The acidic peroxidoxin [also named thiol-specific antioxidant protein (TSA) or protector protein (PRP)], which plays a role in the response against oxidative stress, is one of the major proteins of red blood cells. In this work, we show that this protein is induced at early stages of erythroid differentiation prior to haemoglobin accumulation, which suggests that it may play a role at the erythroblast stage, where haemoglobinized, nucleated and genetically active cells are submitted to a maximally dangerous oxidative stress. The early accumulation of this protein has been demonstrated both on transformed cell systems and on normal differentiating human erythroid cells. This suggests that this protein may play an important role in the differentiation of the erythroid cells.
We show that, in vitro, Ca2+-dependent protein kinase C (PKC) phosphorylates recombinant murine p53 protein on several residues contained within a conserved basic region of 25 amino acids, located in the C-terminal part of the protein. Accordingly, synthetic p53-(357 -381)-peptide is phosphorylated by PKC at multiple Ser and Thr residues, including Ser360, Thr365, Ser370 and Thr377. We also establish that p53-(357 -381)-peptide at micromolar concentrations has the ability to stimulate sequence-specific DNA binding by p53. That stimulation is lost upon phosphorylation by PKC. To further characterise the mechanisms that regulate PKC-dependent phosphorylation of p53-(357-381)-peptide, the phosphorylation of recombinant p53 and p53-(357-381)-peptide by PKC were compared. The results suggest that phosphorylation of full-length p53 on the C-terminal PKC sites is highly dependent on the accessibility of the phosphorylation sites and that a domain on p53 distinct from p53-(357-381)-peptide is involved in binding PKC. Accordingly, we have identified a conserved 27-amino-acid peptide, p53-(320-346)-peptide, within the C-terminal region of p53 and adjacent to residues 357-381 that interacts with PKC in vitro. The interaction between p53-(320-346)-peptide and PKC inhibits PKC autophosphorylation and the phosphorylation of substrates, including p53-(357 -381)-peptide, neurogranin and histone HI.Conventional Ca"-dependent PKC a, p and y and the catalytic fragment of PKC (PKM) were nearly equally susceptible to inhibition by p53-(320-346)-peptide. The Ca2+-independent PKC 6 was much less sensitive to inhibition. The significance of these findings for understanding the in vivo phosphorylation of p53 by PKC are discussed.
A new ferredoxin has been purified from the photosynthetic bacterium Rhodobacter capsulatus. It is the sixth ferredoxin to be isolated from this bacterium and it was called FdVI.Its primary structure was established based on amino acid sequence analysis of the protein and of peptides derived from it. It is composed of 106 residues including five cysteines. The calculated mass of the polypeptide is 11402.6 Da which matches the experimental value determined by electrospray mass spectrometry. Amino acid sequence comparison revealed that ferredoxin VI (FdVI) is strikingly similar to a ferredoxin from Caulobacter crescentus and to the putidaredoxin from Pseudomonas putida.FdVI exhibited an ultraviolet-visible absorption spectrum typical for a [2Fe-2S] ferredoxin. EPR spectroscopy of the reduced protein showed a nearly axial signal similar to that of mitochondria1 and €? putida ferredoxins.FdVI is biosynthesized in cells growing anaerobically under either nitrogen-sufficient or nitrogen-deficient conditions. Although the function of FdVI is unknown, its structural resemblance to [2Fe-2S] ferredoxins known to transfer electrons to oxygenases such as P-450 cytochromes, suggests that FdVI may have a similar role in R. capsulatus.The photosynthetic bacterium Rhodobacter capsulatus is a facultative phototroph endowed with remarkable abilities of metabolic adaptation. It can grow autotrophically or heterotrophically either in the light or in darkness and can choose between five different growth modes (Madigan and Gest, 1979). It is also capable of fixing molecular nitrogen through a metabolic process which has been extensively studied by biochemical (Hallenbeck et al., 1982a; and genetic approaches (Willison et al., 1985;Klipp et al., 1988). Nitrogen fixation is catalyzed by nitrogenase and requires ATP and a low-potential reductant. Two types of electron-carrier proteins, ferredoxins (Fd) and flavodoxins, are known to serve as electron donors to nitrogenase. In R. capsulatus, the identification of the actual physiological reductant of nitrogenase is complicated by the occurence in this bacterium of an exceptional diversity of electron carriers. Five distinct ferredoxins have been isolated and biochemically characterized; three of them, FdI, FdII and FdIII, appear as representative molecular forms of the dicluster ferredoxins found in a wide range of bacteria (Bruschi and Guerlesquin, 1988). FdI and the homodimeric FdIII, both contain two [4Fe-4S] clusters/monomer (Hallenbeck et al.,
