Solution-Processed p-Dopant as Interlayer in Polymer Solar Cells.

We report here an original approach to dope the semiconducting polymer-metal interface in an inverted bulk-heterojunction (BHJ) organic solar cell. Solution-processed 2,3,5,6-tetrafluoro-7,7,8,8-tetracyanoquinodimethane (F4-TCNQ), is deposited on top of a P3HT:PC61BM layer before deposition of the top electrode. Doping of P3HT by F4-TCNQ occurs after thermally induced diffusion at 100 °C of the latter into the BHJ. Diffusion and doping are evidenced by XPS and UV-vis-NIR absorption. XPS highlights the decrease in Fluorine concentration on top of the BHJ after annealing. In the same time, a charge transfer band attributed to doping is observed in the UV-vis-NIR absorption spectrum. Inverted polymer solar cells using solution-processed F4-TCNQ exhibit power conversion efficiency of nearly 3.5% after annealing. This simple and efficient approach, together with the low annealing temperature required to allow diffusion and doping, leads to standard efficiency P3HT:PC61BM polymer solar cells, which are suitable for printing on plastic flexible substrate.

CadA, the Cd2+-ATPase from Listeria monocytogenes, can use Cd2+ as co-substrate.

CadA is a membrane protein of the P-type ATPase family which is the major determinant of the resistance to Cd2+ in Listeria monocytogenes. During its catalytic cycle, CadA undergoes auto-phosphorylation from ATP at Asp398, which allows Cd2+ translocation across the membrane. In the reverse mode, Asp398 is phosphorylated from Pi. From the data obtained so far, the CadA catalytic mechanism is similar to that proposed for the sarcoplasmic reticulum Ca2+-ATPase, the model of the P-type ATPase family. We show here that CadA is sensitive to two different ranges of Cd2+ concentration. The 0.1-10 microM range of added CdCl2 corresponds to Cd2+ binding at the transport site of unphosphorylated CadA which induces the reaction of the enzyme with ATP and impairs its reaction with Pi. The 0.1-1 mM range of added CdCl2 could correspond to Cd2+ binding to the transport site accessible from the extracellular medium. In addition, although it is widely accepted that the actual substrate of P-type ATPases is the MgATP complex, we show here that CadA can also perform its cycle in the absence of Mg2+, using CdATP in the place of MgATP at the catalytic site.

A possible regulatory role for the metal-binding domain of CadA, the Listeria monocytogenes Cd2+-ATPase.

Using the baculovirus/Sf9 expression system, we produced CadA and DeltaMBD, a metal-binding domain, truncated CadA. Both proteins had the expected properties of P-type ATPases: ATP-induced Cd2+ accumulation, Cd2+-sensitive ATP and Pi phosphorylation and ATPase activity. DeltaMBD displayed lower initial transport velocity as well as lower maximal ATPase activity than CadA. MBD truncation flattened the Cd2+ dependence of the ATPase activity and increased apparent Cd2+ affinity, suggesting a positive cooperativity between MBD and membranous transport sites. We propose that occupancy of MBD by Cd2+ modulates CadA activity.

Purification of heterologous sarcoplasmic reticulum Ca2+-ATPase Serca1a allowing phosphoenzyme and Ca2+-affinity measurements.

We describe here a protocol to prepare milligrams of active and stable heterologous sarcoplasmic reticulum Ca(2+)-ATPase (Serca1a). Serca1a was tagged with 6 histidines at its C-terminal end and overexpressed using the baculovirus-Sf9 system. In a first trial, Serca1a accounted for 24% of membrane proteins, 95% of which were inactive. Glucose in the culture medium reduced the production of Serca1a to 3 to 5% of membrane proteins and all Serca1a was active. Seventy-five percent of active Serca1a was solubilized by C(12)E(8) in the presence of phosphatidylcholine under conditions avoiding denaturation. Purification by Ni(2+)-nitrilo-triacetic acid affinity chromatography was tried, but only 3% of active Serca1a remained bound to the column, as if the His-tag were not accessible. Yields of 43% were reached by purification on reactive red 120 columns when eluting with 2 M NaCl. The purity was about 25% and Serca1a was stable for at least 1 week at 0 degrees C. Typically, 500 ml of culture medium produced 3 mg of active Serca1a and 1 mg of purified active Serca1a allowing measurements of phosphoenzyme (2 nmol/mg) or Ca(2+) affinity (2 microM at pH 7).

