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Scientific Reports Apr 2019Citrate synthase (CS, EC 2.3.3.1) catalyses the initial reaction of the tricarboxylic acid (TCA) cycle. Although CSs from heterotrophic bacteria have been extensively...
Citrate synthase (CS, EC 2.3.3.1) catalyses the initial reaction of the tricarboxylic acid (TCA) cycle. Although CSs from heterotrophic bacteria have been extensively studied, cyanobacterial CSs are not well-understood. Cyanobacteria can produce various metabolites from carbon dioxide. Synechocystis sp. PCC 6803 (Synechocystis 6803) is a cyanobacterium used to synthesize metabolites through metabolic engineering techniques. The production of acetyl-CoA-derived metabolites in Synechocystis 6803 has been widely examined. However, the biochemical mechanisms of reactions involving acetyl-CoA in Synechocystis 6803 are poorly understood. We characterised the CS from Synechocystis 6803 (SyCS) and compared its characteristics with other bacterial CSs. SyCS catalysed only the generation of citrate, and did not catalyse the cleavage of citrate. It is suggested that SyCS is not related to the reductive TCA cycle. The substrate affinity and turnover number of SyCS were lower than those of CSs from heterotrophic bacteria. SyCS was activated by MgCl and CaCl, which inhibit various bacterial CSs. SyCS was not inhibited by ATP and NADH; which are typical feedback inhibitors of other bacterial CSs. SyCS was inhibited by phosphoenolpyruvate and activated by ADP, which has not been reported for CSs from heterotrophic bacteria. Thus, SyCS showed unique characteristics, particularly its sensitivity to effectors.
Topics: Acetyl Coenzyme A; Adenosine Triphosphate; Bacterial Proteins; Calcium Chloride; Carbon Dioxide; Citrate (si)-Synthase; Citric Acid; Citric Acid Cycle; Enzyme Activation; Magnesium Chloride; Synechocystis
PubMed: 30988396
DOI: 10.1038/s41598-019-42659-z -
Cytometry. Part a : the Journal of the... May 2010Effects of some detergents-most frequently used in membrane raft studies-on the polymerization properties of actin were examined under in vitro and in vivo conditions,...
Effects of some detergents-most frequently used in membrane raft studies-on the polymerization properties of actin were examined under in vitro and in vivo conditions, for protein and cellular investigations, respectively. Under in vitro conditions the polymerization rates were measured with pyrene-labeled actin. We found that polymerization rate depended on the detergent concentration by following either biphasic characteristics or only decreasing tendency. The strongest effects were observed at relatively low detergent concentrations. SDS-PAGE electrophoresis and dynamic light-scattering measurements provided further evidences for the size distribution of actin filaments formed under the influence of detergents. Comparing the polymerization rates measured in the presence of different detergents to those obtained with various magnesium and KCl concentrations showed that detergents may influence the actin polymerization at three levels by modifying: (i) the monomer-monomer interaction, (ii) the local ionic strength, and (iii) the affinity of actin for various cations. In vivo studies on NIH 3T3MDR1 cells using TRITC-phalloidin detected fast depolymerization of large extent around the critical micellar concentrations of the detergents. We concluded that microdomain insolubility observed in the presence of detergents is hardly to be the result of the stabilization of the submembrane actin cytoskeleton merely; rather inter-lipid and lipid-protein interactions are also involved within the detergent-resistant membranes.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Actin Cytoskeleton; Actins; Animals; Detergents; Electrophoresis, Polyacrylamide Gel; Magnesium Chloride; Mice; NIH 3T3 Cells; Potassium Chloride; Rabbits
PubMed: 20151434
DOI: 10.1002/cyto.a.20855 -
Scientific Reports Feb 2015We report our studies on the adsorption properties of double-stranded DNA molecules on mica surfaces in a confined environment using a surface force apparatus....
