Novel protein and Mg2+ configurations in the Mg2+GDP complex of the SRP GTPase ffh

Ffh is the signal sequence recognition and targeting subunit of the prokaryotic signal recognition particle (SRP). Previous structural studies of the NG GTPase domain of Ffh demonstrated magnesium-dependent and magnesium-independent binding conformations for GDP and GMPPNP that are believed to reflect novel mechanisms for exchange and activation in this member of the GTPase superfamily. The current study of the NG GTPase bound to Mg(2+)GDP reveals two new binding conformations-in the first the magnesium interactions are similar to those seen previously, however, the protein undergoes a conformational change that brings a conserved aspartate into its second coordination sphere. In the second, the protein conformation is similar to that seen previously, but the magnesium coordination sphere is disrupted so that only five oxygen ligands are present. The loss of the coordinating water molecule, at the position that would be occupied by the oxygen of the gamma-phosphate of GTP, is consistent with that position being privileged for exchange during phosphate release. The available structures of the GDP-bound protein provide a series of structural snapshots that illuminate steps along the pathway of GDP release following GTP hydrolysis.     

Effect of Mg2+ during reactivation and refolding of guanidine hydrochloride-denatured creatine kinase

Creatine kinase (ATP: creatine N-phosphotransferase, EC 2.7.3.2) was completely denatured using 3 M guanidine hydrochloride for 2 h as in previous studies [Yao et al. (1982), Sci. Sin. 25B, 1296–1302; Yao et al. (1984), Biochemistry 23, 2740–2744; Yao et al. (1982), Sci. Sin. 25B, 1186–1193]. Under suitable conditions, about 60–70% of the activity can be recovered in the presence of different Mg²⁺ concentrations. Both the reactivation and the refolding processes follow two-phase courses after dilution in the proper solutions. A comparison of the rate constants for the refolding of unfolded creatine kinase with those for the recovery of its catalytic activity at various Mg²⁺ concentrations shows that these are not synchronized. The reactivity of guanidine hydrochloride-denatured creatine kinase can be inhibited by Mg²⁺; however, the rates of reactivation are independent of the Mg²⁺ concentration. In addition, Mg²⁺ affects the fluorescence intensity, but the rate constants of refolding are independent of Mg²⁺ concentration. Although the reactivation of GdHCl-denatured creatine kinase is complete about 3 h after dilution with reactivation solutions, the conformational changes during refolding occur in a much slower reaction. Mg²⁺ can induce complex changes in the relative fluorescence intensity during refolding over a broad range of concentrations.     

Intracellular Mg(2+) enhances the function of BK-type Ca(2+)-activated K(+) channels.

BK channels modulate neurotransmitter release due to their activation by voltage and Ca(2+). Intracellular Mg(2+) also modulates BK channels in multiple ways with opposite effects on channel function. Previous single-channel studies have shown that Mg(2+) blocks the pore of BK channels in a voltage-dependent manner. We have confirmed this result by studying macroscopic currents of the mslo1 channel. We find that Mg(2+) activates mslo1 BK channels independently of Ca(2+) and voltage by preferentially binding to their open conformation. The mslo3 channel, which lacks Ca(2+) binding sites in the tail, is not activated by Mg(2+). However, coexpression of the mslo1 core and mslo3 tail produces channels with Mg(2+) sensitivity similar to mslo1 channels, indicating that Mg(2+) sites differ from Ca(2+) sites. We discovered that Mg(2+) also binds to Ca(2+) sites and competitively inhibits Ca(2+)-dependent activation. Quantitative computation of these effects reveals that the overall effect of Mg(2+) under physiological conditions is to enhance BK channel function.     

Enhancing ethanol tolerance of a self-flocculating fusant of Schizosaccharomyces pombe and Saccharomyces cerevisiae by Mg2+ via reduction in plasma membrane permeability

Mg²⁺ at 3.5 mM increased the tolerance of a self-flocculating fusant of Schizosaccharomyces pombe and Saccharomyces cerevisiae to ethanol. After 9 h of exposure to 20% (v/v) ethanol at 30 °C, all cells died whereas over 50% remained viable for the cells grown with Mg²⁺. The effect of Mg²⁺ is closely related to its ability to decrease plasma membrane permeability of cells subjected to ethanol stress.     

