase, indicating that the enzyme turned over. Furthermore, nitrate reductase molecules continued to be synthesized as nitrate reductase activity decayed under noninducing conditions. Turnover of both pre-existing proteins labeled with 14C and newly synthesized proteins labeled with 3H was also observed.