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

Cosmic-ray pitch-angle scattering in imbalanced mhd turbulence simulations

Pitch-angle scattering rates for cosmic-ray particles in magnetohydrodynamic (MHD) simulations with imbalanced turbulence are calculated for fully evolving electromagnetic turbulence. We compare with theoretical predictions derived from the quasilinear theory of cosmic-ray diffusion for an idealized slab spectrum and demonstrate how cross helicity affects the shape of the pitch-angle diffusion coefficient. Additional simulations in evolving magnetic fields or static field configurations provide evidence that the scattering anisotropy in imbalanced turbulence is not primarily due to coherence with propagating Alfven waves, but an effect of the spatial structure of electric fields in cross-helical MHD turbulence.Comment: 13 pages, 15 figures. Accepted by Ap

Drift effects and the cosmic ray density gradient in a solar rotation period: First observation with the Global Muon Detector Network (GMDN)

We present for the first time hourly variations of the spatial density gradient of 50 GeV cosmic rays within a sample solar rotation period in 2006. By inversely solving the transport equation, including diffusion, we deduce the gradient from the anisotropy that is derived from the observation made by the Global Muon Detector Network (GMDN). The anisotropy obtained by applying a new analysis method to the GMDN data is precise and free from atmospheric temperature effects on the muon count rate recorded by ground based detectors. We find the derived north-south gradient perpendicular to the ecliptic plane is oriented toward the Helioshperic Current Sheet (HCS) (i.e. southward in the toward sector of the Interplanetary Magnetic Field (IMF) and northward in the away sector). The orientation of the gradient component parallel to the ecliptic plane remains similar in both sectors with an enhancement of its magnitude seen after the Earth crosses the HCS. These temporal features are interpreted in terms of a local maximum of the cosmic ray density at the HCS. This is consistent with the prediction of the drift model for the $A<0$ epoch. By comparing the observed gradient with the numerical prediction of a simple drift model, we conclude that particle drifts in the large-scale magnetic field play an important role in organizing the density gradient, at least in the present $A<0$ epoch. We also found that corotating interaction regions did not have such a notable effect. Observations with the GMDN provide us with a new tool for investigating cosmic ray transport in the IMF.Comment: 35 pages, 10 figures, submitted to the Astrophysical Journa

Electron spin resonance in membrane research: protein–lipid interactions from challenging beginnings to state of the art

Conventional electron paramagnetic resonance (EPR) spectra of lipids that are spin-labelled close to the terminal methyl end of the acyl chains are able to resolve the lipids directly contacting the protein from those in the fluid bilayer regions of the membrane. This allows determination of both the stoichiometry of lipid–protein interaction (i.e., number of lipid sites at the protein perimeter) and the selectivity of the protein for different lipid species (i.e., association constants relative to the background lipid). Spin-label EPR data are summarised for 20 or more different transmembrane peptides and proteins, and 7 distinct species of lipids. Lineshape simulations of the two-component conventional spin-label EPR spectra allow estimation of the rate at which protein-associated lipids exchange with those in the bulk fluid regions of the membrane. For lipids that do not display a selectivity for the protein, the intrinsic off-rates for exchange are in the region of 10 MHz: less than 10× slower than the rates of diffusive exchange in fluid lipid membranes. Lipids with an affinity for the protein, relative to the background lipid, have off-rates for leaving the protein that are correspondingly slower. Non-linear EPR, which depends on saturation of the spectrum at high radiation intensities, is optimally sensitive to dynamics on the timescale of spin-lattice relaxation, i.e., the microsecond regime. Both progressive saturation and saturation transfer EPR experiments provide definitive evidence that lipids at the protein interface are exchanging on this timescale. The sensitivity of non-linear EPR to low frequencies of spin exchange also allows the location of spin-labelled membrane protein residues relative to those of spin-labelled lipids, in double-labelling experiments

Global Processes that Determine Cosmic Ray Modulation

The global processes that determine cosmic ray modulation are reviewed. The essential elements of the theory which describes cosmic ray behavior in the heliosphere are summarized, and a series of discussions is presented which compare the expectations of this theory with observations of the spatial and temporal behavior of both galactic cosmic rays and the anomalous component; the behavior of cosmic ray electrons and ions; and the 26-day variations in cosmic rays as a function of heliographic latitude. The general conclusion is that the current theory is essentially correct. There is clear evidence, in solar minimum conditions, that the cosmic rays and the anomalous component behave as is expected from theory, with strong effects of gradient and curvature drifts. There is strong evidence of considerable latitude transport of the cosmic rays, at all energies, but the mechanism by which this occurs is unclear. Despite the apparent success of the theory, there is no single choice for the parameters which describe cosmic ray behavior, which can account for all of the observed temporal and spatial variations, spectra, and electron vs. ion behavior.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43782/1/11214_2004_Article_164792.pd

In vivo NMR as a tool for probing molecular structure and dynamics in intact Chlamydomonas reinhardtii cells

Solid state NMR/Biophysical Organic Chemistr

Estimating the rotation rate in the vacuolar proton-ATPase in native yeast vacuolar membranes.

The rate of rotation of the rotor in the yeast vacuolar proton-ATPase (V-ATPase), relative to the stator or steady parts of the enzyme, is estimated in native vacuolar membrane vesicles from Saccharomyces cerevisiae under standardised conditions. Membrane vesicles are formed spontaneously after exposing purified yeast vacuoles to osmotic shock. The fraction of total ATPase activity originating from the V-ATPase is determined by using the potent and specific inhibitor of the enzyme, concanamycin A. Inorganic phosphate liberated from ATP in the vacuolar membrane vesicle system, during ten min of ATPase activity at 20 °C, is assayed spectrophotometrically for different concanamycin A concentrations. A fit of the quadratic binding equation, assuming a single concanamycin A binding site on a monomeric V-ATPase (our data are incompatible with models assuming multiple binding sites), to the inhibitor titration curve determines the concentration of the enzyme. Combining this with the known ATP/rotation stoichiometry of the V-ATPase and the assayed concentration of inorganic phosphate liberated by the V-ATPase, leads to an average rate of ~10 Hz for full 360° rotation (and a range of 6–32 Hz, considering the ± standard deviation of the enzyme concentration), which, from the time-dependence of the activity, extrapolates to ~14 Hz (8–48 Hz) at the beginning of the reaction. These are lower-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 is not fixed to a solid support; instead it is functioning in its native membrane environment