Search PubMed⌕ Search

Biomedical subjects

J Gaillard

Publications and source records attributed to J Gaillard.

At least 55 records · Page 3Linked to original sources
PubMed1994

Mutated forms of the [2Fe-2S] ferredoxin from Clostridium pasteurianum with noncysteinyl ligands to the iron-sulfur cluster.

The [2Fe-2S] ferredoxin from Clostridium pasteurianum is unique among ferredoxins, both by its sequence and by the distribution of its cysteine residues (in positions 11, 14, 24, 56, 60). Thus, no homologous sequences are available to infer, by comparison, the identity of the ligands of the iron-sulfur cluster. Therefore, in order to obtain information on the latter point, a combination of site-directed mutagenesis and UV-vis, EPR, and resonance Raman spectroscopy has been implemented. All of the cysteine residues have individually been replaced by serine and two of them by alanine. Cysteine 14 could be replaced by either serine or alanine without any modification of the spectroscopic properties of the protein and was therefore dismissed as a ligand of the [2Fe-2S] cluster. The C56S, and C60S-mutated proteins were both found to display UV-vis, EPR, and resonance Raman spectra consistent with serine-coordinated [2Fe-2S] clusters. The C11S-mutated protein was considerably less stable than the wild type ferredoxin. This observation, together with the hypsochromic shifts of UV-visible absorption features upon cysteine 11-->serine mutation, suggested cysteine 11 to be a ligand of the [2Fe-2S] cluster. Cysteine 24 could be replaced by either serine or alanine without decreasing the stability of the protein and without dramatically changing its spectroscopic properties. Thus, either cysteine 24 is not a ligand of the [2Fe-2S] cluster or it is replaced by another ligand in the C24A mutated protein. A [2Fe-2S] cluster was also assembled in the C14A/C24A doubly mutated protein, i.e., in a polypeptide chain containing only three cysteine residues.2+ off

Alanine↗
PubMed1994

Purification and properties of a halophilic catalase-peroxidase from Haloarcula marismortui.

A heme protein, hCP, from the extreme halophile, Haloarcula marismortui, showing both peroxidatic and catalatic activity has been purified and characterized as a catalase-peroxidase. Catalatic activity is enhanced by molar concentrations of NaCl or (NH4)2SO4, while peroxidase activity decreases with increasing salt concentration. Optimal pH values are 6.0 for peroxidatic activity assayed in absence of NaCl and 7.5 for catalatic activity assayed in molar concentrations of NaCl. The two activities present saturation behaviour with increasing H2O2 concentration with apparent Km values of 0.5 and 2.5 mM for the peroxidatic and catalatic activities, respectively. A molecular mass of 81,292 +/- 9 Da was measured for the polypeptide by mass spectroscopy. One heme group (protoporphyrin IX with an iron atom in the ferric state) is associated with one molecule of hCP. Its amino-acid composition shows hCP to contain a high proportion of acidic residues. The EPR spectrum of the NO-compound of reduced (ferrous) hCP strongly suggests that the proximal ligand of the heme is the imidazole group of a histidine residue.

Amino Acids↗
PubMed1994

Purification of a sixth ferredoxin from Rhodobacter capsulatus. Primary structure and biochemical properties.

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 11,402.6 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 mitochondrial and P. 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.

Amino Acid Sequence↗
PubMed1994

On the role of conserved proline residues in the structure and function of Clostridium pasteurianum 2[4Fe-4S] ferredoxin.

The widespread occurrence of Pro residues adjacent to Cys ligands in the sequences of [4Fe-4S] electron transfer proteins has not yet found a functional basis. The two such Pro of Clostridium pasteurianum 2[4Fe-4S] ferredoxin have been probed by site-directed mutagenesis. Any one of them, but not both simultaneously, can be substituted without impairing the proper folding of the protein. The reduction potentials of the ferredoxin variants fall in a narrow range of < 20 mV above the potential of the native protein. The biological activities with C. pasteurianum hydrogenase and pyruvate-ferredoxin oxidoreductase do not change significantly, except when Lys replaces Pro. In these cases, the data suggest that the two clusters of 2[4Fe-4S] ferredoxin may not always be equivalent in the interaction with the redox partners. Destabilization of the structure has been observed as the consequence of the Pro19 or Pro48 substitutions. Using 2-D NMR, this effect has been associated with perturbations of both the hydrogen bond network and one amino acid side chain around the [4Fe-4S] clusters. Thus, the conserved Pro found in the binding motif of [4Fe-4S] clusters in proteins strongly stabilizes the active site but does not play an essential role in the mechanism of electron transfer.

Amino Acid Sequence↗
PubMed1993

Effect of replacing conserved proline residues on the EPR and NMR properties of Clostridium pasteurianum 2[4Fe-4S] ferredoxin.

