Brian A. Notton

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.

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.

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.

Monoclonal antibodies to a higher-plant nitrate reductase: Differential inhibition of enzyme activities.

A set of monoclonal antibodies has been raised against NADH-nitrate reductase (NR; EC 1.6.6.1) from spinach (Spinacea oleracea L.) leaves. Antibodies were screened by enzyme-linked immunosorbent assay and by their ability to inhibit various activities of the enzyme. The six monoclonals selected (AFRC MAC 74 to 79) are all gamma globulins; four (MAC 74 to 77) inhibit all terminal donating activities (NADH-NR; flavin mononucleotide, reduced form (FMNH2)-NR; and methyl viologen, reduced form (MV)-NR) and two (MAC 78 and 79) inhibit the acceptor activities (NADH-NR, and NADH-cytochrome c reductase). MAC 74 to 77 inhibit the NADH-NR activity of crude extracts of a variety of species (mono- and dicotyledoneae) while MAC 78 and 79 are effective against spinach and marrow, but not oil-seed rape, cucumber, oats, wheat and barley.

Formation of nitrate reductase by recombination of apoprotein fractions from molybdenum-deficient plants with a molybdenum-containing complex

Abstract A molybdenum-containing complex (MCC) has been obtained from NADH-nitrate reductase, partially purified from spinach leaves, by treatment at pH 2.5. The apoprotein of nitrate reductase, which was obtained from leaves of molybdenum-deficient plants grown with nitrate, and ammonium or tungsten or both, retains cytochrome c reductase activity and was shown to react in vitro with MCC to form active nitrate reductase. This was used as a method for estimating the apoprotein content of leaves. Temperature, time and pH conditions for maximum stability of MCC and reconstitution with apoprotein to form active enzyme were determined. Cytochrome c reductases, sedimenting with values of 3.7 S or 8.1 S by sucrose density centrifugation or separated by molecular sieve chromatography, were shown to reconstitute nitrate reductase with MCC. The effect of heat in vivo (> 41 °C) or in vitro (35 °C) was less detrimental to gross cytochrome c reductase activity than to its ability to reconstitute nitrate reductase with MCC. Nitrate reductase adsorbed by AMP-Sepharose was found, using a 185 W analogue marker, to release a low molecular weight metal-containing fraction when washed with phosphate. This fraction appeared to reconstitute nitrate reductase with apoprotein and could be further fractionated into a larger and smaller fragment neither of which could reconstitute the enzyme without the other. The structural and possible significance of these results is discussed.

The reactivation of nitrate reductase from spinach (Spinacia oleracea L.) inactivated by NADH and cyanide, using trivalent manganese either generated by illuminated chloroplasts or as manganipyrophosphate

Nitrate reductase of spinach (Spinacia oleracea L.) leaves which had been inactivated in vitro by treatment with NADH and cyanide, was reactivated by incubation with oxidant systems and measured as FMNH2-dependent activity. Reactivation was produced with trivalent manganese compounds represented either by manganipyrophosphate or produced by oxidation of Mn2+ ions in the presence of illuminated chloroplasts and compared with reactivation obtained with ferricyanide. Reactivation in the chloroplast system was equivalent to that with ferricyanide when orthophosphate was used but was variable and weak in the presence of pyrophosphate, although manganipyrophosphate was formed, freely. Reactivation by manganipyrophosphate in dark reaction conditions was less effective than with ferricyanide but was not inhibited by the addition of pyrophosphate. Reactivation with illuminated unheated chloroplasts was dependent on added manganese and oxidation of manganese in the presence of pyrophosphate was abolished by boiling the chloroplasts. In the presence of orthophosphate however, boiled, illuminated chloroplasts reactivated the enzyme in the absence of added manganese. Reactivation occurred spontaneously in air, more slowly than with the other oxidants, but to a similar extent to that produced by manganipyrophosphate. The results provide a possible model for physiological reactivation mechanisms.

Isoelectric focusing of spinach nitrate reductase and its tungsten analogue

Abstract Highly purified nitrate reductase from spinach ( Spinacea oleracea L.) was submitted to isoelectric focusing. Two peaks of enzyme activity occurred at pH 3·5 and 4·9. Both nitrate reductases could use NADH and reduced benzylviologen but not NADPH as electron donors. The enzymically inactive tungsten analogue of nitrate reductase labelled with 185 W during biosynthesis was mixed with the active enzyme before purification and yielded two tungsten protein peaks after isoelectric focusing coincident with those containing nitrate reductase.

Spinach nitrate reductase: Further purification and removal of ‘nicked’ sub-units by affinity chromatography

Abstract NADH-nitrate reductase (EC 1.6.6.1) from spinach (Spinacea oleracea L. v. Noorman) has been purified 3400-fold by a multi-stage procedure involving streptomycin sulphate, (NH4)2SO4, hydroxylapatite, molecular seiving and two stages of affinity chromatography using blue-Sepharose and 5′ AMP-Sepharose. The enzyme has a specific activity of 51 μmol NO2− produced min−1 mg−1 and a functional haem with extinction coefficients (mM) of 127 at 412 nm (oxidized) and 172 at 423 (NADH-reduced). Gel electrophoresis indicates two sub-units of approx. 110 000 and 120 000.

Light transmission into apple fruit and leaves

Abstract Light transmission into tissue of fruit or leaves of apple (Malus pumila Mill.) cultivars ‘Golden Delicious’, ‘Gloster’ and ‘Coxs Orange Pippin’ was studied both quantitatively and qualitatively. For comparative purposes, fruit or leaf was exposed to a constant photosynthetically active radiation (PAR) of 1750 μmol m−2 s−1. Under these conditions, transmission of PAR into the inner tissues of apple fruit ranged between 0.04 and 8.4 μmol m−2 s−1, dependent on development stage, cultivar and position of the apple tissue relative to the light source. Parenchyma of apple fruit transmitted 1.5–2% of the incident PAR 1.5 mm−1 fruit tissue. Peel of apple fruit transmitted 1–3% of incident PAR at 400 nm, increasing to 10–12% at 850 nm. Whole apple leaves transmitted up to 2%, whereas an upper epidermis and mesophyll layer transmitted up to 7% of incident PAR. With green fruit, transmission and action spectra were dominated by chlorophyll absorption at 678 nm, whereas apple fruit with red or yellow peel additionally absorbed at 480 nm.

Inhibition by L-azetidine-2-carboxylic acid of induction of nitrate reductase in plants and its reversal by L-proline

Abstract The effect of L -azetidine-2-carboxylic acid on the induction of nitrate reductase in leaf tissues of radish and cauliflower plants was studied. Plants were grown under conditions of nitrate starvation or with ammonium sulphate either in sterile culture, or in the presence of 2-chloro-6-(trichloromethyl)-pyridine (“N-Serve”) or under conditions of molybdenum deficient sand culture. L -Azetidine-2-carboxylic acid severely inhibited nitrate reductase induction by nitrate in tissues with a low proline content. Inhibition was reversed by simultaneous infiltration of L -proline. Induction of the enzyme in response to molybdenum was less inhibited in tissues of molybdenum deficient plants which had a high endogenous proline content. No reversal was obtained with seventeen other normal protein amino acids.