35S-Atractyloside binding affinity to the inner mitochondrial membrane

AbstractIsolated inner mitochondrial membrane contains a small number of binding sites for atractyloside (of the order of 0.1 nmole/mg of protein) with very high binding affinity (half saturation at 0.014 μM atractyloside). The high affinity binding ability of the inner mitochondrial membrane is markedly decreased upon aging, acidification of the medium or addition of ADP, but remains unchanged in the presence of uncouplers such as FCCP. Added ADP causes a two-step transition from the high affinity binding to low affinity binding (Kd > 0.50 μM) and concomitantly a significant increase of the measured number of binding sites (about a doubling). The half maximum effect in the first step transition is given by 1 μM ADP. The use of 35S-atractyloside as a probe of the inner mitochondrial membrane conformation specifically related to the adenine nucleotide translocation is discussed

Immunological properties of O•−2 generating oxidase from bovine neutrophils

AbstractTwo antisera have been prepared against the O•−2 generating oxidase purified from bovine polymorphonuclear neutrophils (PMNs). The first antiserum was directed against the enzymatically active fraction obtained after isoelectric focusing (pI oxidase), which consisted of a major protein of Mr 65 000 [(1985) Biochemistry 24, 7231–7239]. The second antiserum was directed against the 65 kDa band excised from an SDS-polyacrylamide gel after electrophoresis of the pI oxidase preparation. The pI oxidase antiserum inhibited O•−2 generation by PMN cells, PMN membranes and detergent-solubilized membranes. The 65 kDa band antiserum was virtually non-inhibitory against PMN cells; in contrast, it was nearly as potent as the pI oxidase antiserum on PMN membranes and detergent-solubilized membranes. Inhibition of O•−2 generation by the pI oxidase antiserum was correlated with the immunoreactivity of four membrane-bound proteins of 65, 54, 18 and 16 kDa; the 65 kDa band antiserum reacted only with the two proteins of 65 and 54 kDa. It is concluded that the 18 and 16 kDa proteins, present in trace amounts in the pI oxidase preparation, are probably potent catalysts of the respiratory burst

Etude de la production d'hydrogène en bioréacteur par la bactérie photosynthétique Rhodobacter capsulatus 2. Transformation du lactate et bilans carbonés

La dégradation du lactate par la bactérie photosynthétique Rhodobacter capsulatus cultivée dans un photobioréacteur en anaérobiose, à la lumière, en limitation d'azote, a été mesurée ainsi que la production d'hydrogène par la nitrogénase. En réacteur ouvert à faible taux de dilution (D = 0,04 h-1), le lactate initialement à 30 mM est dégradé à près de 90 % avec une faible accumulation (< 1-2 mM) des produits de dégradation (formiate et acétate). C'est dans ces conditions qu'on observe la meilleure production d'hydrogène (44 ml • h-1 • h-1). Lorsque la culture bactérienne devient trop dense en réacteur fermé ou an recyclage des bactéries, Il y a apparemment passage à un métabolisme de type fermentatif, chute de la production d'hydrogène et accumulation d'acides organiques (formique, acétique, propionique) provenant du lactate. Dans ces conditions, il n'y a pas diminution de la charge de carbone organique. Outre la présence de ces acides à courtes chaînes, les bilans carbonés; font apparaître la présence, dans le surnageant de la culture, de substances carbonées exocellulaires non identifiées pouvant représenter jusqu'à 50 % du flux carboné sortant.A 10 l photobioreactor, consisting of a PVC tubing, spiral-coiled so as to form a plane light captor (DELACHAPELLE et al., 1990), was used to study the degradation of lactate by the photosynthetic bacterium Rhodobacter capsulatus strain B10. The bacterial culture was continuously circulated in the reactor so as to maintain a homogeneous suspension, to optimize illumination of the cells, and make a well-mixed reactor for optimal nutrient transfers and degas ing of the medium. The bacterium was cultivated anaerobically under photoheterotrophic and N-limited conditions. The bioreactor was operated in three modes : as an open system (chemostat), as a closed system (batch), and as an open system with phases of bacteria recycling through the use of an ultrafiltration cell.When the bioreactor functioned as a chemostat, at low dilution rate (D < 0.04 h-1) and at relatively low bacterial density (A660 nm < 2.5), up to 90 % of the added lactate (initial concentration 30 mM) was degraded; the concentration of degradation products (formate, acetate) remained low (<2 mM). Under these conditions nitrogenase-mediated H2 production was maximal (44 ml • h-1 • l-1). In batch cultures or when the cells were « recycled » in the chemostat by ultrafiltration, the increase in absorbancy led to a decrease in cell illumination and the bacteria apparently switched from a photosynthetic of a fermentative type of metabolism; lactate was converted into formate + acetate + propionate, no H2 was produced. From the determination of the carbon balance between the influent and the effluent medium, the presence of non identified extracellular carbon compounds (up to 50 % of the C content of the effluent) was proved

Etude de la production d'hydrogène en bioréacteur par une bactérie photosynthétique Rhodobacter capsulatus 1. Photobioréacteur et conditions optimales de production d'hydrogène

