CORRELATION BETWEEN APPARENT ACTIVATION STATE OF NITRATE REDUCTASE (NR), NR HYSTERESIS AND DEGRADATION OF NR PROTEIN

Nitrate reductase (NR) activity was measured in extracts from spinach leaves exposed to light or prolonged darkness, and to various treatmen ts provoking an artificial activation of the enzyme in the dark. NR ac tivity was determined immediately either in the presence of Mg2+, whic h gives an estimation of the putative (actual) activity in situ (NRact ), or in EDTA without preincubation, which gives an intermediate activ ity (NRint), or after a 30 min preincubation with EDTA plus AMP plus P i, which gives the maximum NR activity (NRmax). NRmax is thought to re flect total NR protein contents. In the dark, NRact was usually very l ow. Dark inactivation was prevented or reversed by feeding AICAR (5-am inoimidazole-4-carboxiamide ribonucleoside), or by anaerobiosis, acid treatment or addition of uncoupler. During prolonged darkness, WRmax d ecreased, indicating net protein degradation with a half-time of 21 h. Conditions which caused an activation (dephosphorylation) of NR in th e dark, slowed down NR protein degradation. This was also confirmed by Western blotting. Blockage of cytosolic protein synthesis with cycloh eximide (CHX) did not accelerate NR protein degradation. In contrast, after 5 h in the dark, NRact increased in CHX-treated leaves. As this increase was sensitive to PPPA-inhibitors, it was probably due to NR d ephosphorylation. However, extractable NR kinase and NR phosphatase ac tivities were not changed by CHX treatment, Apparently, CHX interacted with the NR regulatory system indirectly by affecting turnover of ano ther protein. The increase from NRint to NRmax which occurred during p reincubation of the leaf extract with EDTA plus AMP plus Pi was insens itive to PP2A inhibitors and was interpreted as a hysteretic conversio n of NR from an inactive into an active farm. Hysteretic activation wa s positively correlated to the NR phosphorylation state. A model is pr esented to explain the hysteretic behaviour of NR in relation to NR ph osphorylation/dephosphorylation. Overall, the data indicate that NR pr otein phosphorylation only controls 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-f orm.