Ligand binding to macromolecules or micelles: use of centrifugal ultrafiltration to measure low-affinity binding.

We describe a method for estimating ligand binding to a macromolecular sample under conditions where this binding is of low affinity and must be measured under equilibrium conditions, without removal of the unbound ligand. The method is based on centrifugal ultrafiltration through a membrane with a molecular mass cut-off intermediate between that of the ligand and that of the target, and the amount of bound ligand is calculated from the difference between the (total) ligand in the concentrated sample and the (free) ligand in the ultrafiltrate. Centrifugal ultrafiltration makes it possible to separate free ligand from bound ligand (without changing its concentration) and to simultaneously concentrate the target (such that the proportion of bound ligand becomes significant, even under low-affinity binding conditions). We applied this technique, using Centricon 10 (Amicon) devices, to several cases (soluble proteins, intact membranes, detergent-solubilized proteins, and pure detergent micelles) and assessed its value with respect to the common artifacts that occur in other protocols involving protein retention on nitrocellulose filters (nonspecific ligand adsorption and protein denaturation).

Nickel binding and immunological properties of the C-terminal domain of the Helicobacter pylori GroES homologue (HspA).

Helicobacter pylori synthesizes a heat-shock protein of the GroES class. The gene encoding this protein (heat-shock protein A, HspA) was recently cloned and it was shown to be unique in structure. H. pylori HspA consists of two domains: the N-terminal domain (domain A) homologous with other GroES proteins, and a C-terminal domain (domain B) corresponding to 27 additional residues resembling a metal-binding domain. Various recombinant proteins consisting of the entire HspA polypeptide, the A domain, or the B domain were produced independently as proteins fused to maltose-binding protein (MBP). Comparison of the divalent cation binding properties of the various MBP and MBP-fused proteins allowed us to conclude that HspA binds nickel ions by means of its C-terminal domain. HspA exhibited a high and specific affinity for nickel ions in comparison with its affinity for other divalent cations (copper, zinc, cobalt). Equilibrium dialysis experiments revealed that MBP-HspA binds nickel ions with an apparent dissociation constant (Kd) of 1.8 microM and a stoichiometry of 1.9 ions per molecule. The analysis of the deduced HspA amino acid sequences encoded by 35 independent clinical isolates demonstrated the existence of two molecular variants of HspA, i.e. a major and a minor variant present in 89% and 11% of strains, respectively. The two variants differed from each other by the simultaneous substitution of seven amino acids within the B domain, whilst the A domain was highly conserved amongst all the HspA proteins (99-100% identity). On the basis of serological studies, the highly conserved A domain of HspA was found to be the immunodominant domain. Functional complementation experiments were performed to test the properties of the two HspA variants. When co-expressed together with the H. pylori urease gene cluster in Escherichia coli cells, the two HspA variant-encoding genes led to a fourfold increase in urease activity, demonstrating that HspA in H. pylori has a specialized function with regard to the nickel metalloenzyme urease.

Ca2+ translocation across sarcoplasmic reticulum ATPase randomizes the two transported ions.

