Isolation and Some Properties of a 115-Kilodalton Nitrate Reductase-Inactivator Protein from Spinacia oleracea

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A nitrate reductase inactivator protein in spinach leaves was purified (90-fold). The purification involved precipitation with ammonium sulfate, treatment at pH 4, CM-cellulose chromatography and gel filtration on a Toyopearl HW-55F column. From the Toyopearl HW-55F gel filtration step the molecular weight of the inactivator was estimated to be 115 kDa. The inactivator was particularly sensitive to EDTA,o-phenanthroline and pronase. The inactivator was more stable to heat treatment than NADH-nitrate reductase. Incubation of purified spinach nitrate reductase with the inactivator results in a loss of NADH-nitrate reductase and the associated partial activities, NADH-ferricyanide reductase, NADH-cytochrome c reductase, but not in no loss in nitrate reducing activity with reduced methyl viologen as the electron donor. The molecular weight of the nitrate reductase-inactivator protein complex was estimated by gel filtration on Toyopearl HW-55F to be 460 kDa, compared to an apparent molecular weight of 240 kDa for the untreated control estimated under the same conditions. These results indicate that spinach nitrate reductase inactivator protein acts by binding to nitrate reductase. The stoichiometry of binding is 2 molecules of the inactivator protein to one dimeric molecule of nitrate reductase. The action of the inactivator protein was partially prevented by NADH.
日本植物生理学会の論文