Activation of AMPA/kainate receptors but not acetylcholine receptors causes Mg2+ influx into Retzius neurones of the leech Hirudo medicinalis

In Retzius neurones of the medicinal leech, Hirudo medicinalis, kainate activates ionotropic glutamate receptors classified as AMPA/kainate receptors. Activation of the AMPA/kainate receptor-coupled cation channels evokes a marked depolarization, intracellular acidification, and increases in the intracellular concentrations of Na+ ([Na+]i) and Ca2+. Qualitatively similar changes are observed upon the application of carbachol, an activator of acetylcholine receptor-coupled cation channels. Using multibarrelled ion-selective microelectrodes it was demonstrated that kainate, but not carbachol, caused additional increases in the intracellular free Mg2+ concentration ([Mg2+]i). Experiments were designed to investigate whether this kainate-induced [Mg2+]i increase was due to a direct Mg2+ influx through the AMPA/kainate receptor-coupled cation channels or a secondary effect due to the depolarization or the ionic changes. It was found that: (a) Similar [Mg2+]i increases were evoked by the application of glutamate or aspartate. (b) All kainate-induced effects were inhibited by the glutamatergic antagonist DNQX. (c) The magnitude of the [Mg2+]i increases depended on the extracellular Mg2+ concentration. (d) A reduction of the extracellular Ca2+ concentration increased kainate-induced [Mg2+]i increases, excluding possible Ca2+ interference at the Mg2+-selective microelectrode or at intracellular buffer sites. (e) Neither depolarizations evoked by the application of 30 mM K+, nor [Na+]i increases induced by the inhibition of the Na+/K+ ATPase caused comparable [Mg2+]i increases. (f) Inhibitors of voltage-dependent Ca2+ channels did not affect the kainate-induced [Mg2+]i increases. Moreover, previous experiments had already shown that intracellular acidification evoked by the application of 20 mM propionate did not cause changes in [Mg2+]i. The results indicate that kainate-induced [Mg2+]i increases in leech Retzius neurones are due to an influx of extracellular Mg2+ through the AMPA/kainate receptor-coupled cation channel. Mg2+ may thus act as an intracellular signal to distinguish between glutamatergic and cholinergic activation of leech Retzius neurones.     

Na(+) occupancy and Mg(2+) block of the n-methyl-d-aspartate receptor channel

The effect of extracellular and intracellular Na(+) on the single-channel kinetics of Mg(2+) block was studied in recombinant NR1-NR2B NMDA receptor channels. Na(+) prevents Mg(2+) access to its blocking site by occupying two sites in the external portion of the permeation pathway. The occupancy of these sites by intracellular, but not extracellular, Na(+) is voltage-dependent. In the absence of competing ions, Mg(2+) binds rapidly (>10(8) M(-1)s(-1), with no membrane potential) to a site that is located 0.60 through the electric field from the extracellular surface. Occupancy of one of the external sites by Na(+) may be sufficient to prevent Mg(2+) dissociation from the channel back to the extracellular compartment. With no membrane potential; and in the absence of competing ions, the Mg(2+) dissociation rate constant is >10 times greater than the Mg(2+) permeation rate constant, and the Mg(2+) equilibrium dissociation constant is approximately 12 microM. Physiological concentrations of extracellular Na(+) reduce the Mg(2+) association rate constant approximately 40-fold but, because of the "lock-in" effect, reduce the Mg(2+) equilibrium dissociation constant only approximately 18-fold.     

Influence of monovalent ions on the activity of the (Ca2+ + Mg2+)-ATPase and Ca2+-transport of human red blood cells

In reconstituted human red blood cells a difference was found in (Ca²⁺ + Mg²⁺)-ATPase activity and in Ca²⁺ efflux at 37°C, depending on the side of the membrane at which the monovalent cations K⁺ and Na⁺ were placed. Under the conditions used, (Ca²⁺ + Mg²⁺)-ATPase activity and Ca²⁺ efflux was highest when K⁺ ($\text{35 ± 0.5}\text{mM (± S.E.)}$, mean of four experiments) was at the inside and Na⁺ (130 mM) at the outside of the ghost membrane.     