Cytoplasmic Ca2+ dissociation is sequential, and the Ca2+ ions bound to the nonphosphorylated ATPase are commonly represented as superimposed on each other, so that the superficial Ca2+ is freely exchangeable from the cytoplasm, whereas the deeper Ca2+ is not. Under conditions where ADP-sensitive phosphoenzyme accumulates (leaky vesicles, 5 degrees C, pH 8, 300 mM K+), luminal Ca2+ dissociation is sequential as well, so that the representation of two superimposed Ca2+ ions still holds on the phosphoenzyme, with the superficial Ca2+ facing the lumen freely exchangeable and the deeper Ca2+ blocked by the superficial Ca2+. Under the same conditions, we have investigated whether a prebuilt Ca2+ order is maintained during membrane translocation. Starting from a prebuilt order on the cytoplasmic side, we showed that the Ca2+ ions cannot be identified after translocation to the luminal side. The same result was obtained starting from a prebuilt order on the luminal side and following the luminal to cytoplasmic translocation. We conclude that the two Ca2+ ions are mixed during ATP-induced phosphorylation as well as during ADP-induced dephosphorylation.

Atomic force microscopy study of isolated ivy leaf cuticles observed directly and after embedding in Epon®.

The first results obtained by atomic force microscopy (AFM) of the fine structure of isolated ivy leaf cuticles are reported. Observations of transverse sections embedded in Epon® allow easy recognition of the general shape of cuticles as viewed by light microscopy. The surface profile shows irregularities not revealed by transmission electron microscopy (TEM). The lamellate and reticulate zones, distinguishable by TEM, were also located by AFM. The outer zone appears as an irregularly thick (c. 4 µm) homogeneous region; lamellae can be visualized only after image-processing to subtract relief defects produced by sectioning. The second region or internal zone represents the largest part of the cuticle thickness; it appears heterogeneous and disordered. The cuticle internal surface images show imprints of the epidermal cells. At high magnification, the cell imprint central regions appear to be made up of a network of fibres of c. 50 nm diameter. Some images show that these fibres have a preferential orientation. They disappear after acid-hydrolysis is used to eliminate polysaccharides. This study shows that AFM can produce reproducible images of isolated plant cuticles at a subcellular scale leading to new high-resolution representations of cuticle substructure.

The modulation of Ca2+ binding to sarcoplasmic reticulum ATPase by ATP analogues is pH-dependent.

Excess ATP is known to enhance Ca(2+)-ATPase activity and, among other effects, to accelerate the Ca2+ binding reaction. In previous work, we studied the pH dependence of this reaction and proposed a 3H+/2Ca2+ exchange at the transport sites, in agreement with the H+/Ca2+ counter transport. Here we studied the effect of ADP and nonhydrolyzable ATP analogues on the Ca2+ binding reaction at various pH values. At pH 6, where Ca2+ binding is monophasic and slow, ADP, adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP), or adenyl-5'-yl imidodiphosphate (AMPPNP) increased the Ca2+ binding rate constant 20-fold. At pH 7 and 8, where Ca2+ binding is biphasic, the nucleotides induce fast and monophasic Ca2+ binding. At pH 7, AMP-PCP accelerated Ca2+ binding with an apparent dissociation constant of 10 microM. At acidic pH, ADP, AMPPCP, or AMPPNP increased the equilibrium affinity of Ca2+ for ATPase, whereas at alkaline pH, these nucleotides had no effect. At pH 5.5, AMPPCP increased equilibrium Ca2+ binding with an apparent dissociation constant of 1 microM.

How do Ca2+ ions pass through the sarcoplasmic reticulum membrane.

We propose an overview of the mechanism of Ca2+ transport through the sarcoplasmic reticulum membrane via the Ca(2+)-ATPase. We describe cytoplasmic calcium binding, calcium occlusion in the membrane and lumenal calcium dissociation. A channel-like structure is discussed and related to structural data on the membranous domain of the Ca(2+)-ATPase.

Lumenal Ca2+ dissociation from the phosphorylated Ca(2+)-ATPase of the sarcoplasmic reticulum is sequential.

