E. J. Hewitt

The presence of functional haem in a higher plant nitrate reductase

Abstract Nitrate reductase from spinach (Spinacea oleracea L. v. Noorman) has been purified 2600-fold by a multistep procedure involving streptomycin sulphate treatment, (NH4)2SO4 precipitation, hydroxylapatite adsorption, molecular sieve chromatography and blue-dextran agarose affinity chromatography. Spectral studies of the purified enzyme indicate the presence of a b-type cytochrome associated with the enzyme and involved in both its overall function of reducing nitrate with NADH and its dehydrogenase function using dichlorophenolindophenol (DCPIP) as electron acceptor.

The use ofAspergillus niger (Van Tiegh.) M strain as a test organism in the study of molybdenum as a plant nutrient

The use of l i v ing mate r i a l , in th is i n s t a n c e a mic ro -o rgan i sm, for the b io log ica l a s say of o rgan ic or m i n e r a l ma te r i a l s d e p e n d s on n u m e r o u s a s s u m p t i o n s . I t is b e y o n d the scope of th is pa pe r to r ev iew the p r inc ip l e s of b ioas say which compr ise aspects of g rea t complex i ty . I t wil l be suff ic ient to ou t l i ne the reasons for a d o p t i n g th is procedu re a n d the f u n d a m e n t a l r e q u i r e m e n t s in the re l iable a p p l i c a t i o n of the m e t h o d .

Some effects of nitrate abundance and starvation on metabolism and accumulation of nitrogen in barley (Hordeum vulgare L. cv Sonja)

Nitrate and nitrite reductases were both induced by adding three concentrations of nitrate to the nutrient supply of nitrate-starved barley seedlings. Enzyme induction was not proportional to the amount of nitrate introduced. Glutamine synthetase also increased above a high endogenous activity but the increase did not differ significantly between any of the three nitrate treatments. Nitrate accumulated rapidly in leaves of plants given 4.0 mM or 0.5 mM nitrate but not with 0.1 mM nitrate. In all treatments, amino acids in leaves increased for 2 d, chiefly attributable to glutamine, then declined. Transferring plants from the three nitrate treatments to nitrate-free nutrient produced an immediate decline in nitrate reductase but nitrite reductase continued to increase for 2 d, before declining. Glutamine-synthetase activity was not affected by withdrawal of nitrate, nor did nitrate withdrawal retard plant growth during the 9-d period of the experiment. The disparity between accumulated nitrate and nitrate-reducing capacity and the rapid decrease in leaf nitrate when nutrient nitrate supply was removed, indicated the presence of a nitrate-storage pool that could be called upon to maintain amino-acid production in times of nitrogen starvation.

Nitrate Reductase Systems in Eukaryotic and Prokaryotic Organisms

Publisher Summary This chapter reviews the structure and mechanism of nitrate reductases and presents the distinction between two main types of nitrate reductases on a phylogenetic basis, namely: the eukaryotic type and one (or possibly more) prokaryotic types. The most critical and least understood aspects of the biochemistry of nitrate reduction are the electronic events associated with the production of nitrite from nitrate at the molybdenum center. The eukaryotic forms of nitrate reductases are reported to lie in the molecular weight range between 160,000 and 500,000; although the elliptical shape of the enzyme, as shown by its anomalous behavior on sucrose density centrifugation and gel filtration, requires that more than one method of molecular weight estimation be used to determine the true value. The presence of subunits is characteristic of all the eukaryotic forms of the enzyme studied to date but their sizes and probable numbers differ quite widely. For the prokaryotic, the molecular weight of the purified terminal moieties obtained from prokaryotic sources range from 69,000 to 880,000. Subunit compositions for these are complex and not consistent in terms either of methods of dissociation or of different preparations from separate laboratories.

