Bernard Arrio

Description by quasi elastic laser light scattering of a biological preparation: Sarcoplasmic reticulum vesicles

SummaryQuasi elastic laser light scattering (QELS) was used to describe the size and the homogeneity of a membrane preparation: vesicles from skeletal sarcoplasmic reticulum. The data were compared with results obtained by electron-microscopy. The advantages of each method are discussed. By electron-microscopy the average value for the diameter of the vesicles obtained after negative staining is 0.100±0.040 μm. With the freeze-etching technique this value ranges from 0.13 to 0.25 μm. For the same biological preparation the analysis of the QELS recording curves displaying one correlation time (τ) gives an apparent diameter for the vesicles of 0.17 μm. Although it is difficult to describe by QELS the homogeneity of the preparation, the technique remains a very convenient method for evaluating variation of the size of the vesicles, and thus offers the possibility to follow general modification in the size of a vesicular preparation. The effect of trypsin and phospholipase C treatment on vesicle size is studied: an increase of vesicle size is clearly observed after trypsin treatment.

Electrokinetic and hydrodynamic properties of sarcoplasmic reticulum vesicles: A study by laser doppler electrophoresis and quasi-elastic light scattering☆

The electrical properties of sarcoplasmic reticulum vesicles were studied using electrophoretic mobility measurements by laser Doppler velocimetry. Combined with quasi-elastic light scattering analysis, this method enabled us to monitor changes in membrane surface charge density upon addition of NaCl, CaCl2, and MgCl2 solutions. At low ionic strength (4.7 mM), and in the absence of divalent salts, sarcoplasmic reticulum vesicles displayed a zeta potential value of -32.6 mV and a surface charge density of 5.4 10(-3) C/m2, corresponding to one elementary negative charge per 30 nm2 or about 2900 negative charges per vesicle. Upon addition of NaCl (up to 100 mM) vesicles became more negative (from 2900 to 6200 negative charges per vesicle), suggesting chloride binding or adsorption on the membrane. A noticeable increase of the vesicular size was observed upon addition of calcium or magnesium (25 to 30% increase of the hydrodynamic radius for 2.5 mM CaCl2 or MgCl2). This effect was prevented or suppressed by addition of relatively low concentrations of NaCl (50 mM). At low divalent cation concentrations one-third of the total net charge of the vesicles was neutralized suggesting a specific binding of cation on the membrane. At higher salt concentrations no modification of this parameter was observed. These results support the Gouy and Chapman theory about the electrical properties of sarcoplasmic reticulum vesicles.

Multiple effects of linolenic acid addition to pea thylakoids

Abstract The addition of linolenic acid to thylakoids produces various pH-dependent effects. We have demonstrated a binding site near the Photosystem (PS) II center with a p K a of 6.5: when linolenic acid is unprotonated it induces in the dark a rise of the initial fluorescence level, the latter being similar to the maximum fluorescence obtained during illumination of untreated thylakoids. The comparison of the fluorescence lifetimes in the presence and absence of linolenic acid leads us to conclude that the charge stabilisation on the primary acceptor, Q, is prevented by linolenic acid. A second binding site on the protein carrying B, the secondary acceptor of PS II, has also been demonstrated for linolenic acid. It has a 3-(3,4-dichlorophenyl)-1,1-dimethylurea-type effect both in the protonated and unprotonated forms. Finally, measurements of electrophoretic mobility of the thylakoids indicate several other sites of linolenic acid inclusion with an average p K a of 5.7. At alkaline pH the presence of unprotonated linolenic acid increases the charge density on the membrane. As a result a higher concentration of divalent cations is needed to obtain fluorescence and stacking changes than for untreated thylakoids. The presence, at acidic pH values, of the unprotonated form of linolenic acid leads to the inhibition of cation-induced fluorescence changes, probably by preventing the movement of chlorophyll-protein complexes in the membrane.

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.

Changes of some physical properties of isolated and purified plasma and thylakoid membrane vesicles from the freshwater cyanobacterium Synechococcus 6301 (Anacystis nidulans) during adaptation to salinity