Properties of an ATP-dependent Ca2+ pump and (Ca2+ + Mg2+)-ATPase in brain synaptosome membrane vesicles from the bertha armyworm,Mamestra configurata Wlk

Biochemical and kinetic properties under identical substrate and reaction conditions were obtained for an ATP-dependent Ca²⁺ pump and (Ca²⁺ + Mg²⁺)-ATPase in synaptosome membrane vesicles prepared from the brain of the moth, Mamestra configurata. Both the ATP-dependent Ca²⁺ pump and (Ca²⁺ + Mg²⁺)-ATPase had single, high-affinity binding sites for ATP (K_m = 14 and 116 μM, respectively), Ca²⁺_free (K_m = 0.13 nM and 0.072 nM, respectively), and Mg²⁺ (K_m = 1.1 mM and 0.07 mM, respectively). Both systems were relatively little affected by K⁺ and were insensitive to ouabain, an inhibitor of (Na⁺ + K⁺)-ATPase. The results indicate that the ATP-dependent Ca²⁺ pump and (Ca²⁺ + Mg²⁺)-ATPase are functionally coupled in synaptic membranes and constitute a mechanism for Ca²⁺ transport in the brain of M. configurata. Although moth brain (Ca²⁺ + Mg²⁺)-ATPase is maximally active at nanomolar concentrations of free calcium ion, the enzyme retains at least one-half of its maximal activity at micromolar calcium concentrations, indicating either that the enzyme has two binding sites for calcium (a high-affinity site at nanomolar Ca²⁺_free and a low-affinity site at micromolar Ca²⁺_free), or that there are two enzymes with high and low affinity for calcium, respectively. Calcium extrusion from brain neurones of M. configurata may operate in a two-stage, concentration-dependent process in which a first stage, low-affinity pump reduces intraneuronal calcium to a concentration at which a second stage, high-affinity pump becomes activated.     

SIMS Investigation of the Distribution of K+, Na+, Mg2+ and Ca2+ Cations in Birch Pollen

Two microanalytical techniques were used to investigate the inorganic cation content and distributions in birch (Betula verrucosa Ehrh.) pollen. With intact pollen grains. X-ray microanalysis (EDX) could only give a mean ionic composition. Secondary Ion Microscopy and Spectrometry (SIMS) appeared to be a more suitable technique to image ion distributions in the different pollen structures. This was carried out with samples prepared using a new vapour phase technique designed to improve ion retention. Transmission electron microscopy (TEM)showed good structural preservation of the samples. Monovalent ion (K⁺, Na⁺) distribution showed features different from those of the divalent cations (Ca²⁺, Mg²⁺). In the vegetative cell, the alkaline cations were mainly distributed in the most internal part of the cytoplasm and they were probably associated with starch grains or concentrated in dry vacuoles. Calcium distribution correlated well with the areas in the cytoplasm of the vegetative cell containing a dense network of mitochondria and endoplasmic reticulum. Within the pollen grain, the sperm cell appeared to contain the most calcium. Calcium was also abundant in the sporoderm. These results reveal the potential of SIMS for pollen studies that include germination, the monitoring of air pollutants and the allergens-ion interactions.     