We report our studies on the adsorption properties of double-stranded DNA molecules on mica surfaces in a confined environment using a surface force apparatus. Specifically, we studied the influence of cation species and concentrations on DNA adsorption properties. Our results indicated that divalent cations (Mg(2+) and Co(2+)) preferred to form uniform and moderately dense DNA layers on a mica substrate. By measuring the interactions between DNA-coated mica and bare mica in an aqueous solution, obvious adhesion was observed in a cobalt chloride solution, possibly due to the ion-correlation attraction between negatively charged DNA and the mica surface. Furthermore, the interaction differences that were observed with MgCl2 and CoCl2 solutions reveal that the specific adsorption behaviors of DNA molecules on a mica substrate were mediated by these two salts. Our results are helpful to elucidate the dynamics of DNA binding on a solid substrate.
Topics: Adsorption; Aluminum Silicates; Cations; Cobalt; DNA; Magnesium Chloride; Surface Properties
PubMed: 25676333
DOI: 10.1038/srep08442 -
Nucleic Acids Research Mar 2012A novel isothermal titration calorimetry (ITC) method was applied to investigate RNA helical packing driven by the GAAA tetraloop-receptor interaction in magnesium and...
A novel isothermal titration calorimetry (ITC) method was applied to investigate RNA helical packing driven by the GAAA tetraloop-receptor interaction in magnesium and potassium solutions. Both the kinetics and thermodynamics were obtained in individual ITC experiments, and analysis of the kinetic data over a range of temperatures provided Arrhenius activation energies (ΔH(‡)) and Eyring transition state entropies (ΔS(‡)). The resulting rich dataset reveals strongly contrasting kinetic and thermodynamic profiles for this RNA folding system when stabilized by potassium versus magnesium. In potassium, association is highly exothermic (ΔH(25°C) = -41.6 ± 1.2 kcal/mol in 150 mM KCl) and the transition state is enthalpically barrierless (ΔH(‡) = -0.6 ± 0.5). These parameters are significantly positively shifted in magnesium (ΔH(25°C) = -20.5 ± 2.1 kcal/mol, ΔH(‡) = 7.3 ± 2.2 kcal/mol in 0.5 mM MgCl(2)). Mixed salt solutions approximating physiological conditions exhibit an intermediate thermodynamic character. The cation-dependent thermodynamic landscape may reflect either a salt-dependent unbound receptor conformation, or alternatively and more generally, it may reflect a small per-cation enthalpic penalty associated with folding-coupled magnesium uptake.
Topics: Calorimetry; Kinetics; Magnesium Chloride; Models, Molecular; Potassium Chloride; RNA Folding; Thermodynamics
PubMed: 22058128
DOI: 10.1093/nar/gkr894 -
International Journal of Molecular... Dec 2020HU is a nucleoid-associated protein expressed in most eubacteria at a high amount of copies (tens of thousands). The protein is believed to bind across the genome to...
HU is a nucleoid-associated protein expressed in most eubacteria at a high amount of copies (tens of thousands). The protein is believed to bind across the genome to organize and compact the DNA. Most of the studies on HU have been carried out in a simple in vitro system, and to what extent these observations can be extrapolated to a living cell is unclear. In this study, we investigate the DNA binding properties of HU under conditions approximating physiological ones. We report that these properties are influenced by both macromolecular crowding and salt conditions. We use three different crowding agents (blotting grade blocker (BGB), bovine serum albumin (BSA), and polyethylene glycol 8000 (PEG8000)) as well as two different MgCl conditions to mimic the intracellular environment. Using tethered particle motion (TPM), we show that the transition between two binding regimes, compaction and extension of the HU protein, is strongly affected by crowding agents. Our observations suggest that magnesium ions enhance the compaction of HU-DNA and suppress filamentation, while BGB and BSA increase the local concentration of the HU protein by more than 4-fold. Moreover, BGB and BSA seem to suppress filament formation. On the other hand, PEG8000 is not a good crowding agent for concentrations above 9% (/), because it might interact with DNA, the protein, and/or surfaces. Together, these results reveal a complex interplay between the HU protein and the various crowding agents that should be taken into consideration when using crowding agents to mimic an in vivo system.
