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Book ChapterDOI

Enzymes of Inorganic Nitrogen Metabolism

01 Jan 1964-pp 67-172
TL;DR: The enzymes considered in this chapter include nitrate reduct enzyme, nitrite reductase, hyponitrite reduCTase, hydroxylamine reductases, enzymes of denitrification, and some other enzymes such as aldehyde oxidase with related properties.
Abstract: The enzymes considered in this chapter include nitrate reductase, nitrite reductase, hyponitrite reductase, hydroxylamine reductase, enzymes of denitrification, and some other enzymes such as aldehyde oxidase with related properties. The properties of the various enzymes are described in relation to the different plants and micro-organisms from which they have been obtained1.
Citations
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Journal ArticleDOI
TL;DR: In this article, the enzyme nitrate reductase was found to be active in marine phytoplankton when growing on ammonium or when the nitrogen source was depleted.
Abstract: Certain marine phytoplankton contain the enzyme nitrate reductase when growing on nitrate, but only low levels of enzyme were found during growth with ammonium or when the nitrogen source was depleted. Netted samples of oceanic phytoplankton contained the enzyme when taken from waters with nitrate concentrations 2–10 µm. Ammonium was assimilated in preference to nitrate in phytoplankton cultures supplied with both forms of nitrogen at 5–15 µm. Enzyme synthesis and nitrate use began when ammonium was depleted to 0.5–1.0 µm. Nitrate reductase assay of phytoplankton samples is a useful tool in that a positive result indicates utilization of nitrate and a negative one implies growth on ammonium, nitrogen depletion, or, improbably, growth with other N-sources such as nitrite, urea, or amino acids. The enzyme assay seems especially useful for studying the timecourse of phytoplankton blooms because it provides a sensitive measure of the initiation and cessation of nitrate assimilation.

270 citations

Journal ArticleDOI
01 Sep 1974-Planta
TL;DR: The assimilation of nitrate, nitrite and ammonia in barley, wheat, corn and bean leaves was studied using 15N-labelled molecules and either leaf chamber experiments with the uptake of the nitrogen species in the transpiration stream, or vacuum-infiltration experiments.
Abstract: The assimilation of nitrate, nitrite and ammonia in barley, wheat, corn and bean leaves was studied using 15N-labelled molecules and either leaf chamber experiments with the uptake of the nitrogen species in the transpiration stream, or vacuum-infiltration experiments. The assimilation of 15NO3− into amino nitrogen was strictly dependent on light and ceased abruptly when the light was extinguished. If the leaves were exposed to air, CO2-free air or N2 there was no effect on the rate of NO3− assimilation over 0.5 h. After 1.25 h of CO2-free air, NO3− assimilation into amino acids was sharply reduced. Resupply of air at this time stimulated NO3− assimilation and restored it to the rate observed in leaves exposed to air only. There was no recovery by tissue pretreated for 1.25 h in N2 and subsequently resupplied with air. Incorporation of 15NO2− was also markedly dependent on light with little reduction occurring in the dark. Incorporation of 15NH4+ into amino acids was stimulated 5 fold by light but considerable incorporation occurred in the dark. The presence of 100 mM NO3− had no effect on the rate of incorporation of 15NO2− or 15NH4+. Nitrite at 1 mM had no effect on 15NO3− incorporation but at 10 mM inhibited it completely after 0.5 h. Ammonia at 1 mM had no effect on 15NO3− or 15NO2− incorporation and while 10 mM inhibited incorporation for 0.5 h this inhibition did not persist.

152 citations

Journal ArticleDOI
TL;DR: It is concluded that nitrate reductase is a single moiety with the ability to utilize either NADH or FMNH(2) as cofactor and that in vivo NADH is the electron donor and that nitrates reductases in higher plants should be designated NADH:nitrate reduCTase (E.C.6.1).
Abstract: With respect to cofactor requirements, NADH, and FMNH(2) were equally effective as electron donors for nitrate reductase obtained from leaves of maize, marrow, and spinach, when the cofactors were supplied in optimal concentrations. The concentration of FMNH(2) required to obtain half-maximal activity was from 40- to 100-fold higher than for NADH. For maximal activity with the corn enzyme, 0.8 millimolar FMNH(2) was required. In contrast, NADPH was functional only when supplied with NADP:reductase and exogenous FMN (enzymatic generation of FMNH(2)).All attempts to separate the NADH(2)- and FMNH(2)-dependent nitrate reductase activities were unsuccessful and regardless of cofactor used equal activities were obtained, if cofactor concentration was optimal. Unity of NADH to FMNH(2) activities were obtained during: A) purification procedures (4 step, 30-fold); B) induction of nitrate reductase in corn seedlings with nitrate; and C) inactivation of nitrate reductase in intact or excised corn seedlings. The NADH- and FMNH(2)-dependent activities were not additive.A half-life for nitrate reductase of approximately 4 hours was estimated from the inactivation studies with excised corn seedlings. Similar half-life values were obtained when seedlings were incubated at 35 degrees in a medium containing nitrate and cycloheximide (to inhibit protein synthesis), or when both nitrate and cycloheximide were omitted.In those instances where NADH activity but not FMNH(2) activity was lost due to treatment (temperature, removal of sulfhydryl agents, addition of p-chloromercuribenzoate), the loss could be explained by inactivation of the sulfhydryl group (s) required for NADH activity. This was verified by reactivation with exogenous cysteine.Based on these current findings, and previous work, it is concluded that nitrate reductase is a single moiety with the ability to utilize either NADH or FMNH(2) as cofactor. However the high concentration of FMNH(2) required for optimal activity suggests that in vivo NADH is the electron donor and that nitrate reductase in higher plants should be designated NADH:nitrate reductase (E.C. 1.6.6.1).

