J Integr Plant Biol. ›› 1983, Vol. 25 ›› Issue (4): -.

• Research Articles •    

The Relation Between the Denatured Nitrate Reductase and the Nitrate Reductase-Inhibiting Protein

Chen Wei, Zhang De-yi(Chang Teh-yi) and Tang Yu-wei   

Abstract: Wheat seedlings (Triticum aestivum var. Feng-chan 3 ) were grown on water or KNO3 medium at 24℃. Before the second leaf had grown out, the shoots of the seedlings were cut down and ground with a little quartz sand. The homogenates were filtered through a layer of nylon cloth before centrifugation at 10000g for15 min. The supematant fraction was collected (crude nitrate reductase). Isolation and purification of nitrate reductase (NR) were according to Sherrard et al with a bit modifications. Ammonium sulfate was added to the crude NR and the enzyme protein was precipitated between 20%—40% saturation. After column chromatography on Sephadex G-25, the protein was then subjected to further purification by affinity chromatography on a blue dextran-Sepharose 4B column. The fraction in the NADH (0.1 mM) eluate was the highly purified enzyme. The activity of the isolated NR was assayed in vitro according to the standard method, Nitrate reductase-inhibiting protein (NRIP) was isolated and purified according to Wallace with a little modifications. After fractional precipitation by ammonium sulfate, the protein precipitating between 20%–40% saturation was collected and dissolved in distilled water. Column chromatography on Sephadex G-100 and DEAE (DE-11) cellulose was separately used. After dialysis, condensation of the highly purified NRIP was carried out. Antiserum against NR was prepared by injecting 2 mL purified NR protein (88 nmol NO2-/30 min/0.2 mL) into a rabbit five times with an interval of 10 days. For all five injections, the enzyme was mixed with complete Freund's adjuvant. Bleeding was taken 30 days after the first injection. Antiserum against NRIP was prepared in the same way mentioned above, but purified NRIP was used instead of NR. Rocket immunoelectrophoresis was performed by the method described by Funkhouser. Agarose gels (1.5% W/V). which contained 30 mM Tris and 12.3 mM meleate (pH 8.6) and 0.2% (V/V) crude antiserum were placed on a glass plate. Wells were cut along one side of the plate and filled with 10, 20, 30, 40 μ 1of antigen. Electrophoresis was carried out at 3 mA, 10 V for 2 h at 4 ℃. The antigen-antibody reaction resulted in the formation of rocket shaped immunoprecipitates. After washing overnight in PBS the rockets were visualized by staining with coomassie blue. The procedure of immunodiffusion and immunoelectrophoresis was according to that of Clausen. Nitrate reductase is a very unstable enzyme, Our former paper showed that the crude NR lost its enzyme activity by about one half, after it had been maintained at room temperature for 30 min. In order to study the stability of NR. crude NR was prepared and kept at room temperature. After the enzyme activity had been completely lost, it was added to a fresh NR preparation with high activity. The inhibition effect of denatured enzyme was revealed according to the difference between plus or minus denatured enzymes. About 70%–80% NR activities were lost in the preparation to which 0.1 ml denatured enzyme had been added instead of 0.1 ml H2O. Therefore we think that the denatured enzyme itself behaved like an inhibiting protein of NR. Wallace demonstrated that there was an inactivating enzyme of NR in maize roots. Some characteristics of the enzyme investigated in several labs. According to Wallace's methods we got a purified NR-inactivating-protein (NRIP). Furthermore, a purified NR was obtained by an affinity-chromatography method (table 1). Single of either NR or NRIP appeared on the chromatography and their Rm were the same (fig. 2). It might conclude that the NRIP and denatured NR are the similar protein. The highly purified NR protein incubated for several hours at room tempetature also became an inhibitor (table 2). We, therefore, infer that the activated NR could be converted to NRIP at room temperature. Antiserum against NR was prepared by injecting purified NR into rabbit, and antiserum against NRIP was prepared by injecting purified NRIP. The anti-NR antibody and the anti-NRIP antibody were prepared as reagents to study the immunological relation between these two proteins. The antibody of NR gave a single precipitate band against purified NRIP and the antibody of NRIP had a similar precipitate band against purified NR (fig. 3 and 4). Rocket immunoelectrophoresis was performed. The antiserum against NR were added to agarose gel and 4 wells were filled with different amount of NRIP. The height of the rockets was increased with the amount of NRIP (fig. 5). All these results show the identity of the denatured NR and NRIP. The percent of inhibition of NRIP depended upon the concentration of NADH in the reaction mixture. Fig. 6 shows that the NRIP was a competitive inhibitor. The inhibitor and NR both competed for the same cofactor NADH. The percentage of inhibition was decreased when the concentration of NADH in the reaction system was increased. According to this result, we suggest that the NR protein has two active sites. One site binds with nitrate and the other with NADH. When the site bound with nitrate is damaged or changed, the enzyme protein can not catalyze nitrate reduction. However, the site binding with NADH is less labile and not affected by incubation at room temperature, therefore NADH can still be bound on the denatured NR protein. If the concentration of NADH in this reaction system is limited, the nitrite formation decreases. This explains how the effect of NRIP can be overcome in the reaction system at higher concentration of NADH.

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