Mass spectrometry identification of membrane-bound respiratory nitrate reductase from Bradyrhizobium sp. (Lupinus).

2008 ◽  
Vol 55 (4) ◽  
pp. 753-760 ◽  
Author(s):  
Władysław Polcyn
Keyword(s):  
Mass Spectrometry ◽  
Nitrate Reductase ◽  
Biochemical Properties ◽  
Validation Criteria ◽  
Sds Page ◽  
Mass Spectrometry Identification ◽  
Bradyrhizobium Sp ◽  
Rhizobial Species ◽  
Membrane Bound ◽  
Respiratory Nitrate Reductase

Respiratory nitrate reductase (NR) from Bradyrhizobium sp. (Lupinus) USDA 3045 has biochemical properties of the membrane-bound NR type. However, in the completely sequenced rhizobium genomes only genes for the periplasmic type of dissimilatory NR were found. Therefore purification and identification of the enzyme by tandem mass spectrometry (MS/MS) was undertaken. MS/MS spectra representing 149 unique tryptic peptides derived from purified 137-kDa subunit matched the NCBInr-deposited NarG sequences. MS/MS sequencing of two other SDS/PAGE bands (65- and 59-kDa) identified them as derivatives of the NarH-type protein. Applying additional validation criteria, 73% of the sequence of the NarG subunit (902 aa) and 52% of NarH sequence (266 aa) was assembled (UniProt KB acc. no. P85097 and P85098). This is the first unambiguous identification of an active NarGH-like NR in rhizobia. Moreover, arguments are provided here for the existence of a functional enzyme of this type also among other rhizobial species, basing on immunoblot screening and the presence of membrane-associated NR-active electrophoretic forms.

Nitrate-related down-regulation of respiratory nitrate reductase from Bradyrhizobium sp. (Lupinus).

2008 ◽  
Vol 55 (4) ◽  
pp. 761-766 ◽  
Author(s):  
Władysław Polcyn
Keyword(s):  
Nitrate Reductase ◽  
De Novo ◽  
Fold Increase ◽  
Fold Reduction ◽  
Bradyrhizobium Sp ◽  
Membrane Bound ◽  
Activity Band ◽  
Endogenous Proteases ◽  
Nr Activity ◽  
Respiratory Nitrate Reductase

Previously, we showed that anaerobic induction of respiratory nitrate reductase (NR) activity in Bradyrhizobium sp. (Lupinus) USDA 3045 is strongly enhanced by nitrate or nitrite through de novo synthesis. Here, multiple NR-active soluble forms, ranging from 75 kDa to 190 kDa, were observed under anaerobic conditions. Electrophoretic activity band patterns differed depending on the level and the type of the N oxyanion added. The intensity of the membrane-bound NR activity band of 230 kDa changed with time along with consumption of 2 mM nitrate. It was associated with a parallel 5-fold increase and then 2-fold reduction in the amount of membrane-bound NR protein. In contrast, on 4 mM nitrate, the level of NR protein was much more stable, apparently due to slower nitrate depletion. Moreover, in cells anaerobically grown without nitrate addition, a 42-kDa derivative of NR degradation was immunodetected, which was not observed if nitrate was present in the medium. These findings suggest that the amount of the respiratory NR protein could be negatively regulated by endogenous proteases in relation to the level of nitrate available. It seems, therefore, that multiple native forms might be not different isoenzymes but immature complexes or derivatives of the enzyme protein turnover. This report adds to a modest list of bacterial enzymes apparently regulated by proteolysis, such as GS, MurAA, EnvA, GdhA, and MetA.

scholarly journals Membrane-bound respiratory nitrate reductase functioning: It takes two to tango

2012 ◽  
Vol 1817 ◽  
pp. S127
Author(s):  
R. Arias-Cartin ◽  
S. Grimaldi ◽  
P. Lanciano ◽  
P. Arnoux ◽  
B. Guigliarelli ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Membrane Bound ◽  
Respiratory Nitrate Reductase

Synthesis and sideedness of membrane-bound respiratory nitrate reductase (EC1.7.99.4) in Escherichia coli lacking cytochromes

