Regulation of Chlorella nitrate reductase: Control of enzyme activity and immunoreactive protein levels by ammonia

Abstract

Nitrate reductase catalyzes the initial step in the conversion of nitrate to organic nitrogen and is thought to be repressed by ammonia and induced by nitrate. Induction by nitrate and repression by ammonia were studied by following changes in NADH: nitrate reductase and the associated partial activities NADH:cytochrome c reductase and methylviologen r:nitrate reductase. Immunoreactive protein was assessed by enzyme-linked immunosorbent assay and immunoblotting. Molybdenum cofactor levels were investigated using the nit-1 complementation assay as well as fluorescence of the oxidized cofactor. The results indicate that the NADH:cytochrome c reductase activity is “induced” faster than the nitrate-reducing activity and suggest that incorporation of the molybdo-pterin cofactor may be rate limiting in the expression of activity. Molybdenum cofactor levels are significantly elevated in nitrate-treated cells. Under “repressing” conditions all activities decreased at approximately the same rate. A more rapid conversion of the enzyme to a reversibly inactive form also occurred under these conditions. Changes in immunoreactive protein levels correlated most closely with NADH: cytochrome c reductase activity but appeared to increase faster during induction and decrease slightly slower during repression than the enzyme activities. Removal of exogenous ammonia results in the appearance of nitrate reducing activity, as well as immunoreactive protein (derepression). Studies using protein and RNA synthesis inhibitors indicated that de novo synthesis is required for nitrate reductase induction and were in agreement with the results of the immunoreactive studies.