Glycated bovine serum albumin for use in feeding trials with animal models - In vitro methodology and characterization of a glycated substrate for modifying feed pellets.

This study investigated different methods to produce Nε-carboxymethyl-lysine (CML)-enriched bovine serum albumin (BSA) as alternatives to the classical approach using glyoxylic acid (GA) and sodium cyanoborohydride (NaBH3CN) which results in toxic hydrogen cyanide (HCN). The reaction of GA (6 mmol/L) and NaBH3CN (21 mmol/L) to produce CML remained the most effective with CML yields of 24-35%, followed by 13-24% using 300 mmol/L glyoxal (GO). GA promoted specific modification of lysine to CML, and fewer structural modifications of the BSA molecule compared with GO, as evidenced by fluorescence and proteomic analyses. GO promoted greater arginine modification compared with GA (76 vs 23%). Despite structural changes to BSA with GO, murine fecal clearance of CML was similar to literature values. Hence, BSA glycation with 300 mmol/L glyoxal is a suitable alternative to GA and NaBH3CN for generating CML-enriched protein free of HCN, but a CML-only fortification model remains to be described.

Measurements of Na+-occluded intermediates during the catalytic cycle of the Na+/K+-ATPase provide novel insights into the mechanism of Na+ transport.

The Na+/K+-ATPase is an integral plasma membrane glycoprotein of all animal cells that couples the exchange of intracellular Na+ for extracellular K+ to the hydrolysis of ATP. The asymmetric distribution of Na+ and K+ is essential for cellular life and constitutes the physical basis of a series of fundamental biological phenomena. The pumping mechanism is explained by the Albers-Post model. It involves the presence of gates alternatively exposing Na+/K+-ATPase transport sites to the intracellular and extracellular sides and includes occluded states in which both gates are simultaneously closed. Unlike for K+, information is lacking about Na+-occluded intermediates, as occluded Na+ was only detected in states incapable of performing a catalytic cycle, including two Na+-containing crystallographic structures. The current knowledge is that intracellular Na+ must bind to the transport sites and become occluded upon phosphorylation by ATP to be transported to the extracellular medium. Here, taking advantage of epigallocatechin-3-gallate to instantaneously stabilize native Na+-occluded intermediates, we isolated species with tightly bound Na+ in an enzyme able to perform a catalytic cycle, consistent with a genuine occluded state. We found that Na+ becomes spontaneously occluded in the E1 dephosphorylated form of the Na+/K+-ATPase, exhibiting positive interactions between binding sites. In fact, the addition of ATP does not produce an increase in Na+ occlusion as it would have been expected; on the contrary, occluded Na+ transiently decreases, whereas ATP lasts. These results reveal new properties of E1 intermediates of the Albers-Post model for explaining the Na+ transport pathway.

Enrichment of Quercetin from Winemaking Residual Diatomaceous Earth via a Tailor-Made Imprinted Adsorbent.

Residual diatomaceous earth (RDE) from winemaking activities is a rich and currently underexploited source of phenolic compounds which ought to be recycled from the perspective of circular bioeconomy. In this work, we demonstrate the feasibility of molecularly imprinted polymers (MIPs) for the enrichment of quercetin, a flavonoid at a fairly high content in residual diatomaceous earth. These MIPs were synthesized through free radical polymerization. FTIR confirmed the integration of the functional monomers into the polymeric chains. Batch adsorption experiments were used to assess the retention and selectivity of those MIPs towards quercetin. Commercial resins were compared with the synthesized materials using the same procedures. These adsorption experiments allowed the selection of the best performing MIP for the valorization of RDE extract. This treatment consisted of saturating the selected MIP with the extract and then desorbing the retained compounds using solvents of selected compositions. The desorbed fractions were analyzed using liquid chromatography, and the results demonstrated an increase in quercetin's fractional area from 5% in the RDE extract to more than 40% in some fractions, which is roughly an eightfold enrichment of quercetin. Moreover, other flavonoids of close chemical structure to quercetin have been rather retained and enriched by the MIP.

