Enzymic Assimilation of Nitrate in Tomato Plants. I. Reduction of Nitrate to Nitrite

Abstract

It has been established with considerable certainty that nitrate is assimilated via ammonia in higher plants. Evidence for this comes from several different investigations in which N15-labeled compounds have been utilized. Firstly, it has been shown that nitrate is reduced to anmmonia (7,17). Secondly, it has been shown that nitrate and ammonia have essentially the same effect on the production of free amino acids and proteins (17,26). Finally, ammonia has been shown to be directly incorporated into organic nitrogenous substances via glutamate and glutamine (4,26).It has come to be generally accepted that nitrate reductase is the first enzynme to act on nitrate during its assimilation in higher plants (8). However, the physiological importance of this enzyme must rest, ultimately, on the demnonstrationi that enzymes capable of reducing nitrite to the level of amiiino-nitr-ogen, also exist in higher plants (20).In this investigation a stu(ly wras mlade of the enzymic systems responsible for the entire process of nitrate assimilation in tomato plants. The which was designated "Normal NO,-Level Solution," was 3.2 mA. One set of cultures was growvn in the above nutrient solution nmodified by increasing the level of nitrate containing salts sixfold. The concentration of nitrate in these cultures, which was designatedl "6 X NO,-Level," was 19.2 mai. In these studies the seecds wvere sterilized before germination and all subsequent manip)ulations were carriedI out un(ler aseptic con(litionis.Preparation of E;izvy;ic. Plant tissue wvas ground in 4 times its wseight of 0.1 Mr Tris HCl buffer (pH 7.5) containinig 10 " cysteine. Grinding was done with a cold nmortar and pestle containing approximately as mluclh cold acid washedl sand by weight as plant mlaterial. The macerate wvas pressed through cheese cloth and the filtrate was centrifuged at 1750 X g for 20 minutes. The supernatant solution was either used directly as the crude enzyme preparation or after purification by Sephadex treatment.Treatmlent with Sephadex was carried out by passing 4.0 ml of crude enzyme preparation through a colunin containing 6.0 g of Sephadex G-25 (A. B. Pharmacia, Uppsala, Sweden). Elution was carried out with 0.01 Ni Tris HCl buffer (pH 7.5) containing 10-3 AI cysteine. From 80 % to 100 % of the enzyme was normally recovered virtually free of nitrate in the macerate in the 6 ml of eluate following the first 12 ml. The enzyme containing eluate was used as the purified enzyme preparation. All of the above operations were carried out at 00 to 40 using cold materials and reagents.Enzymitic Assay. Nitrate reductase was assayed using the following reaction mixture: 0.5 ml of 0.1 WI potassium phosphate (pH 7.5), 10,umoles KNO3, 0.27 ,umole DPNH, 0.05 to 0.20 ml enzyme preparation and distilled water to make a final volume of 0.8 ml.Incubations were for 20 minutes at 270. Incubations were stopped by adding 0.2 ml M zinc acetate followedl by 6.0 ml 95 % ethanol. The treated reaction mixtures were centrifuged at 1500 X g for 5 minutes. A suitabl...

Generation of Reduced Nicotinamide Adenine Dinucleotide for Nitrate Reduction in Green Leaves

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Evidence for a complex control system for nitrate reductase in wheat leaves

“…NR was estimated by the following modification of the methods of Sanderson and Cocking [6] and Spencer [7] . The assay mixture contained 0.4 ml potassium phosphate buffer (0.1 M, pH 7.5), 0.1 ml each of the following: alcohol dehydr~enase (Sigma, 0.75 mg per ml potassium phosphate buffer, 0.1 M, pH 7.5), NADH (Sigma, 2.7 mM in 0.005 M Tris), KNOB (0.1 M), ethanol (3 M in potassium phosphate buffer, 0.1 M, pH 7.5) and 0.2 ml of the enzyme extract.…”

Synthesis and Turnover of Nitrate Reductase in Corn Roots

“…The assay mixture for nitrate reductase was as follows: phosphate, pH 7.8, 90 ,moles; KNO,,20 ,umoles; NADH, 1.1 ,tmoles and enzyme, in total volume of 1.5 ml. The colorimetric method for determining nitrite was essentially the same as that described by Sanderson and Cocking (15). A unit of nitrate reductase is defined as that amount of enzyme which can form 1 mpmole of nitrite in 1 hr.…”