A Remarkably Stable Phosphorylated Form of Ca 2+ -ATPase Prepared from Ca 2+ -loaded and Fluorescein Isothiocyanate-labeled Sarcoplasmic Reticulum Vesicles

After the nucleotide binding domain in sarcoplasmic reticulum Ca2+-ATPase has been derivatized with fluorescein isothiocyanate at Lys-515, ATPase phosphorylation in the presence of a calcium gradient, with Ca2+ on the lumenal side but without Ca2+ on the cytosolic side, results in the formation of a species that exhibits exceptionally low probe fluorescence (Pick, U. (1981) FEBS Lett. 123, 131-136). We show here that, as long as the free calcium concentration on the cytosolic side is kept in the nanomolar range, this low fluorescence species is remarkably stable, even when the calcium gradient is subsequently dissipated by ionophore. This species is a Ca2+-free phosphorylated species. The kinetics of Ca2+ binding to it indicates that its transport sites are exposed to the cytosolic side of the membrane and retain a high affinity for Ca2+. Thus, in the ATPase catalytic cycle, an intrinsically transient phosphorylated species with transport sites occupied but not yet occluded must also have been stabilized by fluorescein isothiocyanate (FITC), possibly mimicking ADP. The low fluorescence mainly results from a change in FITC absorption. The Ca2+-free low fluorescence FITC-ATPase species remains stable after addition of thapsigargin in the absence or presence of decavanadate, or after solubilization with dodecylmaltoside. The remarkable stability of this phosphoenzyme species and the changes in FITC spectroscopic properties are discussed in terms of a putative FITC-mediated link between the nucleotide binding domain and the phosphorylation domain in Ca2+-ATPase, and the possible formation of a transition state-like conformation with a compact cytosolic head. These findings might open a path toward structural characterization of a stable phosphorylated form of Ca2+-ATPase for the first time, and thus to further insights into the pump's mechanism

Simulation of consumer exposure to deoxynivalenol according to wheat crop management and grain segregation: Case studies and methodological considerations

International audienceWe combined agronomic data and a model simulating exposure based on consumption data to assess the impact of crop management and grain segregation procedures on consumer exposure to deoxynivalenol. We used three scenarios of soil tillage at a regional scale and three scenarios of grain segregation for a supply area. The soil tillage scenarios were applied to a range of mean crop contamination levels, with various coefficients representing the degree of tillage. The grain segregation scenarios were applied to two real datasets of DON content distributions. We found that the increase in consumer exposure in response to increases in "risky" crop management practices such as direct-drilling depends largely on mean contamination and on the value of the tillage coefficient. The results for grain segregation procedures showed that exposure was most strongly affected by contamination distributions as the segregation procedure minimising risk differed for the two datasets

Structural Studies of a Stabilized Phosphoenzyme Intermediate of Ca 2+ -ATPase

Ca(2+)-ATPase belongs to the family of P-type ATPases and maintains low concentrations of intracellular Ca(2+). Its reaction cycle consists of four main intermediates that alternate ion binding in the transmembrane domain with phosphorylation of an aspartate residue in a cytoplasmic domain. Previous work characterized an ultrastable phosphoenzyme produced first by labeling with fluorescein isothiocyanate, then by allowing this labeled enzyme to establish a maximal Ca(2+) gradient, and finally by removing Ca(2+) from the solution. This phosphoenzyme is characterized by very low fluorescence and has specific enzymatic properties suggesting the existence of a high energy phosphoryl bond. To study the structural properties of this phosphoenzyme, we used cryoelectron microscopy of two-dimensional crystals formed in the presence of decavanadate and determined the structure at 8-A resolution. To our surprise we found that at this resolution the low fluorescence phosphoenzyme had a structure similar to that of the native enzyme crystallized under equivalent conditions. We went on to use glutaraldehyde cross-linking and proteolysis for independent structural assessment and concluded that, like the unphosphorylated native enzyme, Ca(2+) and vanadate exert a strong influence over the global structure of this low fluorescence phosphoenzyme. Based on a structural model with fluorescein isothiocyanate bound at the ATP site, we suggest that the stability as well as the low fluorescence of this phosphoenzyme is due to a fluorescein-mediated cross-link between two cytoplasmic domains that prevents hydrolysis of the aspartyl phosphate. Finally, we consider the alternative possibility that phosphate transfer to fluorescein itself could explain the properties of this low fluorescence species

Etudes structurales et fonctionnelles de l' ATPase-Ca2+ du réticulum sarcoplasmique (SERCA1A) (effets des conditions de cristallisation sur la conformation de l' ATPase-Ca2+)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Effet des acides gras polyinsatures de la serie n-3 sur la plasticité astrocytaire (rôle dans le fonctionnement de l'horloge circadienne (Etude in vitro et in vivo))

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Swillens et al. reply. Saturation and homologous competition studies do have different discriminatory powers

SCOPUS: le.jinfo:eu-repo/semantics/publishe

The Average Conformation at Micromolar [Ca2+] of Ca2+-ATPase with Bound Nucleotide Differs from That Adopted with the Transition State Analog ADP·AlFx or with AMPPCP under Crystallization Conditions at Millimolar [Ca 2+ ]: AMPPCP versus ADP.AlFx, and Ca ion occlusion in SERCA1a

