Nitrogen source-dependent expression of a 126 kDa protein in the plasma membrane of the cyanobacterium Synechococcus PCC 7942.

The expression of a 126 kDa protein in the cytoplasmic membrane of Synechococcus PCC 7942 is shown to be dependent on the nitrogen source. It is absent in ammonium-grown cells and its quantity is inversely related to the concentration of nitrate or nitrite in the growth medium. Addition of ammonium-grown cells to a medium containing nitrate or L-methionine-DL-sulfoximine results in the expression of this protein. It is present in the plasmalemma of the Synechococcus NC3 mutant (nrtC gene deleted) and absent in the NA3 mutant (nrtABCD genes deleted). These results may suggest involvement of the 126 kDa protein in nitrate transport through Synechococcus cytoplasmic membrane.

[Quasi-elastic light scattering. Application to biological problems]. / Diffusion quasi-élastique de la lumière. Application à des problèmes biologiques

The origin of the light scattered from macromolecular solutions is considered and a discussion is made around the use of laser and light beating spectroscopy to analyze the scattered field spectrum. Several experiments are reviewed where quasi elastic light scattering has been applied.

Does a proton-pumping ATPase exist in the tonoplast?

In order to account for the accumulation of metabolites in plant vacuoles, the existence of a proton-pumping ATPase has been widely suggested in the literature. The demonstration of such a tonoplast-bound ATPase was merely based on the characterization of a nitrate-sensitive microsomal fraction. In some examples, this ATPase activity has been evidenced on vacuole preparations obtained under conditions which were criticized by Boller. The application of the reverse phase high-performance liquid chromatography method (RP-HPLC) to the simultaneous separation of adenine nucleotides, in the presence of tonoplast vesicles isolated from Catharanthus roseus, showed results not necessarily correlated with the ATPase hypothesis. Moreover, in light of the H+-quenching of quinacrine fluorescence observed during ATP hydrolysis by vacuoles or tonoplast vesicles, the existence of a proton-pumping ATPase may be questioned.

Design of a chemiluminescent and bioluminescent photometer.

An apparatus for chemi- and bioluminescence measurements is described. The main features are in high sensitivity: for example, 5 10(-12) M adenosine triphosphate, 5 10(-7) M glucose, 2 10(-9) M perhydrol and 4 10(-13) M peroxidase are easily detected; its fast mixing time and the possibility of injecting a reactant, at any time, enabling kinetic studies. Moreover, its compactness and 12 V battery supply allow measurements outside of laboratories.

Studies on the surface properties of human lymphocytes by photon correlation spectroscopy technique.

Electrokinetic behaviour of human lymphocytes was studied by photon correlation spectroscopy technique on laser IR-spectrometer. The electrophoretic mobilities (EPMs) were measured for peripheral blood lymphocytes (PBL) and CEM-C12 T cell line in 1:1 electrolyte at 22 degrees C. Plots of mobility vs ionic strength in the range 0.001-0.1 M were compared with theoretical curves calculated from (i) the Smoluchowsky formula, (ii) the simplified form of the Dukhin-Deryaguin equation which takes into account the fact that the mobility decreases due to the relaxation effect and (iii) the equation suggested by Donath and Pastushenko, which takes into account the influence of cell glycoprotein layer (GPL) on the EPM values. It has been found that the first two equations describe the experimental data with the assumption that surface charge density (sigma) decreases and width of the hydrodynamically immobile layer (L) increases with decreasing ionic strength; the relaxation effect turns out to be insignificant for the cell charges and sizes under consideration. In agreement with these findings, the third equation is approximately consistent with experimental data on the condition that GPL is allowed to expand with decreasing ionic strength, with simultaneous decrease of its full charge density (sigma(f)). The results are compared with relevant evidence for erythrocytes. The possible applications of the inferences arrived at in electrophoretic studies of cell behaviour are also discussed.

Determination of inorganic phosphate by coupling thymidine phosphorylase and reversed-phase high-performance liquid chromatography: application to tonoplast pyrophosphatase activity.

