Expression of the activating enzyme and Fe protein of nitrogenase from Rhodospirillum rubrum

1982 ◽  
Vol 152 (2) ◽  
pp. 786-791
Author(s):  
E W Triplett ◽  
J D Wall ◽  
P W Ludden
Keyword(s):  
Carbon Source ◽  
Rhodospirillum Rubrum ◽  
Nitrogenase Activity ◽  
N Sources ◽  
Fe Protein ◽  
N Source ◽  
In Vitro Activation ◽  
High Ammonia ◽  
Cells Cultured

Activating enzyme (AE) is responsible for the in vitro activation of inactive Fe protein of nitrogenase from Rhodospirillum rubrum cells cultured anaerobically with glutamate as the N source. The expression of Fe protein and AE was examined in R. rubrum cultured photosynthetically or aerobically on media containing malate as the carbon source. One of the following N sources was used in each culture: glutamate, glutamine, limiting ammonia, high ammonia, glutamate plus histidine, and high ammonia plus histidine. Chromatophores from every culture exhibited AE activity; activity was highest in glutamate-grown cells. Fe protein was observed by rocket immunoelectrophoresis in cultures with nitrogenase activity. Several Nif-, Gln-, and His- mutants of R. rubrum were assayed for AE activity, nitrogenase activity, and Fe protein. Every mutant expressed AE activity, and Fe protein was observed in those cultures with nitrogenase activity. AE from every preparation was O2 labile, and each O2-denatured AE preparation inhibited activation by active AE.

Vitamin D modulates the expression of HLA-DR and CD38 after in vitro activation of T-cells

2017 ◽  
Vol 29 (3) ◽  
Author(s):  
Simon Villegas-Ospina ◽  
Wbeimar Aguilar-Jimenez ◽  
Sandra M. Gonzalez ◽  
María T. Rugeles
Keyword(s):  
Vitamin D ◽  
Flow Cytometry ◽  
Healthy Subjects ◽  
Mononuclear Cells ◽  
Activation Markers ◽  
Hla Dr ◽  
In Vitro Activation ◽  
Cells Cultured

AbstractObjective:Vitamin D (VitD) is an anti-inflammatory hormone; however, some evidence shows that VitD may induce the expression of activation markers, such as CD38 and HLA-DR. We explored its effect on the expression of these markers on CD4Materials and methods:CD38 and HLA-DR expression was measured by flow cytometry in PHA/IL-2-activated mononuclear cells cultured under VitD precursors: three cholecalciferol (10Results:Cholecalciferol at 10Conclusion:Although no significant correlations were observed in vivo in healthy subjects, VitD treatment in vitro modulated immune activation by increasing the expression of CD38 and decreasing the proliferation of HLA-DR

scholarly journals Adenine nucleotide levels in Rhodospirillum rubrum during switch-off of whole-cell nitrogenase activity

1984 ◽  
Vol 224 (3) ◽  
pp. 961-969 ◽  
Author(s):  
T D Paul ◽  
P W Ludden
Keyword(s):  
Protein Modification ◽  
Rhodospirillum Rubrum ◽  
Adenine Nucleotide ◽  
Nitrogenase Activity ◽  
Energy Charge ◽  
Whole Cell ◽  
Fe Protein ◽  
Nucleotide Pools ◽  
Phenazine Methosulphate

Adenine nucleotide pools were measured in Rhodospirillum rubrum cultures that contained nitrogenase. The average energy charge [([ATP] + 1/2[ADP])/([ATP] + [ADP] + [AMP])] was found to be 0.66 and 0.62 in glutamate-grown and N-limited cultures respectively. Treatment of glutamate-grown cells with darkness, ammonia, glutamine, carbonyl cyanide m-chlorophenylhydrazone, or phenazine methosulphate resulted in perturbations in the adenine nucleotide pools, and led to loss of whole-cell nitrogenase activity and modification in vivo of the Fe protein. Treatment of N-limited cells resulted in similar changes in adenine nucleotide pools but not enzyme modification. No correlations were found between changes in adenine nucleotide pools or ratios of these pools and switch-off of nitrogenase activity by Fe protein modification in vivo. Phenazine methosulphate inhibited whole-cell activity at low concentrations. The effect on nitrogenase activity was apparently independent of Fe protein modification.