Mechanistic insights from the structures of HincII bound to cognate DNA cleaved from addition of Mg2+ and Mn2+

The three-dimensional X-ray crystal structures of HincII bound to cognate DNA containing GTCGAC and Mn(2+) or Mg(2+), at 2.50A and 2.95A resolution, respectively, are presented. In both structures, the DNA is found cleaved, and the positions of the active-site groups, cleaved phosphate group, and 3' oxygen atom of the leaving group are in very similar positions. Two highly occupied Mn(2+) positions are found in each active site of the four crystallographically independent subunit copies in the HincII/DNA/Mn(2+) structure. The manganese ion closest to the previously identified single Ca(2+) position of HincII is shifted 1.7A and has lost direct ligation to the active-site aspartate residue, Asp127. A Mn(2+)-ligated water molecule in a position analogous to that seen in the HincII/DNA/Ca(2+) structure, and proposed to be the attacking nucleophile, is beyond hydrogen bonding distance from the active-site lysine residue, Lys129, but remains within hydrogen bonding distance from the proRp oxygen atom of the phosphate group 3' to the scissile phosphate group. In addition, the position of the cleaved phosphate group is on the opposite side of the axis connecting the two metal ions relative to that found in the BamHI/product DNA/Mn(2+) structure. Mechanistic implications are discussed, and a model for the two-metal-ion mechanism of DNA cleavage by HincII is proposed.     

Effect of Extracellular Ca2+ and Ca2+-Antagonists on the Movement and Chemoorientation of Male Gametes of Ectocarpus siliculosus (Phaeophyceae)

Chemoorientation in male gametes of Ectocarpus siliculosus in response to sexual pheromones is effected by two distinct mechanisms, chemokinesis and chemoklinotaxis. These are characterized by a strongly asymmetric bending pattern of the anteriorly-directed flagellum and transient unilateral bending of the hind flagellum, respectively. Removal of extracellular Ca²⁺ showed that normal flagellar movement and chemokinesis require millimolar concentrations of Ca²⁺ in the medium. The response to pheromones is strongly inhibited by La³⁺, whereas the Ca²⁺-channel drugs, verapamil and nifedipine, have only little effect. Nifedipine nethertheless effectively inhibited accumulation at pheromone sources. These results are interpreted as an indication for the involvement of two pharmacologically distinct Ca²⁺-channels in chemokinesis and chemoklinotaxis. The calmodulin-antagonist, trifluoperazine, induces, at low concentrations, the same flagellar response in chemokinesis as the pheromone, the mechanism of action remaining unknown.     

Designing a Highly Efficient Refolding System for Alkaline Phosphatase using Combination of Cyclodextrin and Mg<Superscript>2+</Superscript> Ion

Two different folding aids including α-cyclodextrin and Mg²⁺ ions were applied to alkaline phosphatase refolding. The refolding yield depending on concentration of each of the refolding agents reached to 55 and 52% in the presence of 100 mM α-cyclodextrin and/or 5 mM Mg²⁺ ions, respectively. However, the refolding yield, mediated by combination of α-cyclodextrin and Mg²⁺ ions, was more than 96%. Replacement of Mg²⁺ ion with other type of ions which interact with α-cyclodextrin interfere with the function of cyclodextrin resulting in low refolding yields.     

Association and dissociation kinetics for CheY interacting with the P2 domain of CheA

The chemotaxis system of Escherichia coli makes use of an extended two-component sensory response pathway in which CheA, an autophosphorylating protein histidine kinase (PHK) rapidly passes its phosphoryl group to CheY, a phospho-accepting response regulator protein (RR). The CheA-->CheY phospho-transfer reaction is 100-1000 times faster than the His-->Asp phospho-relays that operate in other (non-chemotaxis) two-component regulatory systems, suggesting that CheA and CheY have unique features that enhance His-->Asp phospho-transfer kinetics. One such feature could be the P2 domain of CheA. P2 encompasses a binding site for CheY, but an analogous RR-binding domain is not found in other PHKs. In previous work, we removed P2 from CheA, and this decreased the catalytic efficiency of CheA-->CheY phospho-transfer by a factor of 50-100. Here we examined the kinetics of the binding interactions between CheY and P2. The rapid association reaction (k(assn) approximately 10(8)M(-1)s(-1) at 25 degrees C and micro=0.03 M) exhibited a simple first-order dependence on P2 concentration and appeared to be largely diffusion-limited. Ionic strength (micro) had a moderate effect on k(assn) in a manner predictable based on the calculated electrostatic interaction energy of the protein binding surfaces and the expected Debye-Hückel shielding. The speed of binding reflects, in part, electrostatic interactions, but there is also an important contribution from the inherent plasticity of the complex and the resulting flexibility that this allows during the process of complex formation. Our results support the idea that the P2 domain of CheA contributes to the overall speed of phospho-transfer by promoting rapid association between CheY and CheA. However, this alone does not account for the ability of the chemotaxis system to operate much more rapidly than other two-component systems: k(cat) differences indicate that CheA and CheY also achieve the chemical events of phospho-transfer more rapidly than do PHK-RR pairs of slower systems.     

