Correlation between apparent activation state of nitrate reductase (NR), NR hysteresis and degradation of NR protein

Abstract

Nitrate reductase (NR) activity was measured in extracts from spinach leaves exposed to light or prolonged darkness, and to various treatments provoking an artificial activation of the enzyme in the dark. NR activity was determined immediately either in the presence of Mg2+, which gives an estimation of the putative (actual) activity in situ (NR(act)), or in EDTA without preincubation, which gives an intermediate activity (NR(int), or after a 30 min preincubation with EDTA plus AMP plus Pi, which gives the maximum NR activity (NR(max)). NR(max) is thought to reflect total NR protein contents. In the dark, NR(act) was usually very low. Dark inactivation was prevented or reversed by feeding AICAR (5-aminoimidazole-4-carboxiamide ribonucleoside), or by anaerobiosis, acid treatment or addition of uncoupler. During prolonged darkness, NR(max) decreased, indicating net protein degradation with a half-time of 21 h. Conditions which caused an activation (dephosphorylation) of NR in the dark, slowed down NR protein degradation. This was also confirmed by Western blotting. Blockage of cytosolic protein synthesis with cycloheximide (CHX) did not accelerate NR protein degradation. In contrast, after 5 h in the dark, NR(act) increased in CHX-treated leaves. As this increase was sensitive to PP2A-inhibitors, it was probably due to NR dephosphorylation. However, extractable NR kinase and NR phosphatase activities were not changed by CHX treatment. Apparently, CHX interacted with the NR regulatory system indirectly by affecting turnover of another protein. The increase from NR(int) to NR(max) which occurred during preincubation of the leaf extract with EDTA plus AMP plus Pi was insensitive to PP2A inhibitors and was interpreted as a hysteretic conversion of NR from an inactive into an active form. Hysteretic activation was positively correlated to the NR phosphorylation state. A model is presented to explain the hysteretic behaviour of NR in relation to NR phosphorylation/dephosphorylation. Overall, the data indicate that NR protein phosphorylation not only controls the catalytic activity of NR, but also acts as a signal for NR protein degradation, with phospho-NR probably being a better substrate for protein degradation than the dephospho-form.