Purification and Properties of NADH : Nitrate Reductase from the Red Alga Porphyra yezoensis

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概要

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• Assimilatory nitrate reductase (NADH) (EC 1.6.6.1) from the red alga Porphyra yezoensis was purified 5,700-fold by a combination of polyethylene glycol (PEG) treatment, ammonium sulfate fractionation, chromatography on columns of butyl-Toyopearl 650-M, Blue Sepharose CL-6B. DEAE-cellulose (DE 52), and hydroxyapatite, gel filtration on Sephacryl S-400. The purest preparation of the enzyme had a specific activity of 12.5 units mg^<-1> protein. A single band of protein was detected after polyacrylamide gel electrophoresis under nondenaturing conditions. This band corresponded to a band that stained positive for reduced methyl viologen-nitrate reductase activity. The molecular weight of the native enzyme was estimated to be 220,000. A single band of a protein with a molecular weight of 100,000 was detected after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results indicate that the native nitrate reductase is composed of two identical subunits. The homogeneous preparation of nitrate reductase had a UV/visible spectrum typical of a b-type cyiochrome. The K_m values for NADH and KNO_3 Were 23μM and 64μM, respectively. The pH optimum for the reaction catalyzed by the nitrate reductase was 8.3, while pH values that supported maximum partial activities ranged from 7.0 to 8.5. Sulfhydryl reagents, such as p-HMB and NEM, inhibited full and NADH-utilizing partial activities, while cyanide and azide were effective inhibitors of full and nitrate-reducing partial activities.

• 日本植物生理学会の論文

著者

• Nakamura Yoshiko

  Institute Of Biological Sciences University Of Tsukuba

• Ikawa Tomoyoshi

  Institute Of Biological Sciences The University Of Tsukuba

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