145 citations

Journal ArticleDOI
C. Manzano1, P. Candau1, C. Gomez-Moreno1, A. M. Relimpio1, M. Losada1 
TL;DR: In its presence, the alga particles catalyze the gradual photoreduction of nitrate to nitrite and ammonia, a process that can thus be considered as one of the most simple and relevant examples of Photosynthesis.
Abstract: The dark and light reduction of nitrate and nitrite by cell-free preparations of the blue-green algaAnacystis nidulans has been investigated. The three following methods have been successfully applied to the preparation of active particulate fractions from the alga cells: (a) shaking with glass beads, (b) lysozyme treatment and lysis of the resulting protoplasts, and (c) sonication. The two enzymes of the nitrate-reducing system-namely, nitrate reductase and nitrite reductase-are firmly bound to the isolated pigment-containing particles, and can be easily solubilized by prolonging the vibration or sonication time. Both enzymes-whether solubilized or bound to the particles-depend on reduced ferredoxin as the immediate electron donor. In its presence, the alga particles catalyze the gradual photoreduction of nitrate to nitrite and ammonia, a process that can thus be considered as one of the most simple and relevant examples of Photosynthesis. Some of the properties of nitrate reductase have been studied. Nitrate reductase as well as nitrite reductase are adaptive enzymes repressed by ammonia.

130 citations

Journal ArticleDOI
TL;DR: Ammonium and nitrate are accumulated in the vacuolated cells of the diatom and these intracellular pools serve as substrate for the assimilatory enzymes, while nitrite is either not accumulated or is concentrated, in a very small cellular compartment.
Abstract: SUMMARY Apparent Km values for nitrite reductase, glutamic dehydrogenase, and nitrate reductase are of the order 10−4 molar for nitrite, ammonia, and nitrate, respectively while half-saturation constants for the corresponding uptake mechanisms approximate 10−6 molar. Ammonium and nitrate are accumulated in the vacuolated cells of the diatom (about 10 and 40 mmoles/liter cell volume, respectively) and these intracellular pools serve as substrate for the assimilatory enzymes. Nitrite is either not accumulated or is concentrated, in a very small cellular compartment. Ammonium and nitrate in the external medium exert modifying effects on uptake and assimilatory mechanisms which can be distinguished from effects of the ions accumulated within the cells. Several of these effects are described and fitted into a general scheme of nitrogen assimilation by D. brightwellii.

107 citations

References
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Book
01 Jan 1962
TL;DR: In this article, Microdiffusion analysis and volumetric error was used to detect micro-diffusion errors in the context of micro-scale analysis of the volumetry data.
Abstract: Microdiffusion analysis and volumetric error , Microdiffusion analysis and volumetric error , مرکز فناوری اطلاعات و اطلاع رسانی کشاورزی

2,357 citations

Journal ArticleDOI
TL;DR: The tabulation gives the normal potentials of the various indicators at 30°C.
Abstract: The tabulation gives the normal potentials of the various indicators at 30 degrees C.; referred to the normal hydrogen electrode, the accuracy is estimated to be +/-0.002 volt. Normal potentials of the viologens at 30 degrees C.: Methyl viologen -0.446 volts Ethyl viologen -0.449 volts Betaine viologen -0.444 volts Benzyl viologen -0.359 volts Supposing some solution brings about a coloration of one of these indicators to the extent of A per cent of the maximum color, the oxidation-reduction potential of this solution is E = E(o) - 0.06 log See PDF for Equation where E(o) is the normal potential according to the above tabulation. This normal potential is independent of pH.

339 citations

Journal ArticleDOI
TL;DR: This article describes the identification of the enzyme nitrate reductase (NR) in the leaves of vascular plants, the first report of NR in plants.
Abstract: This article describes the identification of the enzyme nitrate reductase (NR) in the leaves of vascular plants. Although NR had previously been described in Neurospora, this is the first report of NR in plants. The discovery of NR opened up the field of study of plant nitrogen assimilation and led to the publication of hundreds of articles. NR requires reducing power from NADH, NADPH, or both depending on its location within the plant body and the species. This very complicated enzyme took many years to actually purify. After purification, we learned that it is controlled by phosphorylation/ dephosphorylation. Harold Evans and others later showed NR to be one of the few molybdenumcontaining enzymes in vascular plants. After working on NR, Evans turned his attention to another molybdenum-containing enzyme, dinitrogenase, in soybean (Glycine max) root nodule bacteroids. Harold Evans became President of the American Society of Plant Physiologists and a member of the National Academy of Science based on this discovery and many other contributions.

312 citations