Epigallocatechin 3-gallate inhibits the plasma membrane Ca2+-ATPase: effects on calcium homeostasis.

Flavonoids are natural compounds responsible for the health benefits of green tea. Some of the flavonoids present in green tea are catechins, among which are: epigallocatechin, epicatechin-3-gallate, epicatechin, catechin and epigallocatechin-3-gallate (EGCG). The latter was found to induce apoptosis, reduce reactive oxygen species, in some conditions though in others it acts as an oxidizing agent, induce cell cycle arrest, and inhibit carcinogenesis. EGCG also was found to be involved in calcium (Ca2+) homeostasis in excitable and in non-excitable cells. In this study, we investigate the effect of catechins on plasma membrane Ca2+-ATPase (PMCA), which is one of the main mechanisms that extrude Ca2+ out of the cell. Our studies comprised experiments on the isolated PMCA and on cells overexpressing the pump. Among catechins that inhibited PMCA activity, the most potent inhibitor was EGCG. EGCG inhibited PMCA activity in a reversible way favoring E1P conformation. EGCG inhibition also occurred in the presence of calmodulin, the main pump activator. Finally, the effect of EGCG on PMCA activity was studied in human embryonic kidney cells (HEK293T) that transiently overexpress hPMCA4. Results show that EGCG inhibited PMCA activity in HEK293T cells, suggesting that the effects observed on isolated PMCA occur in living cells.

Proactive inter-disciplinary CME to improve medication management in the elderly population.

BACKGROUND: The absence of collaboration between health professionals is known to influence prescriptions' quality, also disadvantaging elderly frail patients' polytherapies. OBJECTIVES: This study aims to improve the adherence to medications of elderly patients suffering from multiple diseases through interpersonal continuing medical education (CME). The CME was organized for general practitioners (GPs) by hospital pharmacists (HPs) from a Territorial Pharmaceutical Centre of Piedmont, in collaboration with pharmacists from the Drug Science and Technology Department of the University of Turin, to enhance awareness on the management of chronic therapies and de-prescription. METHODS: Pharmacists set face-to-face lessons for GPs between April 2018 and November 2018, while therapies' reconciliation and delivery of the Illustrated Therapy Schedules (ITS) lasted until September 2019. Polytherapies were evaluated by pharmacists and GPs in terms of appropriateness (number of potentially inappropriate prescriptions - PIPs according to 2019 Beers Criteria) and number of drug-drug interactions (DDIs), using a clinical decision support system (CDSS - NavFarma©) to help health professionals dealing with the process of review, reconciliation and individuation of possible adverse reactions. RESULTS: From the CME organization it emerged that the collaboration between health professionals supported by a CDSS could improve the quality of elderly patients polytherapies. Two-hundred fifteen patients were enrolled by GPs; patients included were aged - results reported as average (sd) - 76.4 (6.3), mostly men (54.9%), number of daily medications per patient was 8.1 (2.4); 2.1 (1.8) DDIs per patient were individuated, 12.9% of which were solved thanks to the CME. Average number of PIPs found was 2.5 (1.4) per patient. CONCLUSIONS: The CME represented a proactive approach by HPs to the management of elderly patients' polytherapies. Moreover, clinicians' engagement is a mean to enhance quality, safety, professionalism and communication in health processes.

E2P-like states of plasma membrane Ca2+­ATPase characterization of vanadate and fluoride-stabilized phosphoenzyme analogues.

