Elsevier

Methods in Enzymology

Volume 69, 1980, Pages 270-280
Methods in Enzymology

[24] Nitrate reductase from higher plants

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This chapter describes the preparation and extraction of nitrate reductase from higher plants. For preparation of nitrate reductase, chlorophyllous lamina tissue from illuminated plants well supplied with nitrate is normally used as a source material because of its high activity. However, monchlorophyllous organs, such as corn scutella or roots, have been used. Soybean leaves, corn scutella, and cultured rice seedlings are the best known sources of NAD(P)H nitrate reductase. For extraction of the enzyme, the material is homogenized for 30 to 90 seconds in a medium of 1 mM EDTA, 1 to 25 mM cysteine, and 25 mM potassium phosphate, adjusted to a final pH of 8.8 with KOH. Six milliliters of grinding medium are added for each gram of fresh weight of tissue. The homogenate is pressed through four layers of cheesecloth or a single layer of Miracloth and the filtrate centrifuged for 15 minutes at 30,000 g. The supernatant fluid is then decanted through glass wool and used for assays. The homogenates and extracts are kept cold (2° to 3°) throughout. The optimum concentration of cysteine must be verified for each type of tissue and homogenization technique. Glutathione and dithiothreitol are usually slightly more effective (10%) than cysteine.

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