The production of copper, zinc and molybdenum deficiencies in crop plants grown in sand culture with special reference to some effects of water supply and seed reserves

Summary(1) A method is described for the production of acute deficiencies of copper, zinc or molybdenum in large scale sand cultures in which an adequate and satisfactory supply of water was obtained by treating rain water with ion-exchange resins. Results compared favourably with the use of glass distilled water.(2) Visual symptoms of copper, zinc and molybdenum deficiencies in large seeded leguminous crops, and in other species are summarised.(3) Seed reserves played an important part in the incidence of copper and molybdenum deficiencies and to a lesser extent of zinc deficiency in pea, runner bean, dwarf bean and broad bean.(4) The “deficient” seed was obtained by harvesting seed from plants grown in the respective deficiency sand cultures, and was compared with seed obtained similarly from complete nutrient sand cultures or commercially from field-grown plants.(5) Seed reserves of molybdenum were usually sufficient for a complete generation but reserves of copper and zinc were not adequate for the whole copper or zinc requirements of the plants.(6) Analytical data showed a marked decrease in the copper or molybdenum content of “deficient” seed, and a less striking decrease in zinc.(7) Foliage of deficient plants showed decreased content of copper and molybdenum and often of zinc compared with plants given the complete nutrient treatment, but use of “deficient” seed only decreased yields without further appreciably decreasing the mineral content of the foliage.

The role of molybdenum in the synthesis of nitrate reductase in cauliflower (Brassica oleracea L. var. Botrytis L.) and spinach (Spinacea oleracea L.)

Abstract 1. 1.The 185W analogue of nitrate reductase was produced in molybdenum-deficient cauliflower (Brassica oleracea L. var. Botrytis L.) leaves following petiolar uptake of 185W from a molybdenum-free medium. l -Azetidine-2-carboxylic acid in the nutrient solution had no effect on analogue formation, but puromycin decreased by 75% 185W incorporation into protein in the fraction normally associated with nitrate reductase. The analogue co-purified with spinach (Spinacea oleracea L.) nitrate reductase. 2. 2.An antiserum prepared against partially purified spinach nitrate reductase inhibited enzyme activity. 3. 3.Using the antiserum a method was developed, based on 50% inactivation of a “standard” nitrate reductase preparation, to measure the amount of material in solution cross-reacting as nitrate reductase protein but having no enzyme activity. Crude extracts and solutions resulting after purification with respect to nitrate reductase were obtained from molybdenum-deficient spinach plants grown with nitrate either as the sole nitrogen source of supplemented with ammonium salt. These were assayed for their content of cross-reacting material. 4. 4.The results indicate that approximately 30% of the normal complement of nitrate reductase protein found in extracts of healthy spinach plants was present in extracts of molybdenum-deficient spinach plants either as aponitrate reductase orits immunochemically cross-reacting sub-units. Purification of crude extracts with respect to nitrate reductase resulted in a preferential and substantial loss of cross-reacting material relative to the enzyme. It is suggested that breakdown of the apoprotein is rapid in vivo compared with that of the holoenzyme. Continued net increase of nitrate reductase resulting from metal incorporation into apoprotein is dependent on protein synthesis.

Reversible cyanide inhibition of spinach (Spinacea oleracea L.) nitrate reductase and non-exchangeability in vitro of protein bound molybdenum and tungsten

Nicholas and Nason [l] identified molybdenum as the metal constituent of nitrate reductase of soybean by dialysis against cyanide, which supposedly removed the metal, followed by dialysis against purified phosphate buffer and finally reactivation of the intact enzyme specifically with molybdate. Attempts to repeat these results with enzymes obtained from different plants, including spinach, have been unsuccessful [2-51. Nevertheless, contrary to previous doubts [4,6], nitrate reductase of spinach is now shown [7] to be a molybdoprotein. Tungsten inhibits the production of nitrate reductase in barley [8] but still allows the induction by nitrate of an associated NADHcytochrome c reductase protein [9]. The inhibitory effect of Tungsten [8] is explained by the production in uivo of an inactive tungstoprotein analogue of nitrate reductase [lo]. We now report that inhibition of the spinach enzyme by cyanide [4] is reversible without removal of molybdenum from the enzyme. Cyanide is unable also to remove ‘*‘W labelled tungsten from the protein produced when tungstate is given to molybdenum deficient plants. Moreover, exchange in vitro during dialysis between protein bound and free molybdenum or tungsten is insignificant in spite of evidence for occurrence of this reaction in vivo [7, 111.