The plasma membrane Ca2+­ATPase (PMCA) belongs to the family of P-type ATPases, which share the formation of an acid-stable phosphorylated intermediate as part of their reaction cycle. The crystal structure of PMCA is currently lacking. Its abundance is approximately 0.1% of the total protein in the membrane, hampering efforts to produce suitable crystals for X-ray structure analysis. In this work we characterized the effect of beryllium fluoride (BeFx), aluminium fluoride (AlFx) and magnesium fluoride (MgFx) on PMCA. These compounds are known inhibitors of P-type ATPases that stabilize E2P ground, E2·P phosphoryl transition and E2·Pi product states. Our results show that the phosphate analogues BeFx, AlFx and MgFx inhibit PMCA Ca2+­ATPase activity, phosphatase activity and phosphorylation with high apparent affinity. Ca2+­ATPase inhibition by AlFx and BeFx depended on Mg2+ concentration indicating that this ion stabilizes the complex between these inhibitors and the enzyme. Low pH increases AlFx and BeFx but not MgFx apparent affinity. Eosin fluorescent probe binds with high affinity to the nucleotide binding site of PMCA. The fluorescence of eosin decreases when fluoride complexes bind to PMCA indicating that the environment of the nucleotide binding site is less hydrophobic in E2P-like states. Finally, measuring the time course of E → E2P-like conformational change, we proposed a kinetic model for the binding of fluoride complexes and vanadate to PMCA. In summary, our results show that these fluoride complexes reveal different states of phosphorylated intermediates belonging to the mechanism of hydrolysis of ATP by the PMCA.

"I am not promiscuous enough!": Exploring the low uptake of HIV testing by gay men and other men who have sex with men in Metro Manila, Philippines.

The Philippines faces a severe HIV epidemic among gay and other men who have sex with men (MSM). HIV testing uptake remains low. A case series of 12 men from Metro Manila were interviewed to explore barriers to uptake of HIV testing services. Most did not see the need to get tested for HIV despite significant risk, based on the misconception they were feeling well or showed no symptoms. Being of a higher socioeconomic class, feeling morally superior to other gay men, distance of the testing facility, fear of what will happen once infected, fear of HIV- and sexual stigma, fear of side effects of antiretroviral drugs and fear of high health care expenses after testing positive for HIV were key reasons why MSM kept postponing their test. Misconceptions about HIV risk, disease, and treatment and care need to be addressed in order to increase uptake of HIV services in this population.

Aluminum inhibits the plasma membrane and sarcoplasmic reticulum Ca2+-ATPases by different mechanisms.

Aluminum (Al3+) is involved in the pathophysiology of neurodegenerative disorders. The mechanisms that have been proposed to explain the action of Al3+ toxicity are linked to changes in the cellular calcium homeostasis, placing the transporting calcium pumps as potential targets. The aim of this work was to study the molecular inhibitory mechanism of Al3+ on Ca2+-ATPases such as the plasma membrane and the sarcoplasmic reticulum calcium pumps (PMCA and SERCA, respectively). These P-ATPases transport Ca2+ actively from the cytoplasm towards the extracellular medium and to the sarcoplasmic reticulum, respectively. For this purpose, we performed enzymatic measurements of the effect of Al3+ on purified preparations of PMCA and SERCA. Our results show that Al3+ is an irreversible inhibitor of PMCA and a slowly-reversible inhibitor of SERCA. The binding of Al3+ is affected by Ca2+ in SERCA, though not in PMCA. Al3+ prevents the phosphorylation of SERCA and, conversely, the dephosphorylation of PMCA. The dephosphorylation time courses of the complex formed by PMCA and Al3+ (EPAl) in the presence of ADP or ATP show that EPAl is composed mainly by the conformer E2P. This work shows for the first time a distinct mechanism of Al3+ inhibition that involves different intermediates of the reaction cycle of these two Ca2+-ATPases.

Steady-state analysis of enzymes with non-Michaelis-Menten kinetics: The transport mechanism of Na+/K+-ATPase.

