Nitrate reductases from leaves of Ricinus (Ricinus communis L.) and spinach (Spinacia oleracea L.) have different regulatory properties.
The activity of nitrate reductase (+Mg(2+), NR(act)) in illuminated leaves from spinach, barley and pea was 50-80% of the maximum activity (+EDTA, NR(max)). However, NR from leaves of Ricinus communis L. had a 10-fold lower NR(act), while NR(max) was similar to that in spinach leaves. The low NR(act) of Ricinus was independent of day-time and nitrate nutrition, and varied only slightly with leaf age. Possible factors in Ricinus extracts inhibiting NR were not found. NR(act) 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. NR(max) had a broad pH response profile that was similar for the spinach and the Ricinus enzyme. Accordingly, the Mg(2+)-sensitivity of NR from Ricinus was strongly pH-dependent (increasing sensitivity with increasing pH), and as a result, the apparent activation state of NR from a Ricinus extract varied dramatically with pH and Mg(2+)concentration. Following a light-dark transition, NR(act) from Ricinus decreased within 1 h by 40%, but this decrease was paralleled by NR(max). In contrast to the spinach enzyme, Ricinus-NR was hardly 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 contains the same site. Removal of 14-3-3 proteins from Ricinus-NR by anion exchange chromatography, activated spinach-NR but caused little if any activation of Ricinus-NR. It is suggested that Mg(2+)-inhibition of Ricinus-NR does not require 14-3-3 proteins. The rather slow changes in Ricinus-NR activity upon a light/dark transient may be mainly due to NR synthesis or degradation.
Kandlbinder
A
A
Universität Würzburg, Lehrstuhl für Molekulare Pflanzenphysiologie und Biophysik, Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany.
Weiner
H
H
Kaiser
W M
WM
D048948:14-3-3 Proteins; D015153:Blotting, Western; D006863:Hydrogen-Ion Concentration; D008029:Lighting; D008274:Magnesium / Q000378:metabolism; D050895:Nitrate Reductase (NADH); D009565:Nitrate Reductases / Q000378:metabolism*; D010766:Phosphorylation; D018515:Plant Leaves / Q000201:enzymology / Q000378:metabolism; D010947:Plants, Toxic; D012278:Ricinus / Q000201:enzymology* / Q000378:metabolism; D018724:Spinacia oleracea / Q000201:enzymology* / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
D048948:14-3-3 Proteins; D014446:Tyrosine 3-Monooxygenase; D009565:Nitrate Reductases; D050895:Nitrate Reductase (NADH); D008274:Magnesium