scholarly journals Role of the Dinitrogenase Reductase Arginine 101 Residue in Dinitrogenase Reductase ADP-Ribosyltransferase Binding, NAD Binding, and Cleavage

2001 ◽  
Vol 183 (1) ◽  
pp. 250-256 ◽  
Author(s):  
Yan Ma ◽  
Paul W. Ludden
Keyword(s):  
Rhodospirillum Rubrum ◽  
Nitrogenase Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Adp Ribosylation ◽  
Dinitrogenase Reductase ◽  
Adp Ribosyltransferase

ABSTRACT Dinitrogenase reductase is posttranslationally regulated by dinitrogenase reductase ADP-ribosyltransferase (DRAT) via ADP-ribosylation of the arginine 101 residue in some bacteria.Rhodospirillum rubrum strains in which the arginine 101 of dinitrogenase reductase was replaced by tyrosine, phenylalanine, or leucine were constructed by site-directed mutagenesis of thenifH gene. The strain containing the R101F form of dinitrogenase reductase retains 91%, the strain containing the R101Y form retains 72%, and the strain containing the R101L form retains only 28% of in vivo nitrogenase activity of the strain containing the dinitrogenase reductase with arginine at position 101. In vivo acetylene reduction assays, immunoblotting with anti-dinitrogenase reductase antibody, and [adenylate-32P]NAD labeling experiments showed that no switch-off of nitrogenase activity occurred in any of the three mutants and no ADP-ribosylation of altered dinitrogenase reductases occurred either in vivo or in vitro. Altered dinitrogenase reductases from strains UR629 (R101Y) and UR630 (R101F) were purified to homogeneity. The R101F and R101Y forms of dinitrogenase reductase were able to form a complex with DRAT that could be chemically cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The R101F form of dinitrogenase reductase and DRAT together were not able to cleave NAD. This suggests that arginine 101 is not critical for the binding of DRAT to dinitrogenase reductase but that the availability of arginine 101 is important for NAD cleavage. Both DRAT and dinitrogenase reductase can be labeled by [carbonyl-14C]NAD individually upon UV irradiation, but most 14C label is incorporated into DRAT when both proteins are present. The ability of R101F dinitrogenase reductase to be labeled by [carbonyl-14C]NAD suggested that Arg 101 is not absolutely required for NAD binding.

scholarly journals Purification and partial characterization of a pyruvate oxidoreductase from the photosynthetic bacterium Rhodospirillum rubrum grown under nitrogen-fixing conditions

1991 ◽  
Vol 279 (1) ◽  
pp. 155-158 ◽  
Author(s):  
E Brostedt ◽  
S Nordlund
Keyword(s):  
Klebsiella Pneumoniae ◽  
Rhodospirillum Rubrum ◽  
Nitrogenase Activity ◽  
Photosynthetic Bacterium ◽  
Molecular Properties ◽  
Partial Characterization ◽  
Nitrogen Fixing ◽  
Pyruvate Oxidoreductase

A pyruvate oxidoreductase with the capacity to support pyruvate-dependent nitrogenase activity in vitro has been purified from the photosynthetic bacterium Rhodospirillum rubrum. The enzyme requires CoA for activity and is irreversibly inactivated by oxygen. The molecular properties and Km values for the substrates have been studied. In supporting nitrogenase activity addition of ferredoxin is required. Overall the enzyme is similar to the nif-specific pyruvate: flavodoxin oxidoreductase purified from Klebsiella pneumoniae.

Effect of pyruvate on the metabolic regulation of nitrogenase activity in Rhodospirillum rubrum in darkness

Microbiology ◽  
2011 ◽  
Vol 157 (6) ◽  
pp. 1834-1840 ◽  
Author(s):  
Tiago Toscano Selao ◽  
Tomas Edgren ◽  
He Wang ◽  
Agneta Norén ◽  
Stefan Nordlund
Keyword(s):  
Nitrogen Source ◽  
Metabolic Regulation ◽  
Rhodospirillum Rubrum ◽  
De Novo ◽  
Nitrogenase Activity ◽  
Signal Transduction Pathway ◽  
Nitrogen Sources ◽  
Synthetase Activity ◽  
Glutamine Synthetase Activity ◽  
Fe Protein