Most of [4Fe-4S] proteins bind their metallic center by four cysteine residues, three clustered in a single stretch of seven amino acids and a remote fourth generally followed by a proline residue. Two such prolines in Clostridium pasteurianum 2[4Fe-4S] ferredoxin have been substituted by different amino acids and the resulting molecular variants studied with EPR and NMR spectroscopies. The isolated EPR contributions of the [4Fe-4S]+ clusters do not change much in all variants. The exact positions or the number of features composing the fully reduced EPR spectra built by the two interacting [4Fe-4S]+ S = 1/2 systems vary slightly but, in none of the proteins in which either proline 19 or 48 were substituted, do they indicate a major difference either in the folding of the ferredoxin or in the electronic structure of its clusters. A subset of paramagnetically shifted NMR signals is significantly affected by these replacements at both redox levels. The corresponding protons belong to two cysteines liganding the cluster close to the substitution. These data, combined with the presently available three-dimensional information, form the basis for partial assignments of the most shifted resonances in the NMR spectra of such proteins. The positions of intermediate lines in the NMR spectra of semireduced ferredoxins depend on the difference between the redox potentials of the two clusters; this difference is sensitive to the substitutions of either conserved proline residue by lysine.

Amino Acid Sequence↗
PubMed1993

Mutated forms of a [2Fe-2S] ferredoxin with serine ligands to the iron-sulfur cluster.

The [2Fe-2S] ferredoxin from Clostridium pasteurianum contains five cysteine residues in positions 11, 14, 24, 56 and 60. Residues 24, 56 and 60 have been separately mutated into serine. The modified ferredoxins have been purified and were all found to contain a [2Fe-2S]-type cluster. The electronic absorption and EPR spectra of the C24S protein were only slightly different from those of the native one. In contrast, the C56S and C60S ferredoxins displayed spectroscopic features witnessing the presence of an oxygen ligand to the iron-sulfur cluster: the UV-visible absorption bands were shifted to higher energy by ca. 20 nm, and the high field components of the EPR spectra were shifted from gx = 1.92 and gy = 1.95 to gx = 1.88 and gy = 1.92, respectively.

Amino Acid Sequence↗
PubMed1993

Purification and characterization of a novel dimeric ferredoxin (FdIII) from Rhodobacter capsulatus.

A new ferredoxin, called FdIII, has been isolated and purified from the photosynthetic bacterium Rhodobacter capsulatus. Its complete amino acid sequence has been determined. The FdIII polypeptide consists of 100 residues, including 9 cysteines and has a calculated molecular mass of 10,688 Da, which was confirmed by electrospray mass spectrometry. In its native form, FdIII is a homodimer as deduced from molecular sieve chromatography and non-denaturing polyacrylamide gel electrophoresis, as well as cross-linking experiments. The dimeric ferredoxin was found to contain 15.2 +/- 0.6 iron atoms and 13 +/- 1 inorganic sulfur atoms, consistent with the presence of four [4Fe-4S] clusters/molecule. The UV visible absorption spectrum of oxidized FdIII exhibited maxima at 282 and at 386 nm and a shoulder near 314 nm. FdIII was fully reduced by excess dithionite at pH 8.0 or photochemically using 5-deazaflavin. Anaerobic oxidative titration of reduced FdIII with thionin indicated that each FdIII monomer exchanges two electrons. Exposure of FdIII to air resulted in a rapid and irreversible oxidative denaturation of the Fe-S clusters. The EPR spectrum of fully reduced FdIII showed a broad signal with an average g value of 1.94 that integrated to about two spins/monomer. EPR analysis of partially reduced FdIII (approximately 20% reduction) revealed a complex set of signals which was interpreted as being the resulting sum of the contribution of two distinct paramagnetic centres. Based on its biochemical and spectroscopic properties, it is concluded that FdIII is a dimeric ferredoxin containing four [4Fe-4S] clusters. The synthesis of FdIII occurs only under growth conditions allowing the derepression of nif genes. Results of in vitro electron transfer assays indicate that FdIII cannot serve as an electron donor to nitrogenase.

Amino Acid Sequence↗
PubMed1993

An iron-sulfur center and a free radical in the active anaerobic ribonucleotide reductase of Escherichia coli.

Anaerobically grown Escherichia coli contain an oxygen-sensitive ribonucleotide reductase. The enzyme requires anaerobic activation by two E. coli fractions with S-adenosylmethionine, NADPH, dithiothreitol, and KCl. We now find that photochemically reduced deazaflavin can substitute for these two fractions and NADPH. The reductase contained roughly equimolar amounts of iron and sulfide, suggesting the presence of an Fe-S complex. The cluster is characterized by a charge transfer band at 420 nm and a low temperature EPR signal centered at g = 2.01 that is difficult to saturate at 14 K, suggested to be a (3Fe-4S)+ cluster. In five different preparations of essentially protein-pure reductase containing widely different amounts of iron, the catalytic activity correlated well with the iron content. The iron signal disappeared during reductive anaerobic activation, with the appearance of a new EPR signal at g = 2.0033 showing a temperature behavior and microwave power saturability consistent with an organic free radical. The signal disappeared after exposure of the activated enzyme to air. We suggest that activation involves generation of a specific amino acid free radical that is dependent on the reduced Fe-S cluster and S-adenosylmethionine. From other work it appears likely that the free radical is localized on glycine 681 of the polypeptide chain.