Once two radioactive Ca2+ coming from the cytoplasm are bound to the transport sites of the nonphosphorylated ATPase, excess EGTA induces rapid dissociation of both ions, whereas excess nonradioactive Ca2+ only reaches one of the two bound Ca2+. This difference has been explained assuming that the two Ca2+ sites are in a single file channel in which the superficial Ca2+ is freely exchangeable from the cytoplasm, whereas the deeper Ca2+ is exchangeable only when the superficial site is vacant. The same experiment was done using phosphorylated ATPase to determine whether Ca2+ dissociation toward the lumen is sequential as well. Under conditions that allow ADP-sensitive phosphoenzyme to accumulate (leaky vesicles, 5 degrees C, pH 8, 300 mM KC1), we found the same two pools of Ca2+. Excess EGTA induced dissociation of both ions together with dephosphorylation. Excess nonradioactive Ca2+ induced the exchange of half the radioactive Ca2+ without any effect on the phosphoenzyme level. Our results show a close similarity between the transport sites of the nonphosphorylated and the phosphorylated enzymes, although the orientation, affinities, and dissociation rate constants are different.

Ca2+ binding to sarcoplasmic reticulum ATPase revisited. II. Equilibrium and kinetic evidence for a two-route mechanism.

The experiments reported in the present paper were designed to check the model proposed for Ca2+ binding in the preceding paper (Forge, V., Mintz, E., and Guillain, F. (1993) J. Biol. Chem. 268, 10953-10960). The pH dependence of the Mg(2+)-induced variation of the intrinsic fluorescence, as well as that of the phosphorylation by Pi, confirmed that there are several species of Ca(2+)-deprived ATPase. Kinetics of Ca2+ binding as a function of pH suggested that the deprotonated form of the ATPase binds Ca2+ rapidly (k > 50 s-1), whereas the protonated forms bind Ca2+ slowly (1.3-2.7 s-1). At variance with other models which are linear, slow and rapid Ca2+ binding take two different routes, and intermediate pH values and Mg2+, which favors the deprotonated forms, result in biphasic kinetics. Mg2+ binds to all Ca(2+)-deprived species and to species having one bound Ca2+ but does not bind to ECa2. This is the reason why Mg2+ inhibits Ca2+ binding, and this inhibition is removed in the presence of adenosine-5'-O-(3-thiotriphosphate) which drives Mg2+ into the catalytic site.

Ca2+ binding to sarcoplasmic reticulum ATPase revisited. I. Mechanism of affinity and cooperativity modulation by H+ and Mg2+.

H+ and Mg2+ are known to inhibit Ca2+ binding to the transport sites of sarcoplasmic reticulum-ATPase. Evaluation of the affinity for the Ca2+ binding sites requires measurement of the amount of Ca2+ bound to ATPase as a function of the free Ca2+ concentration imposed by a Ca2+ chelator. The choice of the chelator is crucial as it determines the accuracy of the free Ca2+ concentration. At pH > 7, the EGTA affinity for Ca2+ is higher than that of ATPase, inducing artifacts that alter the shape of the binding curves. Thus, we have used 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), whose affinity is unchanged at pH > or = 7. Ca2+ binding was studied at equilibrium, from pH 6 to pH 8 and from 0 to 10 mM Mg2+, using EGTA and/or BAPTA and [45Ca]Ca2+. Under all conditions, the stoichiometry was 2 Ca2+/ATPase. At variance with previous studies, the Hill coefficient was 1.1-2 and higher at pH 6 than at pH 8. In addition, it decreased in the presence of Mg2+. The Ca2+ binding curves were analyzed according to a model in which they result from a sequential binding of two Ca2+, each binding step being modified by H+ and Mg2+. The effect of H+ is described by two steps involving two H+ and one H+, with pK 7 and 7.9, respectively. At pH 6, ATPase must lose two H+ for the first Ca2+ to bind and a third H+ for the second Ca2+ to bind. At pH 9, both Ca2+ bind without any H+ exchange. Mg2+ can bind to all species, except to that saturated with Ca2+. The species having lost two H+ has a higher affinity for Mg2+ (< or = 1 mM) than the species having bound three H+ (4 mM). The above model allows us to analyze the effects of H+ and Mg2+ at each Ca2+ binding step and to explain the changes in the apparent affinity and cooperativity.

Dimethyl sulfoxide favours the covalent phosphorylation and not the binding of Pi to sarcoplasmic reticulum ATPase.

