PARTIAL-PURIFICATION AND CHARACTERIZATION OF A CALCIUM-DEPENDENT PROTEIN-KINASE AND AN INHIBITOR PROTEIN REQUIRED FOR INACTIVATION OF SPINACH LEAF NITRATE REDUCTASE
M. Bachmann et al., PARTIAL-PURIFICATION AND CHARACTERIZATION OF A CALCIUM-DEPENDENT PROTEIN-KINASE AND AN INHIBITOR PROTEIN REQUIRED FOR INACTIVATION OF SPINACH LEAF NITRATE REDUCTASE, Plant physiology, 108(3), 1995, pp. 1083-1091
Evidence is accumulating that the activity of spinach (Spinacia olerac
ea L.) leaf NADH:nitrate reductase (NR) is modulated both in vitro and
in vivo by protein phosphorylation. From the present study we report
the partial purification of the two protein factors needed for NR inac
tivation. We identified NR-protein kinase (NR-PK) as a calcium-depende
nt and metabolite-regulated protein kinase and have provided additiona
l evidence that phosphorylation of NR is necessary but not sufficient
to inactivate the enzyme. The inhibitor protein required for inactivat
ion of phospho-NR was purified 625-fold by polyethylene glycol fractio
nation and sequential column chromatography. Using partially purified
inhibitor protein and NR-PK, we characterized NR inactivation (increas
ed sensitivity to Mg2+ inhibition) in a reconstituted in vitro system.
NR-PK activity was inhibited by a variety of metabolic phosphate este
rs including dihydroxyacetone phosphate, glucose-6-phosphate, and fruc
tose-1,6-bisphosphate. Light-to-dark transition experiments with a sta
rchless tobacco (Nicotiana sylvestris) mutant, which accumulates phosp
hate esters during the photoperiod, indicated that NR inactivation in
vivo might, indeed, be down-regulated by metabolites. Additionally, we
postulate that cytosolic free calcium could play an important role in
the regulation of NR activity in vivo.