Studies On The Regulation Mechanism Of Nitrate Reductase In Wheat Leaves

Nitrate reductase is the key enzyme and it catalyzes the step from NO3" to NO₂". NO₃" has active effects on nitrate reductase. The effects of NO₃" on nitrate reductase often occur latterly. Usually the effects were 3 hours later. NO₃" also has effects on nitrate reductase.Presently, there are two different opinions. One is the synthesis of nitrate reductase protein from beginning. The other is to active the pre-nitrate reductase. We regard the former as reasonable one. Besides NO₃", light shows great effects on nitrate reductase, light makes NADH for the reducing system of NO₃". Light is necessary for the nitrate reductase from yellowing primary seedlings. The changing form of nitrate reductase activity is different in green and etiolated seedlings. The main mechanism of effects on nitrate reductase shows that the synthesis of nitrate reductase depends on photosynthesis. The experiment of cycloheximide on Nitrate reductase induction shows that rapid enhance of Nitrate reductase activity results from systhesis of Nitrate reductase from beginning. Nitrate reductase is an unstable enzyme, it is constantly synthesized and decomposed in vivo. It loses activities in vitro because of unstability. Somebody thinks that there exists NIP or inhibitors in NR extracts. We get the 242-fold nitrate reductase purified by ammonium sulfate precipitation and gel filtration. The stability isdifferent from each other at different temperature.The half life is different at different temperature too.At 15, 25, 30, 40 and 50 °C,The half life are 60,40,29,20, lOminuteSo The activity of nitrate reductase is high in the temperature between 20 °C and 35°C.The optimum temperature of nitrate reductase is about 30 °C. Nitrate reductase uses NADH as the electron donor in wheat primary seedlings.The optimum pH of nitrate reductase is about 7.5.The kinetics of The activity and stability of nitratereductase is different when pH<5.5 or pH>7.5. The Km value of nitrate reductase is3.25X 10⁴mol/L and 8.4X 10-5mol/L with NO3'and NADH as substrates. We draw figures with inK vs 1/T range from 15 to 50 °C .From these figures the Ea about 48.7kj/mol (?* 11.65kcal/mol) is gained at 28.2°C.The higher temperature(above 28.2 °C ),the greater Eais demanded. When a crude extract from wheat leaves is fractionated by a combination of ammonium sulfate (40%⁷0% saturation) precipitation and Sephadex G-75 gel filtration, the presence of nitrate reductase inactivating protein is revealed. The nitrate reductase inactivating protein may be one of the main factors that regulation of nitrate reductase activity. We get the 101.6-fold nitrate reductase inactivating protein. Inactivating protein has a strongly inhibitory effect on nitrate reductase activity. The effect of inactivating protein is much higher in room temperature and neutral. The nitrate reductase inactivating protein has an effect on nitrate reductase by combining to nitrate reductase. The results of some reactions suggest that the inactivating protein may be a competitive inactivating protein for nitrate reductase. The nitrate reductase inactivating protein links to nitrate reductase by a function. This is a time course. The results of some reactions suggest that the inactivating protein may be a competitive inactivating protein for nitrate reductase. From the figure of Guggenheim,we get the binding constant between NIP and NR,that is 0.1875. The mechanisms of the transcriptional and translational level must be involved in the regulation of nitrate reductase synthesis, and The mechanisms of posttranslational must be involved in the regulation of nitrate reductase degradation. At the same time other mechanisms may play important role in the regulation of the activity of nitrate reductase and nitrate metabolism. The stability of nitrate reductase mRNA affects the activity of nitrate reductase smartly. The activity of nitrate reductase is also affected by phosphorylation/dephosphorylation. At the same time, some others factors affect the activity and stability of nitrate reductase. For instance , Ca2+and Mg+...