1975 ◽  
Vol 148 (2) ◽  
pp. 329-333 ◽  
Author(s):  
M B Kemp ◽  
B A Haddock ◽  
P B Garland
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Cytochrome B ◽  
Cytoplasmic Membrane ◽  
Coli Strain ◽  
Respiratory Pathway ◽  
Aminolaevulinic Acid ◽  
Membrane Bound ◽  
Respiratory Nitrate Reductase

The synthesis of nitrate reductase and its incorporation into the cytoplasmic membrane of Escherichia coli strain A1004a (5-aminolaevulinic acid auxotroph) does not require synthesis of cytochrome b. The synthesis of the apoprotein(s) of the cytochrome b of the respiratory pathway from NADH to nitrate appears to be inhibited by the absence of haem. No member of the respiratory pathway from NADH to oxygen is capable of reducing nitrate reductase directly. The site on nitrate reductase that oxidizes FMNH2 is located on the cytoplasmic aspect of the cytoplasmic membrane.

scholarly journals Synthesis of cytoplasmic membrane during growth and division of Escherichia coli. Dispersive behaviour of respiratory nitrate reductase

1979 ◽  
Vol 184 (1) ◽  
pp. 45-50 ◽  
Author(s):  
E Cadenas ◽  
P B Garland
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Nitrate Reductase Activity ◽  
Cytoplasmic Membrane ◽  
Reductase Activity ◽  
Selection Method ◽  
Separation Procedure ◽  
Physical Separation ◽  
Membrane Bound ◽  
Respiratory Nitrate Reductase

We have used the penicillin selection method of Autissier & Kepes [(1972) Biochimie 54, 93–101] to study the segregation of membrane-bound respiratory nitrate reductase (EC 1.9.6.1) in Escherichia coli for the three generations after cessation of nitrate reductase synthesis caused by withdrawal of nitrate from the growth medium. We also included a physical separation procedure that permitted direct assay for nitrate reductase activity among all fractions produced by the penicillin selection method. We conclude that the segregation of nitrate reductase after cell division is dispersive, and not semi-conservative as proposed by Autissier & Kepes (1972).

Rhodobacter capsulatus strain BK5 possesses a membrane bound respiratory nitrate reductase rather than the periplasmic enzyme found in other strains

1990 ◽  
Vol 154 (3) ◽  
pp. 301-303 ◽  
Author(s):  
Anna L. Ballard ◽  
Alastair G. McEwan ◽  
David J. Richardson ◽  
J. Baz Jackson ◽  
Stuart J. Ferguson
Keyword(s):  
Nitrate Reductase ◽  
Rhodobacter Capsulatus ◽  
Membrane Bound ◽  
Periplasmic Enzyme ◽  
Respiratory Nitrate Reductase

Nitrate and (per)chlorate reduction pathways in (per)chlorate-reducing bacteria

2011 ◽  
Vol 39 (1) ◽  
pp. 230-235 ◽  
Author(s):  
Margreet J. Oosterkamp ◽  
Farrakh Mehboob ◽  
Gosse Schraa ◽  
Caroline M. Plugge ◽  
Alfons J.M. Stams
Keyword(s):  
Nitrate Reductase ◽  
Nitrite Reduction ◽  
Type Ii ◽  
Oxygen Production ◽  
Anoxic Conditions ◽  
Oxygen Formation ◽  
Membrane Bound ◽  
Chlorate Reductase ◽  
Reducing Bacteria ◽  
Respiratory Nitrate Reductase

The reduction of (per)chlorate and nitrate in (per)chlorate-reducing bacteria shows similarities and differences. (Per)chlorate reductase and nitrate reductase both belong to the type II DMSO family of enzymes and have a common bis(molybdopterin guanine dinucleotide)molybdenum cofactor. There are two types of dissimilatory nitrate reductases. With respect to their localization, (per)chlorate reductase is more similar to the dissimilatory periplasmic nitrate reductase. However, the periplasmic, unlike the membrane-bound, respiratory nitrate reductase, is not able to use chlorate. Structurally, (per)chlorate reductase is more similar to respiratory nitrate reductase, since these reductases have analogous subunits encoded by analogous genes. Both periplasmic (per)chlorate reductase and membrane-bound nitrate reductase activities are induced under anoxic conditions in the presence of (per)chlorate and nitrate respectively. During microbial (per)chlorate reduction, molecular oxygen is generated. This is not the case for nitrate reduction, although an atypical reaction in nitrite reduction linked to oxygen formation has been described recently. Microbial oxygen production during reduction of oxyanions may enhance biodegradation of pollutants under anoxic conditions.