Topics: Algorithms; Bacterial Proteins; DNA; DNA, Bacterial; DNA-Binding Proteins; Magnesium Chloride; Models, Molecular; Models, Theoretical; Polyethylene Glycols; Protein Binding; Protein Conformation
PubMed: 33334011
DOI: 10.3390/ijms21249553 -
Proceedings of the National Academy of... Jan 2012Dynamic RNA molecules carry out essential processes in the cell including translation and splicing. Base-pair interactions stabilize RNA into relatively rigid...
Dynamic RNA molecules carry out essential processes in the cell including translation and splicing. Base-pair interactions stabilize RNA into relatively rigid structures, while flexible non-base-paired regions allow RNA to undergo conformational changes required for function. To advance our understanding of RNA folding and dynamics it is critical to know the flexibility of these un-base-paired regions and how it depends on counterions. Yet, information about nucleic acid polymer properties is mainly derived from studies of ssDNA. Here we measure the persistence lengths (l(p)) of ssRNA. We observe valence and ionic strength-dependent differences in l(p) in a direct comparison between 40-mers of deoxythymidylate (dT(40)) and uridylate (rU(40)) measured using the powerful combination of SAXS and smFRET. We also show that nucleic acid flexibility is influenced by local environment (an adjoining double helix). Our results illustrate the complex interplay between conformation and ion environment that modulates nucleic acid function in vivo.
Topics: DNA, Single-Stranded; Fluorescence Resonance Energy Transfer; Ions; Magnesium Chloride; Models, Molecular; Osmolar Concentration; Pliability; RNA; Scattering, Small Angle; Sodium Chloride; X-Ray Diffraction
PubMed: 22203973
DOI: 10.1073/pnas.1119057109 -
International Journal of Molecular... May 2024The skin wound healing process consists of hemostatic, inflammatory, proliferative, and maturation phases, with a complex cellular response by multiple cell types in the...
The skin wound healing process consists of hemostatic, inflammatory, proliferative, and maturation phases, with a complex cellular response by multiple cell types in the epidermis, dermis, and immune system. Magnesium is a mineral essential for life, and although magnesium treatment promotes cutaneous wound healing, the molecular mechanism and timing of action of the healing process are unknown. This study, using human epidermal-derived HaCaT cells and human normal epidermal keratinocyte cells, was performed to investigate the mechanism involved in the effect of magnesium on wound healing. The expression levels of epidermal differentiation-promoting factors were reduced by MgCl, suggesting an inhibitory effect on epidermal differentiation in the remodeling stage of the late wound healing process. On the other hand, MgCl treatment increased the expression of matrix metalloproteinase-7 (MMP7), a cell migration-promoting factor, and enhanced cell migration via the MEK/ERK pathway activation. The enhancement of cell migration by MgCl was inhibited by MMP7 knockdown, suggesting that MgCl enhances cell migration which is mediated by increased MMP7 expression. Our results revealed that MgCl inhibits epidermal differentiation but promotes cell migration, suggesting that applying magnesium to the early wound healing process could be beneficial.
Topics: Wound Healing; Humans; Cell Movement; Keratinocytes; Cell Differentiation; Magnesium; Matrix Metalloproteinase 7; Skin; MAP Kinase Signaling System; Cell Line; Epidermis; Magnesium Chloride
PubMed: 38732212
DOI: 10.3390/ijms25094994 -
Nucleic Acids Research Nov 2016Non-coding RNAs must fold into specific structures that are stabilized by metal ions and other co-solutes in the cell's interior. Large crowder molecules such as PEG...
Non-coding RNAs must fold into specific structures that are stabilized by metal ions and other co-solutes in the cell's interior. Large crowder molecules such as PEG stabilize a bacterial group I ribozyme so that the RNA folds in low Mg concentrations typical of the cell's interior. To understand the thermodynamic origins of stabilization by crowder molecules, small angle X-ray scattering was used to measure the folding and helix assembly of a bacterial group I ribozyme at different temperatures and in different MgCl and polyethylene glycol (PEG) concentrations. The resulting phase diagrams show that perturbations to folding by each variable do not overlap. A favorable enthalpy change drives the formation of compact, native-like structures, but requires Mg ions at all temperatures studied (5-55°C). PEG reduces the entropic cost of helix assembly and increases correlations between RNA segments at all temperatures. The phase diagrams also revealed a semi-compact intermediate between the unfolded and folded ensemble that is locally more flexible than the unfolded state, as judged by SHAPE modification. These results suggest that environmental variables such as temperature and solute density will favor different types of RNA structures.
