Estimating the Rotation Rate in the Vacuolar Proton-ATPase in Native Yeast Vacuolar Membranes

The rate of rotation of the rotor of the yeast vacuolar proton-ATPase (V-ATPase), relative to the stator or the steady parts of enzyme, is estimated in native vacuolar membrane vesicles of Saccharomyces cerevisiae under standardised conditions. Membrane vesicles are spontaneously formed after exposing purified yeast vacuoles to osmotic shock. The fraction of the total ATPase activity originating from V-ATPase is determined using the potent and specific inhibi-tor of the enzyme, concanamycin A. Inorganic phosphate liberated from ATP in the vacuolar membrane vesicle system, during 10 min of ATPase activity at 20 °C, is assayed spectrophotometrically for different concanamycin A concentrations. A fit to the quadratic binding equation, assuming a single concanamycin A binding site on a monomeric V-ATPase (our data is incompatible with models assuming more binding sites) to the inhibitor titration curve determines the concentration of the enzyme. Combining it with the known rotation:ATP stoichiometry of V-ATPase and the assayed concentration of inorganic phosphate liberated by V-ATPase leads to an average rate of ~9.53 Hz of the 360 degrees rotation, which, according to the time-dependence of the activity, extrapolates to ~14.14 Hz for the beginning of the reaction. These are low limit estimates. To our knowledge this is the first report of the rotation rate in a V-ATPase that is not subjected to genetic or chemical modification and it is not fixed on a solid support, instead it is functioning in its native membrane environment

Effects of contaminants on biological model membranes: The advantage of the ASAXS method for the study of the location of copper ions and dihalogenated phenol molecules

The features of the location of copper ions and the dihalogenated phenol molecules were investigated at their low concentration in the dipalmitoylphosphatidylcholine (DPPC)-water liposomes by using small-angle X-ray scattering (SAXS) and anomalous small-angle X-ray scattering (ASAXS) methods. Only the ASAXS method can provide information about the structural features of the domains rich in these ions and organic guest molecules. The copper ions are mainly located in large domains between the lamellae and only partly in the characteristic periodic water shells of the liposomes. The dihalogenated phenol molecules are embedded in the layers and do not destroy the lamellar arrangement. Their inhomogeneous distribution appears laterally and phase separation occurs. The local structures induced by the copper ions and the dihalogenated phenols were also visualised by freeze-fracture method. (c) 2005 Elsevier B.V. All rights reserved

Biological Systems as Nanoreactors: Anomalous Small-Angle Scattering Study of the CdS Nanoparticle Formation in Multilamellar Vesicles

The formation of cadmium sulfide (CdS) particles in the gaps between the layers of the multilamellar vesicles is described, introducing a new pathway in the preparation of nanometer-scale particles. The in situ structural characterization of both the CdS particles and the vesicles as a reaction medium was performed in the early and final states of the process by using anomalous small-angle X-ray scattering (ASAXS) and freeze-fracture methods. The ASAXS method provides the separation of the scattering of nanoparticles present in a small amount, whereby the monitoring of their formation and growth in the whole time range of manufacturing has become possible

Unbinding Transition in Lipid Multibilayers Induced by Copper(II) Ions

We describe our observations on an unbinding transition in a multilamellar dispersion of phosphatidylcholine (PC) vesicles induced by copper(II) ions. The small-angle X-ray measurements clearly show that the increasing amount of CuCl2 in the millimolar concentration range continuously increases the amount of the unbound bilayers in the gel phase. Moreover, this phenomenon becomes more pronounced when the samples are heated above the so-called pretransition temperature between the gel and the ripple gel phase. The proposed reason for the latter is the increased repulsive electrostatic interaction due to the appearance of the surface modulation in the ripple gel phase. The observed effects reveal a new aspect of the unbinding phenomena since only the transition induced by the steric repulsion due to the layer fluctuations has been considered so far. Here, we show that the unbinding can also be triggered by the change in the electrostatic interactions. These findings are connected to the physical basis of the crucial role of copper(II) ions in biological processes such as neurodegenerative diseases and cell evolution

Influence of aminoglycoside antibiotics on the thermal behaviour and structural features of DPPE - DPPG model membranes

The effects of three aminoglycosides (AGs), tobramycin, streptomycin and spectinomycin on 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE)-1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-1-glycerol] (DPPG)/water systems with 0.05 and 0.2 DPPG/DPPE+DPPG molar fractions were examined by using differential scanning calorimetry (DSC), simultaneous small- and wide-angle X-ray scattering (SWAXS), and freeze-fracture transmission electronmicroscopy (FF-TE) at 10(-1) AG/lipid molar ratio in the doped systems. The effects induced by the AGs strongly depend on DPPG/DPPE+DPPG molar fraction. In the presence of the AGs regular lyotropic structures form as opposed to the complex and badly correlated layer structures of the pure 0.2 DPPG/DPPE+DPPG molar fraction system. The investigated AGs possess different structural and thermotropic effects: tobramycin and streptomycin which are true aminoglycosides decrease the main transition enthalpy in both model systems and induce the formation of correlated layer arrangements, while the occurrence of spectinomycin, a non-aminoglycoside aminocyclitol still ranked as an AG, results in a hexagonal phase as it can be deduced from the SAXS patterns and visually observed in the electronmicrographs

Vesicles as reactors of nanoparticles: an anomalous small-angle X-ray scattering study of the domains rich in copper ions

The formation of copper hydroxide and copper oxide particles in the gaps among the stacks of multilamellar vesicles is described, illustrating a new pathway in the preparation of nanometre-scale particles. The in situ structural characterization of both the solid particles and the vesicles as a reaction medium was performed in the initial and final states of the process by using anomalous small-angle X-ray scattering (ASAXS) and freeze-fracture methods. The ASAXS method provides a description of the particle-size distribution of the copper nanoparticles, in spite of the fact that they are present in low concentration. This method allows the particle formation and growth to be monitored throughout the whole time range of the synthesis

Localization of dihalogenated phenols in vesicle systems determined by contrast variation X-ray scattering

Localization of 2,4-dichloro- and 2,4-dibromophenol in multilamellar vesicles in a 1/1 dihalogenated phenol/lipid molar ratio was investigated by classical contrast variation X-ray scattering using the isomorphous replacement method. The results were compared with those obtained by anomalous small-angle X-ray scattering from a vesicle system doped with 2,4-dibromophenol. Dissimilarities in the results of the two methods are discussed, taking into account the advantages and disadvantages of both techniques in studying multilamellar systems