Procedures to define kinetic mechanisms from catalytic activity measurements that obey the Michaelis-Menten equation are well established. In contrast, analytical tools for enzymes displaying non-Michaelis-Menten kinetics are underdeveloped, and transient-state measurements, when feasible, are therefore preferred in kinetic studies. Of note, transient-state determinations evaluate only partial reactions, and these might not participate in the reaction cycle. Here, we provide a general procedure to characterize kinetic mechanisms from steady-state determinations. We described non-Michaelis-Menten kinetics with equations containing parameters equivalent to kcat and Km and modeled the underlying mechanism by an approach similar to that used under Michaelis-Menten kinetics. The procedure enabled us to evaluate whether Na+/K+-ATPase uses the same sites to alternatively transport Na+ and K+ This ping-pong mechanism is supported by transient-state studies but contradicted to date by steady-state analyses claiming that the release of one cationic species as product requires the binding of the other (ternary-complex mechanism). To derive robust conclusions about the Na+/K+-ATPase transport mechanism, we did not rely on ATPase activity measurements alone. During the catalytic cycle, the transported cations become transitorily occluded (i.e. trapped within the enzyme). We employed radioactive isotopes to quantify occluded cations under steady-state conditions. We replaced K+ with Rb+ because 42K+ has a short half-life, and previous studies showed that K+- and Rb+-occluded reaction intermediates are similar. We derived conclusions regarding the rate of Rb+ deocclusion that were verified by direct measurements. Our results validated the ping-pong mechanism and proved that Rb+ deocclusion is accelerated when Na+ binds to an allosteric, nonspecific site, leading to a 2-fold increase in ATPase activity.

Profile-QSAR 2.0: Kinase Virtual Screening Accuracy Comparable to Four-Concentration IC50s for Realistically Novel Compounds.

While conventional random forest regression (RFR) virtual screening models appear to have excellent accuracy on random held-out test sets, they prove lacking in actual practice. Analysis of 18 historical virtual screens showed that random test sets are far more similar to their training sets than are the compounds project teams actually order. A new, cluster-based "realistic" training/test set split, which mirrors the chemical novelty of real-life virtual screens, recapitulates the poor predictive power of RFR models in real projects. The original Profile-QSAR (pQSAR) method greatly broadened the domain of applicability over conventional models by using as independent variables a profile of activity predictions from all historical assays in a large protein family. However, the accuracy still fell short of experiment on realistic test sets. The improved "pQSAR 2.0" method replaces probabilities of activity from naïve Bayes categorical models at several thresholds with predicted IC50s from RFR models. Unexpectedly, the high accuracy also requires removing the RFR model for the actual assay of interest from the independent variable profile. With these improvements, pQSAR 2.0 activity predictions are now statistically comparable to medium-throughput four-concentration IC50 measurements even on the realistic test set. Beyond the yes/no activity predictions from a typical high-throughput screen (HTS) or conventional virtual screen, these semiquantitative IC50 predictions allow for predicted potency, ligand efficiency, lipophilic efficiency, and selectivity against antitargets, greatly facilitating hitlist triaging and enabling virtual screening panels such as toxicity panels and overall promiscuity predictions.

Uncoiling collagen: a multidimensional mass spectrometry study.

Mass spectrometry can be used to determine structural information about ions by activating precursors and analysing the resulting series of fragments. Two-dimensional Fourier transform ion cyclotron resonance mass spectrometry (2D FT-ICR MS) is a technique that correlates the mass-to-charge (m/z) ratio of fragment and precursor ions in a single spectrum. 2D FT-ICR MS records the fragmentation of all ions in a sample without the need for isolation. To analyse specific precursors, horizontal cross-sections of the spectrum (fragment ion scans) are taken, providing an alternative to conventional tandem mass spectrometry (MS/MS) experiments. In this work, 2D FT-ICR MS has been used to study the tryptic digest of type I collagen, a large protein. Fragment ion scans have been extracted from the 2D FT-ICR MS spectrum for precursor m/z ratios: 951.81, 850.41, 634.34, and 659.34, and 2D FT-ICR MS spectra are compared with a set of 1D MS/MS spectra using different fragmentation methods. The results show that two-dimensional mass spectrometry excells at MS/MS of complex mixtures, simplifying spectra by eliminating contaminant peaks, and aiding the identification of species in the sample. Currently, with desktop computers, 2D FT-ICR MS is limited by data processing power, a limitation which should be alleviated using cluster parallel computing. In order to explore 2D FT-ICR MS for collagen, with reasonable computing time, the resolution in the fragment ion dimension is limited to 256k data points (compared to 4M data points in 1D MS/MS spectra), but the vertical precursor ion dimension has 4096 lines, so the total data set is 1G data points (4 Gbytes). The fragment ion coverage obtained with a blind, unoptimized 2D FT-ICR MS experiment was lower than conventional MS/MS, but MS/MS information is obtained for all ions in the sample regardless of selection and isolation. Finally, although all 2D FT-ICR MS peak assignments were made with the aid of 1D FT-ICR MS data, these results demonstrate the promise of 2D FT-ICR MS as a technique for studying complex protein digest mixtures.

