Nitrate reductases from leaves of Ricinus (Ricinus communis L.) and spinach (Spinacia oleracea L.) have different regulatory properties

The activity of nitrate reductase (+Mg2+, NRact) in illuminated leaves from spinach, barley and pea was 50-80% of the maximum activity (+EDTA, NRmax), However, NR from leaves of Ricinus communis L. had a 10-fold lower NRact, while NRmax was similar to that in spinach leaves, The low NRact of Ricinus was independent of day-time and nitrate nutrition, and varied only slightl y with leaf age. Possible factors in Ricinus extracts inhibiting NR were no t found, NRact from Ricinus, unlike the spinach enzyme, was very low at pH 7.6, but much higher at more acidic pH with a distinct maximum at pH 6.5. N Rmax had a broad pH response profile that was similar for the spinach acid the Ricinus enzyme. Accordingly, the Mg2+ -sensitivity of NR from Ricinus w as strongly pH-dependent (increasing sensitivity with increasing pH), and a s a result, the apparent activation state of NR from a Ricinus extract vari ed dramatically with pH and Mg2+ concentration. Following a light-dark tran sition, NRact from Ricinus decreased within 1 h by 40%, but this decrease w as paralleled by NRmax. In contrast to the spinach enzyme, Ricinus-NR was h ardly inactivated by incubating leaf extracts with ATP plus okadaic acid. A competition analysis with antibodies against the potential 14-3-3 binding site around ser 543 of the spinach enzyme revealed that Ricinus-NR containe s the same site. Removal of 14-3-3 proteins from Ricinus-NR by anion exchan ge chromatography, activated spinach-NR but caused little if any activation of Ricinus-NR. It is suggested that Mg2+-inhibition of Ricinus-NR does not require 14-3-3 proteins. The rather slow changes in Ricinus-NR activity up on a light/dark transient may be mainly due to NR synthesis or degradation.