Jean Rigaud

Incorporation of bacteriorhodopsin into large unilamellar liposomes by reverse phase evaporation

The reverse phase evaporation procedure was used to prepare large unilamellar liposomes containing bacteriorhodopsin. Electron microscopy showed that proteoliposomes were unilamellar and fairly uniform in size provided the preparation was extruded through calibrated nucleopore membranes : the vesicles have diameters around 200 nm. The spectral properties of the bacteriorhodopsin in the large liposomes resembled those of bacteriorhodopsin in purple membrane. Furthermore, the chromoprotein in the reconstituted vesicles had an inside-out orientation and on illumination, translocated protons efficiently from the external medium into the vesicles in the presence of the ionophore valinomycin. In the absence of the latter, a light-independent transmembrane potential of about 60 mV was measured from thiocyanate distribution. In the presence of valinomycin, this transmembrane electrical potential was abolished and then a light-dependent transmembrane pH gradient of about 2 pH units could be generated.

Changes in intracellular pH in French-bean nodules induced by senescence and nitrate treatment

Abstract Intracellular pH of Phaseolus nodules was determined by using radioactive 5,5-dimethyloxazolidine-2,4-dione (DMO) as a probe. A continuous decline in the intracellular pH was observed when nodule age increased. The extracellular volume of nodules, required for the pH calculation, was the lowest in mature nodules and increased in senescing nodules. Low concentrations of nitrate in the culture medium induced a drop in nitrogen fixation associated with a decrease in the intracellular pH. Acidic proteolysis measured by in vitro hydrolysis of leghemoglobin was strongly stimulated under these conditions. In Phaseolus nodules, senescence and nitrate treatment were similarly characterized by a lower intracellular pH and an active proteolysis which both contributed to a decline in nitrogen fixation capacities.

O2 consumption and superoxide dismutase content in purified mitochondria from soybean root nodules

Abstract Soybean nodule mitochondria have been separated from cotnaminating organella on discontinuous Percoll gradients. The preparations appeared highly purified and at least 80% of the mitochondria were estimated to be derived from infected cells. Percoll-purified mitochondria showed important respiratory activity; in the case of succinate, the rate of O 2 consumption was 185 nmol O 2 min −1 mg −1 and the respiratory control and ADP/O ratio reached 2.72 and 1.16, respectively. These organelles also exhibited an active manganese containing superoxide dismutase (9.7 U mg −1 ), whose purification is reported. These results are consistent with a significant O 2 consumption by host cell mitochondria in vivo and the possibility of a competition for O 2 supply with the bacteroids is discussed.

The effect of hydration of lecithin-water lamellar phases: A comparative study of solute diffusion and ESR spectroscopic measurements

Abstract An analysis of the paramagnetic resonance spectra of spin labels in the lipidic region of lecithin-water lamellar phases as a function of phase water content has been carried out. The observed variation of the local organization and mobility of the lipids is consonant with previous results obtained from solute diffusion measurements. The previously observed sudden changes of solute diffusion for hydration of 9 and 18 molecules water per lecithin molecule are compared with the concomitant sudden changes as seen by ESR spectroscopy. The results also indicate that there is a gradient of fluidity across the lipid leaflets which are therefore not homogeneous to diffusing molecules.

Δ-aminolevulinate uptake by Rhizobium bacteroids and its limitation by the peribacteroid membrane in legume nodules

Heme is overproduced during Rhizobium-Legume symbiosis and delta-aminolevulinate (ALA) is a common precursor in both bacterial and plant synthesis pathways of this molecule. ALA uptake by bacteroids from French bean and soybean nodules was characterized. The action of several metabolic inhibitors and the competition effect of malate on this uptake were studied. ALA transport appeared to be mediated by the dicarboxylate carrier system. Purified symbiosomes--bacteroids surrounded by the peribacteroid membrane--failed to accumulate significant amount of ALA. These experiments rule out the possibility for the plant cytosol to provide the bacteroid with ALA and strengthen the restrictive role of the peribacteroid membrane for exchanges between the two symbiotic partners.

Alternative energy-yielding substrates for bacteroids isolated from stem and root nodules of Sesbania rostrata submitted to O2 restricted conditions

Abstract After 3 days of flooding, root and stem nodules of Sesbania rostrata maintained a 50% C 2 H 2 reduction capacity whereas the activity was 80% inhibited in soybean nodules. At the bacteroid level, the efficiency of malate and lactate to support C 2 H 2 reduction was largely increased by flooding which also induced the ability for bacteroids to use ethanol. Malate and lactate were respectively oxidized by bacteroid malate dehydrogenase and lactate dehydrogenase, whose activities were strongly enhanced by nodule immersion. These enzymes also occuring in nodule cytosol, but working in an opposite way, exhibited a higher activity under O 2 restricted conditions. In addition to the presence of higher level of malate and lactate, the generation of ethanol provided a range of energy-yielding substrates requiring different O 2 tensions for optimal bacteriod C 2 H 2 reduction. The existence of these fermentative processes could be related to the adaptation of Sesbania nodules to flooding.

Ferrochelatase activities and heme contents in purified mitochondria from soybean roots and root nodules

Summary Mitochondria extracted from soybean root nodules contained a significantly higher ferrochelatase (EC 4.99.1.1.) activity than root organelles. However, the heme contents of mitochondria of both origins appeared of the same order of magnitude. Self-assembly of apoleghemoglobin with heme synthesized in vitro by purified nodule mitochondria was observed and the possibility that these organelles can be involved in leghemoglobin synthesis in vivo is discussed.