We developed an HPLC method for the measurement of inorganic phosphate using thymidine phosphorylase (EC 2.4.2.4). This enzyme catalyzes the phosphorolysis of thymidine to 2-deoxyribose 1-phosphate and thymine. Thymine release was measured at 265 nm after separation by reverse-phase HPLC. The assay was sensitive enough to detect as little as 10 pmol of Pi. The response to the phosphate concentration was linear from 1 to 100 microM. The value of this method was demonstrated in an analysis of the kinetics of Pi release from PPi in the presence of Catharanthus roseus tonoplast pyrophosphatase.

Pyrocystis lunula bioluminescence: physicochemical characterization of the luciferin precursor.

The luminescence of the dinoflagellate Pyrocystis lunula is controlled by the reduction state of the luciferin precursor. This molecule (P630) is a chromopeptide more stable than luciferin in methanolic solutions at low temperature. Cations may oxidize P630 or cleave the bond between the peptidic chain and the extended tetrapyrrole. Reduction of P630 is performed enzymatically by a NAD(P)H-dependent oxidoreductase or chemically by 2-mercaptoethanol or dithiothreitol. The state of reduction is monitored by the absorption and fluorescence emission which reveal a conformational change of the chromopeptide depending on the pH. These data will be useful for forthcoming studies on intracellular reducing power regulation and luminescence rhythms of these cells.

Influence of the surface potential on the purple membrane structure and activity.

The role of the divalent cations in the purple membrane is generally understood as the release mechanism of the blue form appearance. The reconstitution by cation addition leads to the recovery of the initial spectral properties. Numerous data are available in the literature on this matter but they are scattered, so that synthetic understanding is not easy. The role of divalent cations was studied through spectrophotometric titrations and electrophoretic mobility measurements, i.e., zeta potential valuations. Thus, correlations between the bacteriorhodopsin (bR) state and the whole membrane in equilibrium with a definite medium could be made. Deionization was not a fully reversible process. The absence of cations affect neither the rate of the M412 formation nor its lifetime but the yield of M412/bR was 50% lower. The number of protons involved in the blue to purple transition of both membranes was different and the reconstitution did not erase this difference. It was observed that the number of protons dissociated upon cation addition corresponded approximately to the number of positive charges removed by deionization. Electrophoretic mobility titrations showed large differences between the membranes, illustrating the influence of the surface charge density on the pK of the transition. Taking advantage of the reversible light adaptation process, the reciprocal influence of the charge density of the membrane surface and the retinal state in bR was shown. Specificity of the divalent cations was questioned by a direct substitution of them by imidazol, which left the membrane intact. The partial reversibility of the deionization, the decrease of the M412 yield, the differences in the titratable protons, and the nonstrict specificity toward divalent cations suggested that another unknown factor could be removed from the membrane.

Un nouveau colorant fluorescent pour la mesure du temps de relaxation brownienne des proteines.

Several authors have described the NBD-chloride (7-chloro-4-nitrobenzo-2-oxa-1,3-diazole) as a fluorogenic reagent of the amino groups and the sulfhydryl groups. It reacts rapidly with the trypsin to produce a stable and highly fluorescent conjugate. The determination of the labelled residues was made with thin layer chromatographies; the lysin residues were preferentially labelled.Fluorescence depolarization measurements of the conjugate were used to determine the brownian relaxation of the enzyme. The temperature dependence of the life-time is very strong and the corrected Perrin's plot demonstrates that the relaxation time of the enzyme decreases rapidly with the temperature.The conclusion is that the NBD-chloride can be used, like the DNS-chloride but is more reactive and stable and its fixation, on trypsin can be followed by measuring the absorbance of the dye at 470 nm.

Photoionization used as a tool for the study of biomembranes: fate of tetramethylbenzidine photocation in purple membrane.

Photoionization of hydrophobic probes has been developed in micelles or synthetic vesicles. Studies of the yields, compartmentation, and lifetimes of the photo-produced charged species have gathered reliable information on the interfacial and structural properties of these assemblies. Such an approach has never been applied to biological membranes. The present system is tetramethylbenzidine as the probe in native or modified (deionized and/or bleached) purple membrane from halobacteria. The data on photocation formation yields (phi ion) and lifetimes (tau 1/2) allow two main conclusions to be made: (1) tetramethylbenzidine, as the cation, is buried in the membrane core, and (2) its incorporation does not alter the biological activity of the protein. In this biological membrane the photocation lifetime and yield present the same trend of variation with the surface potential but to less of an extent than in model membranes. Bleaching of purple membrane completely modifies the photoionization process and the photocation decay. In addition, these experiments reveal a tight correlation between membrane structure and probe photoionization. Further evidence for structural modification of purple membrane, either by deionization or by bleaching is pointed out.