International audienceCrystalline forms of detergent-solubilized sarcoplasmic reticulum Ca 2+-ATPase, obtained in the presence of either a substrate analog, AMPPCP, or a transition state complex, ADP.fluoroaluminate, were recently described to share the same general architecture despite the fact that, when studied in a test tube, these forms show different functional properties. Here, we show that the differences in the properties of the E1.AMPPCP and the E1.ADP.AlFx membraneous (or solubilized) forms are much less pronounced when these properties are examined in the presence of 10 mM Ca 2+ (the concentration prevailing in the crystallisation media) than when they are examined in the presence of the few µM Ca 2+ known to be sufficient to saturate the transport sites. This concerns various properties including ATPase susceptibility to proteolytic cleavage by Proteinase K, ATPase reactivity towards SH-directed Ellman's reagent, ATPase intrinsic fluorescence properties (here described for the E1.ADP.AlFx complex for the first time), and also the rates of 45 Ca 2+-40 Ca 2+ exchange at site "II". These results solve the above paradox at least partially, and suggest that the presence of a previously unrecognized Ca 2+ ion in the Ca 2+-ATPase.AMPPCP crystals should be re-investigated. A contrario, they emphasize the fact that the average conformation of the E1.AMPPCP complex under usual conditions in the test tube differs from that found in the crystalline form. The extended conformation of nucleotide revealed by the E1.AMPPCP crystalline form might be only indicative of the requirements for further processing of the complex, towards the transition state leading to phosphorylation and Ca 2+ occlusion

Phosphorylated Ca 2+ -ATPase Stable Enough for Structural Studies

The atomic structure of sarcoplasmic reticulum Ca(2+)-ATPase, in a Ca(2+)-bound conformation, has recently been elucidated (Toyoshima, C., Nakasako, M., Nomura, H. & Ogawa, H. (2000) Nature 405, 647-655). Important steps for further understanding the mechanism of ion pumps will be the atomic structural characterization of different key conformational intermediates of the transport cycle, including phosphorylated intermediates. Following our previous report (Champeil, P., Henao, F., Lacapère, J.-J. & McIntosh, D. B. (2000) J. Biol. Chem. 276, 5795-5803), we show here that it is possible to prepare a phosphorylated form of sarcoplasmic reticulum Ca(2+)-ATPase (labeled with fluorescein isothiocyanate) with a week-long stability both in membranes and in mixed lipid-detergent micelles. We show that this phosphorylated fluorescein isothiocyanate-ATPase can form two-dimensional arrays in membranes, similar to those that were used previously to reconstruct from cryoelectron microscopy images the three-dimensional structure of Ca(2+)-free unphosphorylated ATPase. The results also provide hope that crystals of phosphorylated Ca(2+)-ATPase suitable for x-ray crystallography will be achieved

Does a radiolabelled ligand bind to a homogeneous population of non-interacting receptor sites?

Receptor subtypes of pharmacological interest are often characterized by studies in which a particular radiolabelled ligand is competitively displaced from its binding sites, either by its unlabelled analogue (homologous displacement), or by different, unlabelled, ligands (heterologous displacement). In this article, Stephane Swillens, Magali Waelbroeck and Philippe Champeil emphasize the fact that a single homologous displacement curve is generally unable to reveal that the radioligand binds to a heterogeneous receptor population. Overlooking this heterogeneity may, in turn, dramatically affect interpretation of heterologous displacement curves displaying more than one receptor type for the unlabelled ligand: neither the binding affinities of the ligands for the different receptors, nor the proportion of these receptors, can be estimated reliably.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Stochastic simulation of a single inositol 1,4,5-trisphosphate-sensitive Ca2+ channel reveals repetitive openings during 'blip-like' Ca2+ transients

Confocal microscope studies with fluorescent dyes of inositol 1,4,5-trisphosphate (InsP,)-induced intracellular Ca*+ mobilization recently established the existence of ‘elementary’ events, dependent on the activity of individual InsP,-sensitive Ca2+ channels. In the present work, we try by theoretical stochastic simulation to explain the smallest signals observed in those studies, which were referred to as Ca 2+ ‘blips’ [Parker I. Yao Y. Ca*+ transients associated with openings of inositol trisphosphate-gated channels in Xenopus oocytes. J Physiol Land 1996; 491: 663-6681. For this purpose, we assumed a simple molecular model for the InsP,-sensitive Ca*+ channel and defined a set of parameter values accounting for the results obtained in electrophysiological bilayer experiments [Bezprozvanny I. Watras J. Ehrlich B.E. Bell-shaped calcium-response curves of lns(1,4,5)P,- and calcium- gated channels from endoplasmic reticulum of cerebellum. Nature 1991; 351: 751-754; Bezprozvanny I. Ehrlich B.E. lnositol (1,4,5)-trisphosphate (InsP,)-gated Ca channels from cerebellum: conduction properties for divalent cations and regulation by intraluminal calcium. J Gen Physiol1994; 104: 821-8561. With a stochastic procedure which considered cytosolic Ca*+ diffusion explicitly, we then simulated the behaviour of a single channel, placed in a realistic physiological environment. An attractive result was that the simulated channel exhibited bursts of activity, arising from repetitive channel openings, which were responsible for transient rises in Ca*+ concentration and were reminiscent of the relatively long-duration experimental Ca2+ blips. The influence of the values chosen for the various parameters (affinity and diffusion coefficient of the buffers, luminal Ca*+ concentration) on the kinetic characteristics of these theoretical blips is analyzed.info:eu-repo/semantics/publishe