Topics: Azoarcus; Entropy; Magnesium Chloride; Nucleic Acid Conformation; Nucleotides; Polyethylene Glycols; RNA; RNA Folding; RNA, Bacterial; Scattering, Small Angle; Solutions; Temperature; Thermodynamics; X-Ray Diffraction
PubMed: 27378777
DOI: 10.1093/nar/gkw597 -
British Journal of Haematology Jan 2008Platelet protein disulphide isomerase (PDI) has a role in platelet aggregation, probably targeting a thiol-containing platelet surface protein. The thiol-containing...
Platelet protein disulphide isomerase (PDI) has a role in platelet aggregation, probably targeting a thiol-containing platelet surface protein. The thiol-containing P2Y(12) ADP receptor is involved in aggregation induced by most agonists and may be the target of PDI. By excluding the P2Y(12) pathway and using the anti-PDI antibody RL90 this study showed that PDI targets a non-P2Y(12) thiol-protein in aggregation. Anti-PDI inhibited signalling-independent activation of the thiol-containing fibrinogen receptor alphaIIbbeta3 by Mn(2+), suggesting that PDI directly interacts with alphaIIbbeta3. The thiol-containing form of PDI increased on the platelet surface with platelet activation, suggesting that active PDI readily becomes available for redox regulation of alphaIIbbeta3. Finally, using purified proteins PDI had greater ability to isomerize disulphide bonds than the alphaIIbbeta3 integrin, which also has PDI-like activity. In summary, a mechanism exists in platelets to increase the functional form of surface PDI and this PDI has a non-P2Y(12) target that may be alphaIIbbeta3.
Topics: Antibodies, Monoclonal; Blood Platelets; Cells, Cultured; Humans; Magnesium Chloride; Platelet Activation; Platelet Aggregation; Platelet Glycoprotein GPIIb-IIIa Complex; Protein Disulfide-Isomerases; Ribonucleases; Signal Transduction
PubMed: 18028487
DOI: 10.1111/j.1365-2141.2007.06898.x -
Calcified Tissue International Sep 1996The present study further characterizes an extract from immature, human tooth apicies from which an intact dentin phosphoprotein has been identified. Third molar apicies... (Comparative Study)
Comparative Study
The present study further characterizes an extract from immature, human tooth apicies from which an intact dentin phosphoprotein has been identified. Third molar apicies from developing roots were decalcified in 10% EDTA until Ca2+ was undetectable in the decalcifying solution. The crude extract was run on 7.5% SDS-PAGE and stained with "Stains-All." Four distinct bands were found and the molecular weights were 140, 60, 50, and 34 k. When run on a SDS-PAGE under nonreducing conditions the 60, 50, and 34 k bands were absent. These results suggest that the lower molecular weight bands may be subunits of the larger protein. The extract was then further purified by adding CaCl2 and MgCl2 to precipitate the phosphoprotein. The precipitate was subjected to a DEAE-Sepharose CL6B column and eluted by 0-0.7 M NaCl gradient solution. The amino acid composition of the purified phosphoprotein was determined and the extract was found to be rich in serine and aspartic acid residues. The N-terminal peptide Asp-Asp-Pro was identified. The sequence of the three amino acids is identical to rat incisor phosphoprotein.
Topics: Amino Acid Sequence; Amino Acids; Animals; Calcium Chloride; Carbohydrate Metabolism; Dentin; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Humans; Incisor; Magnesium Chloride; Molar, Third; Molecular Sequence Data; Molecular Weight; N-Acetylneuraminic Acid; Phosphoproteins; Rats; Sialic Acids; Staining and Labeling; Tooth Root
PubMed: 8694890
DOI: 10.1007/s002239900101