Un réacteur de 10 litres, automatisé pour la culture continue en anaérobiose de bactéries photosynthétiques, a été réalisé et mis au point. Ce réacteur parfaitement agité a été utilisé dans différentes conditions de fonctionnement en système fermé (batch), en système semi-ouvert (atouts de substrats concentrés en discontinu), en système ouvert (chémostat) avec et sans recyclage de la biomasse, afin d'étudier la consommation, par les bactéries, d'un substrat carboné, le lactate. La production d'hydrogène par la bactérie photosynthétique Rhodobacter capsulatus, souche B10, résultant de la dégradation du lactate, est optimale pour des cultures en continu diluées, limitées en source azotée. Ainsi, à un taux de dilution de 0,04 h-1, avec 5 mM glutamate dans le milieu nutritif, la densité bactérienne étant de 2,1 è 660 nm, on a observé une production continue moyenne de 65 ml • h-1• l-1 pendant une période de 200 heures. Pour des concentrations bactériennes élevées, la limitation d'énergie lumineuse entrain une perte d'activité nitrogénase et, de ce fait, une chute de la production d'hydrogène.A photobioreactor was set up to cultivate a photosynthetic bacterium in continuous cultures. The bioreactor was designed so as to 1) allow the capture of light energy by bacteria through a spiral transparent flexible tube placed under the light, in a water bath maintaining the growth temperature at 30 °C; 2) male the suspension of bacteria circulate continuously in the reactor with a volumetric pump to maintain the medium homogeneous; 3) allow degassing of the suspension in a degassing chamber; 4) feed the culture with nutritive media, add neutralizing solution (pH 7) and withdrax aliquots white maintaining constant the volume of the culture; 5) recycle the bacteria by filtration when the bioreactor was used as e closed system (batch).The photosynthetic bacterium was Rhodobacter capsulatus strain B10 is known to lie a good H2 producer [Hillmer and Gest (1977) J. Bacteriol. 129, 724-731]. The bioreactor was run using 10 l of a synthetic medium containing lactate as carbon source and glutamate as nitrogen source. It was studied for its capacity to degrade lactate. Glutamate was the growth-liliting substrate allowing a maximum derepression of nitrogenase, the enzyme catalysing the reduction of protons to H2. The bacterial suspension was continuously circulated in the photoreactor, conceived as a plane light captor of 1 m2, to avoid bacterial self-shading and allow regeneration of ATP by photophosphorylation at high rates. The circuit was tightly closed to avoid air entry, which would prevent H2 production due to respiration of the bacteria.To run it under automated conditions, the bioreactor was equipped with two temperature sensors, two pH electrodes, a water level detector, a manometer and a computer-controlled electric valve. The bioreactor, of the well-mixed type, was used under various working conditions, namely as a closed (batch) system, as a fed-batch system (discontinuous additions of concentrated substrates), and as an open system (chemostat) with or without biomass recycling.Control of key parameters (pH, temperature, dilution rates) allowed us to define the culture conditions producing maximal amounts of molecular hydrogen. The production of H2 accompanying lactate degradation was maximal in diluted nitrogen-limited continuous cultures. It was observed at a dilution rate of 0.04 h-1 with 5 mM glutamate in the influent medium, the optical density of the culture being 2.1 at 660 nm. Under these conditions an average production H2 of 85 ml • h-1 • l-1 was observed over a 200 h period. At higher bacterial concentrations, the limitation of light energy resulted in a decrease in nitrogenase activity and therefore in a drop in the production of hydrogen.The interdependence of various parameters (pH, dilution rates, N and C sources light intensity) renders the system complex and not easily controlled by computer. Indeed, we observed that during recycling of the bacteria by the use of an ultrafiltration cell, the bacteria became a fermentative-type of metabolism accompanied by a decrease in nitrogenase activity and therefore in a drop in the production of hydrogen

Science expérimentale : naissance, métamorphose et limites

Percer les mystères de Dame Nature… Avec la science expérimentale, une nouvelle façon de « faire la science » fait son apparition en Occident au XVIIe siècle et n’aura de cesse d’évoluer et de progresser. Nous publions ici des extraits d’un panorama historique que Pierre Vignais, tôt disparu en 2006, avait dressé de cette révolution scientifique dans son livre Science expérimentale et connaissance du vivant. La méthode et les concepts (EDP Sciences, 2006).Understanding Nature mechanisms… A new way of “making science”, experimental science, born in Occident during the XVIIth century had never stopped evolving and improving. We publish here some parts of an historical panorama about this scientific revolution written by Pierre Vignais in his book Experimental Science and Knowledge of Life (EDP Sciences, 2006) and published a few months before his death in 2006

The Hyb Hydrogenase Permits Hydrogen-Dependent Respiratory Growth of Salmonella enterica Serovar Typhimurium

Salmonella enterica serovar Typhimurium contains three distinct respiratory hydrogenases, all of which contribute to virulence. Addition of H2 significantly enhanced the growth rate and yield of S. Typhimurium in an amino acid-containing medium; this occurred with three different terminal respiratory electron acceptors. Based on studies with site-specific double-hydrogenase mutant strains, most of this H2-dependent growth increase was attributed to the Hyb hydrogenase, rather than to the Hya or Hyd respiratory H2-oxidizing enzymes. The wild type strain with H2 had 4.0-fold greater uptake of 14C-labeled amino acids over a period of minutes than did cells incubated without H2. The double-uptake hydrogenase mutant containing only the Hyb hydrogenase transported amino acids H2 dependently like the wild type. The Hyb-only-containing strain produced a membrane potential comparable to that of the wild type. The H2-stimulated amino acid uptake of the wild type and the Hyb-only strain was inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone but was less affected by the ATP synthase inhibitor sodium orthovanadate. In the wild type, proteins TonB and ExbD, which are known to couple proton motive force (PMF) to transport processes, were induced by H2 exposure, as were the genes corresponding to these periplasmic PMF-coupling factors. However, studies on tonB and exbD single mutant strains could not confirm a major role for these proteins in amino acid transport. The results link H2 oxidation via the Hyb enzyme to growth, amino acid transport, and expression of periplasmic proteins that facilitate PMF-mediated transport across the outer membrane