Cellular expression and alternative splicing of SLC25A23, a member of the mitochondrial Ca2+-dependent solute carrier gene family

Phospholipase D (PLD) is a ubiquitous enzyme in eukaryotes that participates in various cellular processes. Its catalytic domain is characterized by two HKD motifs in the C-terminal part. Until now, two subfamilies were recognized based on their N-terminal domain structure. The first has a PX domain in combination with a PH domain and is designated as PXPH-PLD. Members of the second subfamily, named C2-PLD, have a C2 domain and have, so far, only been found in plants. Here we describe a novel PLD subfamily that we identified in Phytophthora, a genus belonging to the class oomycetes and comprising many important plant pathogens. We cloned Pipld1 from Phytophthora infestans and retrieved full-length sequences of its homologues from Phytophthora sojae and Phytophthora ramorum genome databases. Their promoters contain two putative regulatory elements, one of which is highly conserved in all three genes. The three Phytophthora pld1 genes encode nearly identical proteins of around 1807 amino acids, with the two characteristic HKD motifs in the C-terminal part. Homology of the predicted proteins with known PLDs however is restricted to the two catalytic HKD motifs and adjacent domains. In the N-terminal part Phytophthora PLD1 has a PX-like domain, but it lacks a PH domain. Instead the N-terminal region contains five putative membrane spanning domains suggesting that Phytophthora PLD1 is a transmembrane protein. Since Phytophthora PLD1 cannot be categorized in one of the two existing subfamilies we propose to create a novel subfamily named PXTM-PLD.     

烟草TTG2蛋白质对NPRI介导的抗病防卫反应与ARF8影响的生长发育进行交叉调控的机制

NPR1蛋白是水杨酸信号和系统获得性抗性的转录调节因子,它的功能受蛋白质降解酶体CUL3．E3的控制。植物的发育主要受生长素信号通路的控制,生长素反应因（ARF）参与生长素信号转导转录调控。植物转录因于NPR1和ARF8分别在蛋白质降解酶体CUL3-E3与CUL1-E3控制下,调控抗病防卫与生长发育。烟草TTG2促进ARF8从细胞质向细胞核转运及其转录调控作用,因此促进生长发育;相反,TTG2把NPR1扣留在细胞质,阻止它对防卫反应基因的转录调控作用,从而抑制抗病性。TTG2与NPR1或ARF8并不直接互作,说明存在协助因子。     

Ca2+激活Cl-通道功能及分子基础研究进展

自从1983年Barish在爪蟾卵母细胞中发现钙激活的Cl–通道以来,此种类型Cl–通道一直在被广泛的研究,其在不同组织中的重要作用也被不断报道。但是,钙激活氯电流的分子机制一直未被阐明。直到2008年,由三个实验室分别发现了构成钙激活Cl–通道的分子基础为跨膜蛋白16A(transmembrane protein 16A,TMEM16A),这一发现使得人为通过基因手段调控钙激活Cl–通道的功能与表达成为可能。该文综述了钙激活Cl–通道在不同组织中的作用、TMEM16A的电生理和药理学特性以及TMEM16A在心肌肥厚和心衰中的可能作用,以及以Cl–通道作为药物作用靶点的研究进展。     

Characterization of the C-terminal half of human juvenile myoclonic epilepsy protein EFHC1: dimer formation blocks Ca2+ and Mg2+ binding to its functional EF-hand