The E2P-like state induced by magnesium fluoride complexes in the Na,K-ATPase. Kinetics of formation and interaction with Rb(+).

The first X-ray crystal structures of the Na,K-ATPase were obtained in the presence of magnesium and fluoride as E2(K2)Mg-MgF4, an E2∙Pi-like state capable to occlude K(+) (or Rb(+)). This work presents a functional characterization of the crystallized form of the enzyme and proposes a model to explain the interaction between magnesium, fluoride and Rb(+) with the Na,K-ATPase. We studied the effect of magnesium and magnesium fluoride complexes on the E1-E2 conformational transition and the kinetics of Rb(+) exchange between the medium and the E2(Rb2)Mg-MgF4 state. Our results show that both in the absence and in the presence of Rb(+), simultaneous addition of magnesium and fluoride stabilizes the Na,K-ATPase in an E2 conformation, presumably the E2Mg-MgF4 complex, that is unable to shift to E1 upon addition of Na(+). The time course of conformational change suggests the action of fluoride and magnesium at different steps of the E2Mg-MgF4 formation. Increasing concentrations of fluoride revert along a sigmoid curve the drop in the level of occluded Rb(+) caused by Mg(2+). Na(+)-induced release of Rb(+) from E2(Rb2)Mg-MgF4 occurs at the same rate as from E2(Rb2) but is insensitive to ADP. The rate of Rb(+) occlusion into the E2Mg-MgF4 state is 5-8 times lower than that described for the E2Mg-vanadate complex. Since the E2Mg-MgF4 and E2Mg-vanadate complexes represent different intermediates in the E2-P→E2 dephosphorylation sequence, the variation in occlusion rate could provide a tool to discriminate between these intermediates.

Crystallographic and kinetic study of riboflavin synthase from Brucella abortus, a chemotherapeutic target with an enhanced intrinsic flexibility.

Riboflavin synthase (RS) catalyzes the last step of riboflavin biosynthesis in microorganisms and plants, which corresponds to the dismutation of two molecules of 6,7-dimethyl-8-ribityllumazine to yield one molecule of riboflavin and one molecule of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. Owing to the absence of this enzyme in animals and the fact that most pathogenic bacteria show a strict dependence on riboflavin biosynthesis, RS has been proposed as a potential target for antimicrobial drug development. Eubacterial, fungal and plant RSs assemble as homotrimers lacking C3 symmetry. Each monomer can bind two substrate molecules, yet there is only one active site for the whole enzyme, which is located at the interface between two neighbouring chains. This work reports the crystallographic structure of RS from the pathogenic bacterium Brucella abortus (the aetiological agent of the disease brucellosis) in its apo form, in complex with riboflavin and in complex with two different product analogues, being the first time that the structure of an intact RS trimer with bound ligands has been solved. These crystal models support the hypothesis of enhanced flexibility in the particle and also highlight the role of the ligands in assembling the unique active site. Kinetic and binding studies were also performed to complement these findings. The structural and biochemical information generated may be useful for the rational design of novel RS inhibitors with antimicrobial activity.

Conformational changes produced by ATP binding to the plasma membrane calcium pump.