Rhodospirillum rubrum, a photosynthetic diazotroph, is able to regulate nitrogenase activity in response to environmental factors such as ammonium ions or darkness, the so-called switch-off effect. This is due to reversible modification of the Fe-protein, one of the two components of nitrogenase. The signal transduction pathway(s) in this regulatory mechanism is not fully understood, especially not in response to darkness. We have previously shown that the switch-off response and metabolic state differ between cells grown with dinitrogen or glutamate as the nitrogen source, although both represent poor nitrogen sources. In this study we show that pyruvate affects the response to darkness in cultures grown with glutamate as nitrogen source, leading to a response similar to that in cultures grown with dinitrogen. The effects are related to PII protein uridylylation and glutamine synthetase activity. We also show that pyruvate induces de novo protein synthesis and that inhibition of pyruvate formate-lyase leads to loss of nitrogenase activity in the dark.

scholarly journals The Nitrogenase Regulatory Enzyme Dinitrogenase Reductase ADP-Ribosyltransferase (DraT) Is Activated by Direct Interaction with the Signal Transduction Protein GlnB

2012 ◽  
Vol 195 (2) ◽  
pp. 279-286 ◽  
Author(s):  
Vivian R. Moure ◽  
Karamatullah Danyal ◽  
Zhi-Yong Yang ◽  
Shannon Wendroth ◽  
Marcelo Müller-Santos ◽  
...  
Keyword(s):  
Rhodospirillum Rubrum ◽  
Metal Cluster ◽  
Reductase Activity ◽  
Direct Interaction ◽  
Ammonium Concentration ◽  
Content Type ◽  
Fe Protein ◽  
Dinitrogenase Reductase ◽  
Adp Ribosyltransferase

ABSTRACTFe protein (dinitrogenase reductase) activity is reversibly inactivated by dinitrogenase reductase ADP-ribosyltransferase (DraT) in response to an increase in the ammonium concentration or a decrease in cellular energy inAzospirillum brasilense,Rhodospirillum rubrum, andRhodobacter capsulatus. The ADP-ribosyl is removed by the dinitrogenase reductase-activating glycohydrolase (DraG), promoting Fe protein reactivation. The signaling pathway leading to DraT activation by ammonium is still not completely understood, but the available evidence shows the involvement of direct interaction between the enzyme and the nitrogen-signaling PIIproteins. InA. brasilense, two PIIproteins, GlnB and GlnZ, were identified. We used Fe protein fromAzotobacter vinelandiias the substrate to assess the activity ofA. brasilenseDraTin vitrocomplexed or not with PIIproteins. Under our conditions, GlnB was necessary for DraT activity in the presence of Mg-ADP. The PIIeffector 2-oxoglutarate, in the presence of Mg-ATP, inhibited DraT-GlnB activity, possibly by inducing complex dissociation. DraT was also activated by GlnZ and by both uridylylated PIIproteins, but not by a GlnB variant carrying a partial deletion of the T loop. Kinetics studies revealed that theA. brasilenseDraT-GlnB complex was at least 18-fold more efficient than DraT purified fromR. rubrum, but with a similarKmvalue for NAD+. Our results showed that ADP-ribosylation of the Fe protein does not affect the electronic state of its metal cluster and prevents association between the Fe and MoFe proteins, thus inhibiting electron transfer.

scholarly journals Change in subunit composition of the iron protein of nitrogenase from Rhodospirillum rubrum during activation and inactivation of iron protein

1982 ◽  
Vol 205 (3) ◽  
pp. 489-494 ◽  
Author(s):  
G G Preston ◽  
P W Ludden
Keyword(s):  
Nitrogen Source ◽  
Gel Electrophoresis ◽  
Rhodospirillum Rubrum ◽  
Subunit Composition ◽  
Iron Protein ◽  
Fe Protein ◽  
Lower Band

The subunit composition of the Fe protein of nitrogenase from Rhodospirillum rubrum during activation and inactivation was investigated. It was found that the upper subunit (on gel electrophoresis) of the two-subunit Fe protein was converted into the lower subunit during activation in vitro. When the Fe protein was inactivated in vivo by the addition of NH4Cl and alpha-oxoglutarate to the cells, a phosphate-labelled upper band appeared. During activation in vitro by the activating enzyme, some of the phosphate of the upper band remained with the protein and appeared in the lower band. Activations in vitro were performed on inactive Fe protein obtained from cells grown with glutamate as the nitrogen source. Both native and oxygen-denatured Fe protein exhibited the loss of upper band during treatment with activating enzyme.

Sign in / Sign up

Export Citation Format

Share Document