Anaerobiosis↗
PubMed1992

Expression in Escherichia coli and characterization of a recombinant 7Fe ferredoxin of Rhodobacter capsulatus.

The 7Fe ferredoxin of Rhodobacter capsulatus (FdII) could be expressed in Escherichia coli by cloning the fdxA gene coding for FdII downstream from the lac promoter. The expressed recombinant ferredoxin appeared as a brown protein which was specifically recognized in E. coli cell-free extracts by anti-FdII serum. The purified recombinant ferredoxin was indistinguishable from R. capsulatus FdII on the basis of its molecular, redox and spectroscopic properties. These results indicate that the [3Fe-4S] and [4Fe-4S] clusters were correctly inserted into the recombinant ferredoxin.

Cloning, Molecular↗
PubMed1992

Sequence-specific assignments of the 1H nuclear magnetic resonance spectra of reduced high-potential ferredoxin (HiPIP) from Chromatium vinosum.

The 1H resonances of the high-potential [4Fe-4S]2+ ferredoxin from Chromatium vinosum have been assigned through conventional sequential methodology applied to 2D NMR spectra. Almost 80% of the residues were identified using standard 2D COSY, HOHAHA, and NOESY pulse sequences. These residues correspond to four segments of the primary structure that do not interact strongly with the iron-sulfur cluster. A minor correction to the amino acid sequence is strongly suggested by these NMR data. Additional protons more sensitive to the proximity of the cluster were assigned by a combination of NOESY experiments with fast repetition rates and short mixing times and of HOHAHA spectra recorded with reduced spin-lock duration aimed at compensating for the short relaxation rates. Hence, the contributions of 79 residues out of 85 were identified in NMR spectra, among which the assignments of 64 residues were completed. Even the fastest relaxing protons, like those of the cysteine ligands, could be correlated, partly because the strong hyperfine shifts isolate them from the crowded diamagnetic region. However, other protons, in particular those involved in NH-S hydrogen bonds with the iron-sulfur cluster, were more difficult to identify, most probably because their relatively broad signals overlapped with those of protons not or less perturbed by the active site. The availability of the major part of the 1H NMR assignments has enabled the detection and identification of many interresidue NOESY cross peaks. These data are in full agreement with the elements of secondary structure previously revealed by X-ray crystallographic analysis of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Rapid inactivation of plant aconitase by hydrogen peroxide.

Preincubation of potato (Solanum tuberosum) tuber mitochondria with 300 microM-H2O2 for 10 min nearly stopped the State 3 rate of citrate oxidation. Addition of isocitrate resulted in resumption of O2 uptake. The State 3 rates of succinate, external NADH and 2-oxoglutarate oxidation were unaffected by H2O2 over the dose range 50-500 microM. Preincubation of mitochondria with 300 microM-H2O2 for 5 min unmasked in the matrix space a paramagnetic signal with a peak at a g value of approx. 2.03. Aconitase was purified over 135-fold to a specific activity of 32 mumol/min per mg (with isocitrate as substrate) from the matrix of potato tuber mitochondria. The native enzyme was composed of a single polypeptide chain (molecular mass 90 kDa). Incubation of purified aconitase with small amounts of H2O2 caused the build up of a paramagnetic 3Fe cluster with a low-field maximum of g = 2.03 leading to a progressive inhibition of aconitase activity. The results show that aconitase present in the matrix space was the major intramitochondrial target for inactivation by H2O2.

Aconitate Hydratase↗
PubMed1990

Purification and characterization of a 7Fe-ferredoxin from Rhodobacter capsulatus.

A ferredoxin was purified anaerobically from Rhodobacter capsulatus grown photoheterotrophically with excess ammonia. This ferredoxin, called ferredoxin II (FdII), had a molecular weight of approximatively 15,000 by gel filtration and 14,000 by SDS polyacrylamide gel electrophoresis indicating that it is monomeric. Its absorption spectrum (oxidized form) exhibited maxima at 280 nm and 400 nm; the A400/A280 ratio had a calculated value of 0.55. Chemical determination of its iron and sulfur atom content, the value of the extinction coefficient at 400 nm (epsilon 400 = 26.8 mM-1 cm-1) and EPR spectra indicated that ferredoxin II contained one [3Fe-4S] and one [4Fe-4S] cluster. Upon reduction with excess dithionite only the [3Fe-4S] cluster became reduced. The reduction of both clusters was achieved by using 5-deazaflavin as photocatalyst. Ferredoxin II was also purified from bacteria grown under nitrogen limiting (nif derepressing) conditions. In in vitro assays, ferredoxin II catalyzed electron transport between illuminated chloroplasts and nitrogenase.