Competition between Ca2+ binding and Pi phosphorylation showed that the affinity of Ca(2+)-ATPase for Pi was not changed by 20% DMSO. Thus, the enhancement of Pi phosphorylation in the presence of DMSO should not be attributed to a solvent effect on the affinity for Pi, but rather on the phosphorylation reaction itself.

The reaction mechanism of Ca(2+)-ATPase of sarcoplasmic reticulum. Direct measurement of the Mg.ATP dissociation constant gives similar values in the presence or absence of calcium.

Combining rapid filtration and rapid acid quenching, we have directly measured, at pH 7.0 and 5 degrees C, the association and dissociation rate constants of Mg.ATP binding to the sarcoplasmic reticulum (SR) ATPase in the presence of 50 microM calcium and 5 mM MgCl2 (3-4 x 10(6) M-1.s-1 and 9 s-1, respectively). Therefore, we have determined the true affinity for Mg.ATP (Kd = 3 microM) in the presence of calcium, which can not be measured at equilibrium because of spontaneous and fast phosphorylation. At low concentrations, Mg.ATP binding is the rate limiting step in the phosphorylation process, and Mg.ATP dissociation is slower than dephosphorylation. The kinetics of Ca2+ binding measured by rapid filtration are biphasic, reflecting a two-step mechanism, both steps being accelerated by Mg.ATP. Combining rapid filtration and rapid monitoring of the intrinsic fluorescence of SR Ca(2+)-ATPase, we showed that rate constants for calcium binding are always lower than those of Mg.ATP binding to an EGTA-incubated enzyme. We measured dissociation and association rate constants of Mg.ATP binding in the absence of calcium (k-1 = 25 s-1 and k1 = 7.5 10(6) M-1.s-1). This gives a Kd similar to that obtained by equilibrium measurements (3-4 microM). Both non-phosphorylated conformations of the enzyme have similar affinity for Mg.ATP. Therefore, activation of ATPase activity by an excess of ATP cannot be explained by a change in affinity of the non-phosphorylated enzyme for Mg.ATP. In conjunction with previous results, these data are used to discuss the molecular mechanism for the Ca(2+)-ATPase cycle, in which ATP is sequentially substrate and activator on a multiple-function single site.

Crosslinking the active site of sarcoplasmic reticulum Ca(2+)-ATPase completely blocks Ca2+ release to the vesicle lumen.

Intramolecular crosslinking of the active site of the sarcoplasmic reticulum Ca(2+)-ATPase with glutaraldehyde results in substantial inhibition of ATPase activity and stabilization of the ADP-sensitive E1 approximately P(2Ca) intermediate (E, enzyme) with occluded Ca2+ [Ross, D. C., Davidson, G. A. & McIntosh, D. B. (1991) J. Biol. Chem. 266, 4613-4621]. We show here, using conditions of low passive vesicle permeability and absence of ADP, that Ca2+ "deoccludes" more rapidly than it leaks out of the vesicle lumen. Deocclusion is paralleled by dephosphorylation. Therefore, turnover of crosslinked E1 approximately P(Ca) (approximately 5 nmol/min per mg of protein at 25 degrees C) involves Ca2+ release to the vesicle exterior and concomitant phosphoenzyme hydrolysis. Ca2+ release to the lumen, the normal pathway, is apparently blocked completely. In the presence of ADP, Ca2+ is also released to the vesicle exterior, and this release is coupled to the synthesis of ATP. The results suggest that a tertiary structural change at the active site follows phosphorylation and is an absolute requirement for Ca2+ release from the native enzyme to the vesicle lumen.

Ca2+ gradient and drugs reveal different binding sites for Pi and Mg2+ in phosphorylation of the sarcoplasmic reticulum ATPase.