The aim of this work was to study the plasma membrane calcium pump (PMCA) reaction cycle by characterizing conformational changes associated with calcium, ATP, and vanadate binding to purified PMCA. This was accomplished by studying the exposure of PMCA to surrounding phospholipids by measuring the incorporation of the photoactivatable phosphatidylcholine analog 1-O-hexadecanoyl-2-O-[9-[[[2-[(125)I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine to the protein. ATP could bind to the different vanadate-bound states of the enzyme either in the presence or in the absence of Ca(2+) with high apparent affinity. Conformational movements of the ATP binding domain were determined using the fluorescent analog 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate. To assess the conformational behavior of the Ca(2+) binding domain, we also studied the occlusion of Ca(2+), both in the presence and in the absence of ATP and with or without vanadate. Results show the existence of occluded species in the presence of vanadate and/or ATP. This allowed the development of a model that describes the transport of Ca(2+) and its relation with ATP hydrolysis. This is the first approach that uses a conformational study to describe the PMCA P-type ATPase reaction cycle, adding important features to the classical E1-E2 model devised using kinetics methodology only.

Proteomics applied to transfusion plasma: the beginning of the story.

'Safe blood' is and has always been the major concern in transfusion medicine. Plasma can undergo virus inactivation treatments based on physicochemical, photochemical or thermal methodologies for pathogen inactivation. The validation of these treatments is essentially based on clottability assays and clotting factors' titration; however, their impact on plasma proteins at the molecular level has not yet been evaluated. Proteomics appears as particularly adapted to identify, to localize and, consequently, to correlate these modifications to the biological activity change. At the crossroads of biology and analytical sciences, proteomics is the large-scale study of proteins in tissues, physiological fluids or cells at a given moment and in a precise environment. The proteomic strategy is based on a set of methodologies involving separative techniques like mono- and bidimensional gel electrophoresis and chromatography, analytical techniques, especially mass spectrometry, and bioinformatics. Even if plasma has been extensively studied since the very beginning of proteomics, its application to transfusion medicine has just begun. In the first part of this review, we present the principles of proteomics analysis. Then, we propose a state of the art of proteomics applied to plasma analysis. Finally, the use of proteomics for the evaluation of the impact of storage conditions and pathogen inactivation treatments applied to transfusion plasma and for the evaluation of therapeutic protein fractionated is discussed.

Alternative cycling modes of the Na(+)/K(+)-ATPase in the presence of either Na(+) or Rb(+).

A comprehensive study of the interaction between Na(+) and K(+) with the Na(+)/K(+)-ATPase requires dissecting the incidence of alternative cycling modes on activity measurements in which one or both of these cations are absent. With this aim, we used membrane fragments containing pig-kidney Na(+)/K(+)-ATPase to perform measurements, at 25°C and pH=7.4, of ATPase activity and steady-state levels of (i) intermediates containing occluded Rb(+) at different [Rb(+)] in media lacking Na(+), and (ii) phosphorylated intermediates at different [Na(+)] in media lacking Rb(+). Most relevant results are: (1) Rb(+) can be occluded through an ATPasic cycling mode that takes place in the absence of Na(+) ions, (2) the kinetic behavior of the phosphoenzyme formed by ATP in the absence of Na(+) is different from the one that is formed with Na(+), and (3) binding of Na(+) to transport sites during catalysis is not at random unless rapid equilibrium holds.

Differential effects of G- and F-actin on the plasma membrane calcium pump activity.

We have previously shown that plasma membrane calcium ATPase (PMCA) pump activity is affected by the membrane protein concentration (Vanagas et al., Biochim Biophys Acta 1768:1641-1644, 2007). The results of this study provided evidence for the involvement of the actin cytoskeleton. In this study, we explored the relationship between the polymerization state of actin and its effects on purified PMCA activity. Our results show that PMCA associates with the actin cytoskeleton and this interaction causes a modulation of the catalytic activity involving the phosphorylated intermediate of the pump. The state of actin polymerization determines whether it acts as an activator or an inhibitor of the pump: G-actin and/or short oligomers activate the pump, while F-actin inhibits it. The effects of actin on PMCA are the consequence of direct interaction as demonstrated by immunoblotting and cosedimentation experiments. Taken together, these findings suggest that interactions with actin play a dynamic role in the regulation of PMCA-mediated Ca(2+) extrusion through the membrane. Our results provide further evidence of the activation-inhibition phenomenon as a property of many cytoskeleton-associated membrane proteins where the cytoskeleton is no longer restricted to a mechanical function but is dynamically involved in modulating the activity of integral proteins with which it interacts.