Molybdenum and the fixation of nitrogen inCasuarina andAlnus root nodules

Summary1. The significance of molybdenum in the metabolism and growth ofCasuarina andAlnus was investigated, both for nodulated plants dependent on the fixation of elemental nitrogen and for non-nodulated plants supplied with nitrate nitrogen.2. In both genera molybdenum was found to be necessary for each type of plant. Nitrate plants deprived of molybdenum showed abnormal visual features in the shoot and an accumulation of unreduced nitrate in the tissues. Nodulated plants developed marked nitrogen deficiency, shown to be due to a decrease in the effectiveness of the nodule tissues in fixation of nitrogen.3. The evidence indicates that in Alnus at least molybdenum is of greater significance to nodulated plants than to those utilising nitrate nitrogen.4. The nodules have a high accumulative capacity for molybdenum.5. For nodulated plants the observations are wholly consistent with the view that molybdenum fulfils some essential role in the fixation of nitrogen proceeding in these non-legume nodules, and indicate that in non-nodulated plants supplied with nitrate, molybdenum is concerned with the reduction of nitrate subsequent to absorption.

The regulation of nitrate reductase activity from spinach Spinacea oleracea L. leaves by thiol compounds in the presence of adenosine-5′-diphosphate

The concept of a carbon-nitrogen balance in plants stimulated speculation [l-3] that nitrate reductase (EC 1.6.6.1) may be controlled by kinetic mechanisms associated with photosynthesis or carbohydrate metabolism. Kinetic regulation is indicated by the discovery [4] that nitrate reductase activity in viva may be less than that found after extraction. ADP is a negative effector of nitrate reductases from tomato [5] and spinach [6] leaves. The mechanism involves competition with NADH and noncompetitive inhibition both in a non-linear manner [6]. Inhibition of the tomato enzyme was erratic [5] and ageing of the spinach enzyme caused desensitization [6] . We report that inhibition by ADP is reversibly determined by the thiol and NADH concentrations in the system.

Preparation, crystallization and properties of Cucurbita pepo plastocyanin and ferredoxin

Abstract Homogeneous plastocyanin was obtained from Cucurbita pepo L. The MW was 11 360 daltons based on 0.56% copper. Values of S°20W = 1.69 S and D°20W = 1.46 x10−6 cm2 sec−1 and V = 0.73 (amino acid analysis) or 0.74 (pycnometry) indicated 10 300 to 10 900 daltons. Gel chromatography on Sephadex G75 suggested 12 200 daltons. Amino acid analyses of two cultivars indicated 10928 to 11 000 daltons for 102 residues and one atom of copper, excluding 1.0% protein-bound carbohydrate. The single thiol group reacted slowly with 2-chloromercuri-4-nitrophenol but rapidly with mercuric acetate. The redox potential was +350 mV between pH 6.5 and 9.0 with a one-electron change and an ionizable group of pK 5.6. The isoelectric point was at pH 4.2. Light absorption maxima occurred at 253, 259, 264, 269, 278, 284, 460 (very weak), 597 and 775 nm. The best ratio E597:E278 was 0.87. Extinction coefficient at 597 nm was 4.75 x 103 l.mol−1 cm−1. Copper was reversibly removed and 83% restored to apoprotein during successive treatments with mercuric acetate, Sephadex G25 and glutathione. The oxidized protein was crystallized from 58 or 60%-saturated ammonium sulphate containing 2% dioxan at pH 4.0 to 4.5. Oxidized crystals in ammonium sulphate were reduced by ascorbic acid or photochemically with glycerol and then dissolved. Homogeneous ferredoxin from the same source had a MW of 11 400 daltons based on 0.98% iron and 11 045 by amino acid analysis. Two atoms each of iron and labile sulphide were recorded. Methionine was present. Aggregation occurred during sedimentation which indicated 20 500 daltons from S°20W = 2.44S; D°20W = 1 .0 x 10−6 cm2 sec−1 and V = 0.71. Gel chromatography indicated 12 000 to 16 000 daltons depending on media. Redox potential at 25° was −404 mV at pH 7.5. Light absorption maxima were at 277, 333, 422 and 462 nm. The best ratio E422:E277 nm was 0.51. The extinction coefficient at 422 nm was 9.8 x 10−3 l.mol−1 cm−1. The protein crystallized as red needles from 78% or 80%-saturtted (NH4)2SO4.