Chlamydomonas reinhardtii nitrate reductase complex has 105 kDa subunits in the wild-type strain and a structural mutant

Kalakoutskii, Kyrill; Fernández, Emilio

doi:10.1016/0168-9452(94)04052-4

Cited by 17 publications

(15 citation statements)

References 35 publications

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“…The enzyme could be assayed with NADH or reduced viologens and bromophenol blue as electron donors. The ability of bromophenol blue as an electron donor for nitrate reduction has been previously shown in the assimilatory nitrate reductase from eukaryotic organisms [7, 44]and seems to be a characteristic of the assimilatory nitrate reductases. Actually, the periplasmic enzyme from Rhodobacter sphaeroides , a dimer composed of a cytochrome c subunit and a catalytic nitrate reductase moiety of 92 kDa [17], does not use reduced bromophenol blue as an electron donor (unpublished results).…”

Section: Resultsmentioning

confidence: 67%

“…In polyacrylamide gels, the nitrate reductase and the diaphorase activities were detected as previously described [7, 36].…”

Section: Methodsmentioning

confidence: 99%

“…The assimilation of nitrate is a key process of the nitrogen cycle that is an object of current research in higher plants, fungi, green algae, cyanobacteria and heterotrophic bacteria [1–8]. Nevertheless, the assimilatory nitrate and nitrite reductases have only been scarcely studied in phototrophic bacteria [9, 10].…”

Section: Introductionmentioning

confidence: 99%

“…The electron donor for the assimilatory nitrate reduction is NAD(P)H in eukaryotic organisms [1, 2, 7]and ferredoxin in cyanobacteria [8, 19]. In spite of the assimilatory nitrate reduction having been studied in bacteria, the physiological electron donor has remained unidentified.…”

Section: Introductionmentioning

confidence: 99%

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The assimilatory nitrate reductase from the phototrophic bacterium, Rhodobacter capsulatus E1F1, is a flavoprotein

1997

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The assimilatory nitrate reductase from the phototrophic bacterium Rhodobacter capsulatus has been purified to electrophoretic homogeneity and its molecular and kinetic parameters determined. The native nitrate reductase is a dimer of 144 kDa composed of two subunits of 46 and 95 kDa. The purified enzyme catalyzes the electron transfer from NADH, reduced bromophenol blue or reduced viologens to nitrate. The nitrate reductase contains 1 mol FAD per mole of enzyme and also reduces cytochrome c or dichlorophenol indophenol with NADH as the electron donor. The diaphorase activity is located in the small subunit.

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Section: Resultsmentioning

confidence: 67%

“…In polyacrylamide gels, the nitrate reductase and the diaphorase activities were detected as previously described [7, 36].…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The assimilatory nitrate reductase from the phototrophic bacterium, Rhodobacter capsulatus E1F1, is a flavoprotein

1997

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“…Standard expression of ARC and NR proteins was performed in freshly transformed Escherichia coli TP1000 (mobA mutant) strain (Temple et al 2000) as previously described (Chamizo-Ampudia et al 2011;Sanz-Luque et al 2015). Protein purification was performed with the AKTA Start system (GE Healthcare Life Sciences) using columns of anion exchange (HiTrap Q XL) and gel filtration (HiPrep 16/60), and checked after loading in SDS-polyacrylamide gels (Supplementary Figure S6) (Kalakoutskii & Fernandez 1995). The protein concentration was determined by UV absorption measurements using the calculated extinction coefficient of the analysed polypeptides and by Bradford (Bradford 1976).…”

Section: Expression and Purification Of Recombinant Proteinsmentioning

confidence: 99%

A dual system formed by the ARC and NR molybdoenzymes mediates nitrite‐dependent NO production in Chlamydomonas

Chamizo-Ampudia

Sanz-Luque

Llamas

et al. 2016

Plant Cell & Environment

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130

115

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Nitric oxide (NO) is a relevant signal molecule involved in many plant processes. However, the mechanisms and proteins responsible for its synthesis are scarcely known. In most photosynthetic organisms NO synthases have not been identified, and Nitrate Reductase (NR) has been proposed as the main enzymatic NO source, a process that in vitro is also catalysed by other molybdoenzymes. By studying transcriptional regulation, enzyme approaches, activity assays with in vitro purified proteins and in vivo and in vitro NO determinations, we have addressed the role of NR and Amidoxime Reducing Component (ARC) in the NO synthesis process. N\R and ARC were intimately related both at transcriptional and activity level. Thus, arc mutants showed high NIA1 (NR gene) expression and NR activity. Conversely, mutants without active NR displayed an increased ARC expression in nitrite medium. Our results with nia1 and arc mutants and with purified enzymes support that ARC catalyses the NO production from nitrite taking electrons from NR and not from Cytb5-1/Cytb5-Reductase, the component partners previously described for ARC (proposed as NOFNiR, Nitric Oxide-Forming Nitrite Reductase). This NR-ARC dual system would be able to produce NO in the presence of nitrate, condition under which NR is unable to do it.

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Nitrogen Assimilation and its Regulation

Fernández¹,

Galván²,

Quesada³

The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas

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Chlamydomonas reinhardtii nitrate reductase complex has 105 kDa subunits in the wild-type strain and a structural mutant

Cited by 17 publications

References 35 publications

The assimilatory nitrate reductase from the phototrophic bacterium, Rhodobacter capsulatus E1F1, is a flavoprotein

The assimilatory nitrate reductase from the phototrophic bacterium, Rhodobacter capsulatus E1F1, is a flavoprotein

A dual system formed by the ARC and NR molybdoenzymes mediates nitrite‐dependent NO production in Chlamydomonas

Nitrogen Assimilation and its Regulation

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