Human EFHC1 is a member of the EF-hand superfamily of Ca²⁺-binding proteins with three DM10 domains of unclear function. Point mutations in the EFHC1 gene are related to juvenile myoclonic epilepsy, a fairly common idiopathic generalized epilepsy. Here, we report the first structural and thermodynamic analyses of the EFHC1C-terminus (residues 403-640; named EFHC1C), comprising the last DM10 domain and the EF-hand motif. Circular dichroism spectroscopy revealed that the secondary structure of EFHC1C is composed by 34% of α-helices and 17% of β-strands. Size exclusion chromatography and mass spectrometry showed that under oxidizing condition EFHC1C dimerizes through the formation of disulfide bond. Tandem mass spectrometry (MS/MS) analysis of peptides generated by trypsin digestion suggests that the Cys575 is involved in intermolecular S-S bond. In addition, DTNB assay showed that each reduced EFHC1C molecule has one accessible free thiol. Isothermal titration calorimetry (ITC) showed that while the interaction between Ca²⁺ and EFHC1C is enthalpically driven (ΔH = −58.6 to −67 kJ/mol and TΔS = −22.5 to −31 kJ/mol) the interaction between Mg²⁺ and EFHC1C involves an entropic gain, and is ∼5 times less enthalpically favorable (ΔH = −11.7 to −14 kJ/mol and TΔS = 21.9 to 19 kJ/mol) than for Ca²⁺ binding. It was also found that under reducing condition Ca²⁺ or Mg²⁺ ions bind to EFHC1C in a 1/1 molar ratio, while under oxidizing condition this ratio is reduced, showing that EFHC1C dimerization blocks Ca²⁺ and Mg²⁺ binding.     

Heparan sulfate is required for interaction and activation of the epithelial cell fibroblast growth factor receptor-2IIIb with stromal-derived fibroblast growth factor-7

Summary Fibroblast growth factor-7 (FGF-7) and a specific splice variant of the FGF tyrosine kinase receptor family (FGFR2IIIb) constitute a paracrine signaling system from stroma to epithelium. Different effects of the manipulation of cellular heparan sulfates and heparin on activities of FGF-7 relative to FGF-1 in epithelial cells suggest that pericellular heparan sulfates may regulate the activity of FGF-7 by a different mechanism than other FGFs. In this report, we employ the heparan sulfate-binding protein, protamine sulfate, to reversibly block cellular heparan sulfates. Protamine sulfate, which does not bind significantly to FGF-7 or FGFR2IIIb, inhibited FGF-7 activities, but not those of epidermal growth factor. The inhibition was overcome by increasing the concentrations of FGF-7 or heparin. Heparin was essential for binding of FGF-7 to recombinant FGFR2IIIb expressed in insect cells or FGFR2IIIb purified away from cell products. These results suggest that, similar to other FGF polypeptides, heparan sulfate within the pericellular matrix is required for activity of FGF-7. Differences in response to heparin and alterations in the BULK heparan sulfate content of cells likely reflect FGF-specific differences in the cellular repertoire of multivalent heparan sulfate chains required for assembly and activation of the FGF signal transduction complex.     

Two members of the TRPP family of ion channels, Pkd1l3 and Pkd2l1, are co-expressed in a subset of taste receptor cells

Taste receptors cells are responsible for detecting a wide variety of chemical stimuli. Several molecules including both G protein coupled receptors and ion channels have been shown to be involved in the detection and transduction of tastants. We report on the expression of two members of the transient receptor potential (TRP) family of ion channels, PKD1L3 and PKD2L1, in taste receptor cells. Both of these channels belong to the larger polycystic kidney disease (PKD or TRPP) subfamily of TRP channels, members of which have been demonstrated to be non-selective cation channels and permeable to both Na(+) and Ca(2+). Pkd1l3 and Pkd2l1 are co-expressed in a select subset of taste receptor cells and therefore may, like other PKD channels, function as a heteromer. We found the taste receptor cells expressing Pkd1l3 and Pkd2l1 to be distinct from those that express components of sweet, bitter and umami signal transduction pathways. These results provide the first evidence for a role of TRPP channels in taste receptor cell function.