Amino Acids↗
PubMed1989

[Value of hemodynamics in the surgical indications of emphysema].

The authors present the results of a preliminary study designed to define the surgical indications in diffuse pulmonary emphysema based on the data of haemodynamic investigations. This series consists of 10 patients identified for disabling dyspnoea, diffuse emphysema and dynamic expiratory compression documented by the data of right micro-catheterisation by a DEP/DIP ratio greater than 2. The surgical operation consisted of reduction in the lung volume, the results of which were defined by pre- and postoperative examinations. In this way, it is possible to distinguish 3 groups of patients: one group was markedly improved, another was moderately improved and the third group continued to deteriorate analysis of the results suggests the determinant role of dynamic factors, in particular haemodynamic factors. The role of the diaphragm is demonstrated by modifications in its radius or curvature.

Diaphragm↗
PubMed1989

Lymphoproliferative syndrome associated with c-myc expression driven by a class I gene promoter in transgenic mice.

We have produced transgenic mice carrying an H-2K/human c-myc fusion gene. In this construct, the human c-myc proto-oncogene expression is driven by the 5' flanking sequences (including promoter) of the class I H-2Kb gene, which have previously been shown to direct the expression of a marker gene, the human growth hormone (hGH), in most tissues of H-2K/hGH transgenic mice. Comparative analysis, by S1 nuclease mapping, of the H-2K and human c-myc gene expression in different organs of the H-2K/myc mice shows that exogenous c-myc and endogenous H-2K expression is found in most organs examined. However, the liver is a notable exception, for here c-myc expression is very weak. The exogenous c-myc expression is maximal in lymphoid organs of all H-2K/myc transgenic strains. One strain, H-2K/myc 27, hereditarily develops a lymphoproliferative syndrome which eventually leads to death. The H-2K/myc 27 lymphoid tissues are profoundly abnormal: pre-B cells as well as mature B cells are underrepresented in the bone marrow but the thymus as well as lymph nodes are largely infiltrated by B cells. Moreover, in the thymus, the proportions of the different thymic cell populations are altered. However, in the other H-2K/myc transgenic strains, even in those expressing a comparable or even higher level of myc, no pathology has been observed over a period of 20 months. Our results, therefore, demonstrate that constitutive enforced c-myc expression might disturb lymphocyte development, but does not directly lead to malignancy.

Animals↗
PubMed1989

[Cervicofacial non-Hodgkin's malignant lymphoma. Classification, prognosis and treatment apropos of 65 cases].

The approach to non hodgkins malignant lymphomas (NHML) has changed in recent years, notably because of advances in pathological evaluation due to histo-immuno-labelling leading to a more accurate classification. Such pathology being relatively common in the cervical facial region, with a figure of 10 to 25%, this report concerns the personal experience of the authors based upon 65 cases treated in the department between 1975 and 1985.

Adolescent↗
PubMed1988

Hydrogen-1 nuclear magnetic resonance of the nitrogenase iron protein (Cp2) from Clostridium pasteurianum.

Proton NMR spectra (250 MHz) of the nitrogenase iron protein from Clostridium pasteurianum (Cp2) were found to display 9 or 10 paramagnetically shifted resonances in the 15-50 ppm range. The most shifted resonances belonged to two approximately equal subsets having temperature dependences of opposite sign. The latter occurrence is consistent with the interaction of the corresponding protons with an antiferromagnetically coupled metal center. The number of proton resonances of Cp2, their positions, and their temperature dependences were similar to those observed in spectra of (4Fe-4S)+ ferredoxins, particularly those of the latter that contain a single tetranuclear cluster, such as the ferredoxin from Bacillus stearothermophilus. The effects of several adenine nucleotides on the paramagnetically shifted proton resonances of Cp2 have been investigated. Whereas MgAMP had no effect at all, MgADP and MgATP were found to induce different modifications, which in both cases involved approximately half only of the shifted proton resonances. These data suggest that nucleotide binding affects mainly one part of the iron-sulfur cluster. A remarkable feature of the spectra of Cp2 in the presence of MgATP is the grouping of the shifted proton resonances in sets of two or four having identical chemical shifts and temperature dependences. A nearly perfect 2-fold symmetry is thus suggested for the arrangement of the cysteine protons around the active site. These observations lend support to the proposal that the (4Fe-4S) cluster is held symmetrically between the two identical subunits and are consistent with the existence of two MgATP binding sites on nitrogenase iron proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