PARTIAL-PURIFICATION AND CHARACTERIZATION OF A CALCIUM-DEPENDENT PROTEIN-KINASE AND AN INHIBITOR PROTEIN REQUIRED FOR INACTIVATION OF SPINACH LEAF NITRATE REDUCTASE

Citation
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
Citations number
39
Categorie Soggetti
Plant Sciences
Journal title
ISSN journal
00320889
Volume
108
Issue
3
Year of publication
1995
Pages
1083 - 1091
Database
ISI
SICI code
0032-0889(1995)108:3<1083:PACOAC>2.0.ZU;2-4
Abstract
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.