scholarly journals Structural investigation of the molybdenum site of the periplasmic nitrate reductase from Thiosphaera pantotropha by X-ray absorption spectroscopy

1996 ◽  
Vol 317 (2) ◽  
pp. 557-563 ◽  
Author(s):  
Brian BENNETT ◽  
John M. CHARNOCK ◽  
Heather J. SEARS ◽  
Ben C. BERKS ◽  
Andrew J. THOMSON ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Absorption Spectroscopy ◽  
X Ray ◽  
Periplasmic Nitrate Reductase ◽  
Thiosphaera Pantotropha ◽  
Treated Sample ◽  
Membrane Bound ◽  
X Ray Absorption ◽  
Best Fit ◽  
Respiratory Nitrate Reductase

The molybdenum centre of the periplasmic respiratory nitrate reductase from the denitrifying bacterium Thiosphaera pantotropha has been probed using molybdenum K-edge X-ray absorption spectroscopy. The optimum fit of the Mo(VI) EXAFS suggests two =O, three –S– and either a fourth –S– or an –O–/–N– as molybdenum ligands in the ferricyanide-oxidized enzyme. Three of the –S– ligands are proposed to be the two sulphur atoms of the molybdopterin dithiolene group and Cys-181. Comparison of the EXAFS of the ferricyanide-oxidized enzyme with that of a nitrate-treated sample containing 30% Mo(V) suggests that the Mo(VI) → Mo(V) reduction is accompanied by conversion of one =O to –O–. The best fit to the Mo(IV) EXAFS of dithionite-reduced enzyme was obtained using one =O, one –O– and four –S–/–Cl ligands. The periplasmic nitrate reductase molybdenum co-ordination environment in both the Mo(VI) and Mo(IV) oxidation states is distinct from that found in the membrane-bound respiratory nitrate reductase.

scholarly journals Effect of Ocular Hypertension on D-β-Aspartic Acid-Containing Proteins in the Retinas of Rats

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
Takashi Kanamoto ◽  
Takashi Tachibana ◽  
Yasushi Kitaoka ◽  
Toshio Hisatomi ◽  
Yasuhiro Ikeda ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Ocular Hypertension ◽  
Aspartic Acid ◽  
Atp Synthase ◽  
Wistar Rats ◽  
Protein Band ◽  
Mass Spectrometry Analysis ◽  
Liquid Chromatography Mass Spectrometry ◽  
Spectrometry Analysis ◽  
Sds Page

Purpose. To investigate the effect of ocular hypertension-induced isomerization of aspartic acid in retinal proteins. Methods. Adult Wistar rats with ocular hypertension were used as an experimental model. D-β-aspartic acid-containing proteins were isolated by SDS-PAGE and western blot with an anti-D-β-aspartic acid antibody and identified by liquid chromatography-mass spectrometry analysis. The concentration of ATP was measured by ELISA. Results. D-β-aspartic acid was expressed in a protein band at around 44.5 kDa at much higher quantities in the retinas of rats with ocular hypertension than in those of normotensive rats. The 44.5 kDa protein band was mainly composed of α-enolase, S-arrestin, and ATP synthase subunits α and β, in both the ocular hypertensive and normotensive retinas. Moreover, increasing intraocular pressure was correlated with increasing ATP concentrations in the retinas of rats. Conclusion. Ocular hypertension affected the expression of proteins containing D-β-aspartic acid, including ATP synthase subunits, and up-regulation of ATP in the retinas of rats.

Sign in / Sign up

Export Citation Format

Share Document