Characterization of tonoplast-enriched vesicles isolated by gradient density fractionation of suspension-cultured cells.

A tonoplast-enriched microsomal fraction was isolated from Catharanthus roseus cells. It was characterized by structural and functional criteria. This fraction presented a homogeneous size distribution as shown by quasi-elastic light scattering and a homogeneous density on self-generated gradients of Percoll. The mean diameter of the vesicles was estimated to be 0.30 micron and the buoyant density around 1.04 g/ml. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of its polypeptide pattern was in good agreement with the one obtained for tonoplast purified from isolated vacuoles. According to enzymatic assays and inhibition tests, this fraction possessed pyrophosphate and ATP-dependent proton pumps and very low contamination by submitochondrial particles, endoplasmic reticula and Golgi membranes. In light of our previous published results on tonoplast purified from isolated vacuoles, the very low extent of AMP hydrolysis by the microsomal fraction is interpreted as supplementary proof in favor of a tonoplast-enriched fraction.

Characterization of the plasmalemma ATPase from the cyanobacteria Synechococcus PCC 6311 and PCC 7942.

Biochemical properties of the ATPase from the plasma membrane of the cyanobacteria Synechococcus PCC 6311 and PCC 7942 were examined. ATPase activity associated with purified plasma membrane vesicles was strongly inhibited by 100 microM vanadate (87%), 100 microM diethylstilbestrol (70%) and 100 mM fluoride ions (83%). No inhibition was observed in the presence of dicyclohexylcarbodiimide, nitrate, azide, or molybdate. A 50% activation was observed in the presence of 50 mM KCl but none was observed in the presence of NaCl or NH4Cl. This ATPase was able to form a pH gradient, the amplitude of which was decreased by the presence of 100 microM vanadate. On Western blot of the plasmalemma proteins, no labeling was observed with a monoclonal antibody against the beta subunit of the F0-F1 ATPase, although staining was observed with the 55-kDa subunit of the thylakoid membrane ATPase. After phosphorylation of plasmalemma vesicles, by [gamma-32P]ATP, the autoradiograms of the electrophoreses, performed under acid conditions, exhibited labeling of a 110-kDa protein. The results indicated that the Synechococcus plasma membrane ATPase can be classified as a H+ translocating P-type ATPase and compared to the plant plasmalemma ATPase.

Surface charge of purple membranes measured by laser Doppler velocimetry.

Laser Doppler velocimetry measurements on purple membrane suspensions from Halobacterium halobium showed a linear correlation between electrophoretic mobility and applied electric field, electrokinetic responses could be rapidly monitored. Native membranes are less charged than white membrane preparations (from the R1mW mutant). Chemical modification of carboxyl residues reduces surface charge, and nitrotyrosine modified membranes are more or less charged than native membranes at pH greater than or less than 6.5, respectively. Changes in surface charge are found upon actinic illumination and are greatest (Ca 5 X 10(-4)/A2) under conditions where decay of the M412 intermediate of the photoreaction cycle is inhibited, such as at high pH or after chemical modification.

Enzyme-catalyzed gel proteolysis: an anomalous diffusion-controlled mechanism.

Enzyme-catalyzed proteolysis of gelatin gels has been studied. We report a gel degradation rate varying as the square of the enzyme concentration. The diffusion motion of enzymes in the gel has been measured by two-photon fluorescence correlation spectroscopy and identified as being anomalously slow. These experimental results are discussed from a theoretical point of view and interpreted in terms of a diffusion-controlled mechanism for the gel degradation. These results make a step toward the understanding of enzyme-catalyzed gel degradation and give new insight on biological processes such as the action of metalloproteinases in the extracellular matrix involved in cellular invasion.