Photoautotrophically growing cultures of the freshwater cyanobacterium Anacystis nidulans (Synechococcus sp.) became adapted to the presence of 0.4-0.5 M NaCl in the growth medium (about seawater level) with a lag phase of 2 days after which time the growth rate resumed at 80-90% of the control. Major changes in structure and function of the plasma membranes (and, to a much lesser extent, of the thylakoid membranes) were found to accompany the adaptation process. Plasma and thylakoid membranes were separated from crude cell-free extracts of French pressure cell-treated Anacystis by discontinuous sucrose density gradient centrifugation and purified by repeated recentrifugation on fresh gradients. Concentrations of copper, iron, calcium, and magnesium ions were determined by inductively coupled plasma atomic emission spectrometry with EDTA-washed and dialyzed membrane preparations; salt adaptation was found to increase (decrease) the concentration of membrane-bound calcium in plasma (thylakoid) membranes, qualitatively reciprocal results being obtained for magnesium. Levels of plasma membrane-bound copper and iron roughly tripled during the adaptation process; by contrast, corresponding effects on thylakoid membranes were negligible. The size of the membrane vesicles was measured by quasi-elastic laser light-scattering and the electric surface charge of the membranes was measured by laser Doppler velocimetry. Salt adaptation decreased the mean diameter of plasma membrane vesicles to a much higher extent than that of thylakoid membrane vesicles. Overall surface charge densities of resting vesicles were only slightly affected by the salt treatment as was also seen from titration of the electrophoretic mobility of the vesicles with electrolytes. Yet, induction of (photosynthetic or respiratory) electron transport provoked a charge separation across the membrane which was easily measurable in terms of electrophoretic mobility. The results will be discussed with particular emphasis on the stimulated cytochrome c oxidase activity of plasma (but not thylakoid) membranes from salt-adapted cells compared to control cells and also with respect to the decreased ion permeability of the plasma membrane of salt grown cells.

[14] High-performance liquid chromatography for simultaneous kinetic measurements of adenine nucleotides in isolated vacuoles

Publisher Summary The chapter discusses high-performance liquid chromatography (HPLC) for simultaneous kinetic measurements of adenine nucleotides in isolated vacuoles. The chromatograms presented are a typical example of adenine nucleotide separation during adenosine 5’-diphosphate (ADP) hydrolysis in the presence of tonoplast vesicles. Kinetic analyses obtained from such chromatograms are presented. Besides nucleotide hydrolysis, a concomitant ATP synthesis occurs when ADP is the initial substrate. This synthesis may be because of an adenylate kinase-like activity. These phenomena are observed, whatever the cells (Acer or Catharanthus), in the presence or in the absence of 1 mM molybdate. Using the reversed-phase (RP)-HPLC technique for simultaneous kinetic measurements of adenosine 5’-triphosphate (ATP), ADP, and adenosine 5’-monophosphate (AMP), several enzymes involved in hydrolysis and synthesis of these nucleotides are presented. All these observations emphasize the fact that complex kinetics brings different conclusions, whether they are monitored by specific techniques. From the data, the titration of phosphate, usually at a single reaction time, cannot obviously describe the involvement of the nucleotides. This chromatographic technique can be useful in characterizing the adenine nucleotide-related enzymes during tonoplast preparations.

ATP synthesis and pyrophosphate-driven proton transport in tonoplast-enriched vesicles isolated from Catharanthus roseus

In the presence of PPi as an energy source, the tonoplast‐bound inorganic pyrophosphatase from Catharanthus roseus cells is able to create a proton‐gradient which can drive the synthesis of ATP from ADP and Pi. ATP synthesis is linked to the pH‐gradient dissipation as monitored by the recovery of the fluorescence intensity of quinacrine and by the amount of synthesized ATP measured by the bioluminescent luciferin/luciferase assay. Proton gradient and ATP synthesis were suppressed by the protonic ionophore gramicidin D.

[77] Laser light scattering techniques for determining size and surface charges of membrane vesicles from cyanobacteria

Publisher Summary This chapter illustrates the application of quasi-elastic laser light scattering (QELS) and laser Doppler velocimetry (LDV) to the determination of volumes and surface charges of plasma (CM) and thylakoid (intracellular, ICM) membrane vesicles prepared from normal and salt-stressed cyanobacteria according to a recently established method. Laser light scattering techniques allow accurate determination not only of the diffusion coefficient and, hence, the volume of membrane vesicles but also of intact cells. The surface potential is also obtained by these techniques which have the great advantage of being noninvasive. When a beam of monochromatic light is scattered by a suspension of identical particles the scattered light spectrum has a Lorentzian shape. This is because of the Doppler frequency shift provoked by the random motion of the particles in suspension. Electrophoretic mobility measurements lead to the determination of the electrokinetic potential which reflects the surface charge at the shear plane of the particle. It is emphasized that of all different methods, QELS is the only one that provides immediate results without any modification of the biological sample.

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.

ATP:AMP phosphotransferase activity, a new characteristic of Catharanthus roseus tonoplasts

The ATPase activity of Catharanthus roseus tonoplasts was examined using HPLC separation and quantification of adenine nucleotides. ATP seemed to be degraded into ADP and AMP by tonoplast vesicles. When ADP was the initial substrate, the appearance of AMP and concomitant ATP synthesis were observed; these reactions were inhibited by Ap5A. The apparent degradation of ATP into AMP was also inhibited by Ap5A. These results indicated that AMP arose from an ATP:AMP phosphotransferase activity and excluded the possibility of the hydrolysis of ADP into AMP by the tonoplast ATPase. AMP was degraded by the microsomal fraction from protoplasts or by the cytosol while the tonoplast vesicles did not hydrolyze it. This observation was used to assess the purity of tonoplasts.