To improve the efficiency of one- and two-dimensional electrophoresis for micropreparative purposes, the use of gels polymerized with other initiators than the standard N,N,N',N'-tetramethylethylenediamine (TEMED)/persulfate systems for sodium dodecyl sulfate electrophoresis has been investigated. We show here that the recently described photoinitiator system, composed of methylene blue, toluene sulfinate and diphenyliodonium chloride, leads to a decreased resolution. Resolution can be restored if methylene blue is replaced by riboflavin. Two-dimensional electrophoresis with mg loadings of proteins has also been evaluated with these systems. Independently of the polymerization system, resolution for the first dimension is low with rod gels, increases with gel strips and is further improved when immobilized pH gradients are used. Here too, only the riboflavin/sulfinate/iodonium system results in a resolution that matches the one obtained with the standard TEMED/persulfate system. Gels polymerized with the riboflavin/sulfinate/iodonium system yield better results upon N-terminal microsequencing after blotting than gels polymerized with the standard TEMED/persulfate system.
We describe a method for rapid purification of the integration host factor (IHF) homolog of Rhodobacter capsulatus that has allowed us to obtain microgram quantities of highly purified protein. R. capsulatus IHF is an a,3 heterodimer similar to IHF of Escherichia coli. We have cloned and sequenced the hip gene, which encodes the 0i subunit. The deduced amino acid sequence (10.7 kDa) has 46% identity with the I8 subunit of IHF from E. coli. In gel electrophoretic mobility shift DNA binding assays, R. capsulatus IHF was able to form a stable complex in a site-specific manner with a DNA fragment isolated from the promoter of the structural hupSL operon, which contains the IHF-binding site. The mutated IHF protein isolated from the Hup-mutant IR4, which is mutated in the himA gene (coding for the a subunit), gave a shifted band of greater mobility, and DNase I footprinting analysis has shown that the mutated IHF interacts with the DNA fragment from the hupSL promoter region differently from the way that the wild-type IHF does.The purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus possesses a membrane-bound hydrogenase that functions in H2 uptake under physiological conditions (31). This enzyme enables R. capsulatus cells to grow autotrophically with H2 as the source of electrons (17). It is also synthesized under heterotrophic growth conditions, in particular in H2-evolving cultures in which hydrogenase synthesis is stimulated by H2 (9).The level of hydrogenase expression is closely coupled to environmental and growth conditions (9). Regulatory genes necessary for hydrogenase synthesis in R. capsulatus have recently been identified in the cluster of hup and hyp genes (8 (30,33) were grown anaerobically in the light in minimal salts (RCV) medium (15, 32) supplemented with DL-malate (30 mM) and L-glutamate (7 mM) as C and N sources, respectively, as previously described (7). DNA manipulations. Standard recombinant DNA techniques were performed as described previously (25). Plasmids pI,B2 and pI,B3 were constructed by inserting into the polylinker of pUC18 (34) the 5-kbp PstI-PstI and 2.2-kbp HindIll-HindIII fragments, respectively, of genomic R. capsulatus DNA that hybridized with the IHF13 probe. DNA sequencing on both strands was performed by the dideoxy chain termination method (26). DNA sequence analysis and homology searching were performed with LASERGENE programs (DNASTAR, Madison, Wis.). Gel retardation assays were performed as previously described (30), using the 274-bp EcoRI-EcoRI DNA fragment containing the IHF-binding site isolated from the hupS promoter.Purification of IHF. R capsulatus strains were grown anaerobically in 40-liter cultures until the A660 reached 1.2, typically yielding 120 g of wet cells. The cells were harvested by centrifugation and resuspended in 100 mM Tris HCl-20 mM EDTA, and 0.2 mg of lysosyme per ml was added. The cell extract (150 ml), obtained after sonication and ultracentrifugation (150,000 x g, 2 h) and containing 3 g of protein, was applied to a 5-ml Econo-Pac he...
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