E2→E1 transition and Rb(+) release induced by Na(+) in the Na(+)/K(+)-ATPase. Vanadate as a tool to investigate the interaction between Rb(+) and E2.

This work presents a detailed kinetic study that shows the coupling between the E2→E1 transition and Rb(+) deocclusion stimulated by Na(+) in pig-kidney purified Na,K-ATPase. Using rapid mixing techniques, we measured in parallel experiments the decrease in concentration of occluded Rb(+) and the increase in eosin fluorescence (the formation of E1) as a function of time. The E2→E1 transition and Rb(+) deocclusion are described by the sum of two exponential functions with equal amplitudes, whose rate coefficients decreased with increasing [Rb(+)]. The rate coefficient values of the E2→E1 transition were very similar to those of Rb(+)-deocclusion, indicating that both processes are simultaneous. Our results suggest that, when ATP is absent, the mechanism of Na(+)-stimulated Rb(+) deocclusion would require the release of at least one Rb(+) ion through the extracellular access prior to the E2→E1 transition. Using vanadate to stabilize E2, we measured occluded Rb(+) in equilibrium conditions. Results show that, while Mg(2+) decreases the affinity for Rb(+), addition of vanadate offsets this effect, increasing the affinity for Rb(+). In transient experiments, we investigated the exchange of Rb(+) between the E2-vanadate complex and the medium. Results show that, in the absence of ATP, vanadate prevents the E2→E1 transition caused by Na(+) without significantly affecting the rate of Rb(+) deocclusion. On the other hand, we found the first evidence of a very low rate of Rb(+) occlusion in the enzyme-vanadate complex, suggesting that this complex would require a change to an open conformation in order to bind and occlude Rb(+).

Effect of glare on reaction time for peripheral vision at mesopic adaptation.

When a bright light is present in the field of view, visibility is dramatically reduced. Many studies have investigated the effect of glare on visibility considering foveal vision. However, the effects on peripheral vision have received little attention. In a previous work [J. Opt. Soc. Am. A 25, 1790 (2008)], we showed that the effect of glare on reaction time (RT) for foveal vision at mesopic adaptation depends on the stimulus spatial frequency. In this work, we extend this study to peripheral vision. We measured the RT of achromatic sinusoidal gratings as a function of contrast for a range of spatial frequency, and eccentricity, and for two glare levels, in addition to the no-glare condition. Data were fitted with Piéron's law, following a linear relationship. We found that glare increases the slope of these lines for all conditions. These slopes seem to depend critically on eccentricity for 4 cycles/degree (c/deg), but not for 1 and 2 c/deg. We explain our results in terms of the contrast sensitivity (gain) of the underlying detection mechanisms.

Calcium occlusion in plasma membrane Ca2+-ATPase.

In this work, we set out to identify and characterize the calcium occluded intermediate(s) of the plasma membrane Ca(2+)-ATPase (PMCA) to study the mechanism of calcium transport. To this end, we developed a procedure for measuring the occlusion of Ca(2+) in microsomes containing PMCA. This involves a system for overexpression of the PMCA and the use of a rapid mixing device combined with a filtration chamber, allowing the isolation of the enzyme and quantification of retained calcium. Measurements of retained calcium as a function of the Ca(2+) concentration in steady state showed a hyperbolic dependence with an apparent dissociation constant of 12 ± 2.2 µM, which agrees with the value found through measurements of PMCA activity in the absence of calmodulin. When enzyme phosphorylation and the retained calcium were studied as a function of time in the presence of La(III) (inducing accumulation of phosphoenzyme in the E(1)P state), we obtained apparent rate constants not significantly different from each other. Quantification of EP and retained calcium in steady state yield a stoichiometry of one mole of occluded calcium per mole of phosphoenzyme. These results demonstrate for the first time that one calcium ion becomes occluded in the E(1)P-phosphorylated intermediate of the PMCA.