Characteristics of in vivo nitráte reductase (NR, EC 1.6.6.1) activity in leaf tissue of the crassulacean acid metabolism (CAM) plant Notonia grandiflora DC. were determined. The pH optimum for the enzyme was 7.0. Among the five low molecular mass solvents tested, n-propanol was more effective in promoting NR activity. Supply of 1.0 % (v/v) M-propanol along with 0.5 % Triton X-100 was the most appropriate assay condition to bring about maximum NR activity (NRA), that was proportionally enhanced with duration of the incubation and mass of the leaf tissue. Leaf fragments of 2 to 4 mm slice width had maximal NRA. The highest NRA was obtained with 200 mM of nitráte supplied in the infiltration medium. NRA was higher in fully expanded, mature leaves than in tender and senescing leaves. The cellular nitráte content was strongly positively correlated with NRA in leaves of various physiological ages. The NRA/N03’ content ratio (index of nitráte utilization efQciency) was the highest for the tender leaves and the lowest for the senescing leaves. However, maximum total titratable acidity (malate production) was obtained for the fully expanded mature leaves in comparison with the other leaves. A positive and significant correlation (r = 0.92) was obtained between nitráte reduction and titratable acid content. The results are indicative of a possible role of nitráte reduction in the regulation of noctumal acid production in the CAM plants.
The kinetics and other characteristics of nitrate reductase (NR, EC 1.6.6.1) in cowpea [Vigna unguiculata (L.) Walp.] seedlings irradiated with biologically effective UV-B radiation (280-320 nm, 3.2 W m-2 s-1) were recorded. The in vivo and in vitro NR activities were inhibited by 34 and 41 % under UV-B treatment, respectively. Both Vmax and Km for the substrate were enhanced by UV-B radiation. The Km for nitrate increased from 1.2 to 1.7 mM after the UV-B irradiation. The change in Km for NADH was from 0.12 to 0.17 mM. The increases in Km indicate that UV-B radiation seriously changes the topology of NR, particularly with respect to the nitrate and NADH binding sites. The rate of NR turnover indicates the extent of damage inflicted by UV-B radiation on the nitrate metabolism. The half-life (t1/2) of NR was reduced from 7 to 4 h in the UV-B treated seedlings. UV-B also inhibited the kinetics of nitrate uptake by plants: its Km increased from 0.08 to 0.12 mM. and T. Balakumar ... [et al.].
Cowpea [Vigna unguiculata (L.) Walp. cv. Co 4] seedlings were subjected to a weighted irradiance of 3.2 W m-2 s-1 of biologically effective ultraviolet-B radiation (UV-B, 280-320 nm) and the changes in the kinetic and other characteristics of nitrite reductase (NiR) were recorded. The activity of NiR was hampered by 19 % under UV-B irradiation compared to the control. The UV-B treated plants required higher concentrations of nitrate for the induction of NiR synthesis than the controls. The NiR activity decay kinetics showed that the UV-B treatment significantly lowers the t1/2 of the enzyme, thereby indicating a reduced rate of enzyme turnover. The comparison of kinetic characteristics of nitrate reductase (NR) and NiR under UV-B treatment showed that NiR was not so sensitive to UV-B radiation as NR. As shown by enzyme turnover rates, NiR extracted from plants irradiated by UV-B in situ was less sensitive to UV-B radiation than the enzyme extract subjected to in vitro UV-B irradiation. Though NiR was less damaged by UV-B treatment than NR, subtle changes occurred in its kinetic characteristics. and T. Balakumar ... [et al.].