The first step towards ATP synthesis by the Ca2-ATPase of sarcoplasmic reticulum is the phosphorylation of the enzyme by Pi. Phosphoenzyme formation requires both Pi and Mg2+. At 35 degrees C, the presence of a Ca2+ gradient across the vesicle membrane increases the apparent affinity of the ATPase for Pi more than 10-fold, whereas it had no effect on the apparent affinity for Mg2+. In the absence of a Ca2+ gradient, the phosphorylation reaction is inhibited by both K+ and Na+ at all Mg2+ concentrations used. However, in the presence of 1 mM Mg2+ and of a transmembrane Ca2+ gradient, the reaction is still inhibited by Na+, but the inhibition promoted by K+ is greatly decreased. When the Mg2+ concentration is raised above 2 mM, the enzyme no longer discriminates between K+ and Na+, and the phosphorylation reaction is equally inhibited by the two cations. Trifluoperazine, ruthenium red and spermidine were found to inhibit the phosphorylation reaction by different mechanisms. In the absence of a Ca2+ gradient, trifluoperazine competes with the binding to the enzyme of both Pi and Mg2+, whereas spermidine and ruthenium red were found to compete only with Mg2+. The data presented suggest that the enzyme has different binding sites for Mg2+ and for Pi.

Reversal of the sarcoplasmic reticulum ATPase cycle by substituting various cations for magnesium. Phosphorylation and ATP synthesis when Ca2+ replaces Mg2+.

Reversal of the cycle of sarcoplasmic reticulum ATPase starts from ATPase phosphorylation by Pi, in the presence of Mg2+, and leads to ATP synthesis. We show here that ATP can also be synthesized when Ca2+ replaces Mg2+. In the absence of a calcium gradient and in the presence of dimethyl sulfoxide, ATPase phosphorylation from Pi and Ca2+ led to the formation of an unstable phosphoenzyme. This instability was due to a competition between the phosphorylation reaction induced by Pi and Ca2+ and the transition induced by Ca2+ binding to the transport sites, which led to a conformation that could not be phosphorylated from Pi. Dimethyl sulfoxide and low temperature stabilized the calcium phosphoenzyme, which under appropriate conditions, subsequently reacted with ADP to synthesize ATP. Substitution of Co2+, Mn2+, Cd2+, or Ni2+ for Mg2+ induced ATPase phosphorylation from Pi, giving phosphoenzymes of various stabilities. However, substitution of Ba2+, Sr2+, or Cr3+ produced no detectable phosphoenzymes, under the same experimental conditions. Our results show that ATPase phosphorylation from Pi, like its phosphorylation from ATP, does not have a strict specificity for magnesium.

Reaction mechanism of Ca2+ ATPase of sarcoplasmic reticulum. Equilibrium and transient study of phosphorylation with Ca.ATP as substrate.

At pH 7.0 and 5 degrees C, in the absence of potassium and magnesium, the Ca-ATPase of the sarcoplasmic reticulum slowly hydrolyzes the Ca.ATP at a rate of 0.05 s-1. During turnover 4 nmol of phosphoenzyme per mg of total protein accumulate with a Km value of 10(-8) M. Combining rapid filtration and rapid acid quenching, we took advantage of the above properties to study the early steps of phosphorylation. (formula; see text) Direct measurements by rapid filtration of the rate constant of Ca.ATP binding (k1 = 3.5 x 10(6) M-1 s-1) and dissociation (k-1 = 2.5 s-1) enable us to estimate Ca.ATP affinity (Kd = 7 x 10(-7) M). Rapid acid quenching shows that covalent phosphoenzyme forms slowly with a rate constant of 0.6 s-1 (k2), and parallel filtration experiments indicate that phosphorylation and ADP release occur simultaneously. If the reaction is reversed (toward ATP synthesis) 70% of the phosphoenzyme is ADP-sensitive and ADP-induced dephosphorylation is fast (k-2 = 15 s-1), whereas the ADP-insensitive phosphoenzyme slowly hydrolyzes in the forward direction of the ATPase cycle at a rate of 0.05 s-1. Combination of rapid filtration and rapid acid quenching and the use of a mixture of [14C]ATP and [32P]ATP allow us to study the kinetics of several transient species. These data are used to develop a molecular mechanism for Ca-ATPase phosphorylation.