Activation of membrane-bound high-affinity calcium ion-sensitive adenosine triphosphatase of human erythrocytes by bivalent metal ions.
Ontology highlight
ABSTRACT:
SUBMITTER: Pfleger H
PROVIDER: S-EPMC1165450 | biostudies-other | 1975 May
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Within the three-dimensional architectures of RNA molecules, divalent metal ions populate specific locations, shedding their water molecules to form chelates. These interactions help the RNA adopt and maintain specific conformations and frequently make essential contributions to function. Defining the locations of these site-bound metal ions remains challenging despite the growing database of RNA structures. Metal-ion rescue experiments have provided a powerful approach to identify and distinguish catalytic metal ions within RNA active sites, but the ability of such experiments to identify metal ions that contribute to tertiary structure acquisition and structural stability is less developed and has been challenged. Herein, we use the well-defined P4-P6 RNA domain of the Tetrahymena group I intron to reevaluate prior evidence against the discriminatory power of metal-ion rescue experiments and to advance thermodynamic descriptions necessary for interpreting these experiments. The approach successfully identifies ligands within the RNA that occupy the inner coordination sphere of divalent metal ions and distinguishes them from ligands that occupy the outer coordination sphere. Our results underscore the importance of obtaining complete folding isotherms and establishing and evaluating thermodynamic models in order to draw conclusions from metal-ion rescue experiments. These results establish metal-ion rescue as a rigorous tool for identifying and dissecting energetically important metal-ion interactions in RNAs that are noncatalytic but critical for RNA tertiary structure.
| S-EPMC3358636 | biostudies-literature
Project description:The binding of MgATP to purified Ca2+Mg2+-dependent adenosine triphosphatase from rabbit muscle sarcoplasmic reticulum was studied by using a flow-dialysis method. Phosphoryl-enzyme formation and catalytic activity were also measured, and all three processes demonstrated negative co-operativity, with half-saturation of all three parameters at a MgATP concentration of 40-50muM, and a Hill coefficient (h) of 0.8. The variation of the binding constant with with pH was measured and showed tighter binding of MgATP with increasing pH over the range 6.8-8.5. Binding parameters for ATP analogues were also measured. The binding of Ca2+ in the presence and absence of ATP analogues gave half saturation at a Ca2+ concentration of 1.2-1.3muM. Hill plots of Ca2+-binding data gave a slope of 0.8. These results show that the binding of MgATP and Ca2+ can occur in a random manner, with neither substrate influencing the affinity of the enzyme for the other.
| S-EPMC1164174 | biostudies-other
Project description:The K+-stimulated ATPase associated with the purified gastric microsomal fraction can be completely inactivated by treatment with 15% (v/v) ethanol for 60s at 37 degrees C, but not at 25 degrees C. Sequential exposure of the microsomal fraction to 15% ethanol at 25 degrees C and 37 degrees C caused release of 2.5% and 2.9% of the total membrane phospholipids respectively. Restoration of the enzyme activity was achieved by sonication with phosphatidylcholine in the presence of Mg2+, K+ and ATP, which were essential for the reconstitution. Our data suggest that the phospholipids extracted by 15% ethanol at 37 degrees C are derived primarily from the immediate lipid environment of the enzyme, and ATP, together with the metal ions, helps the partially delipidated enzyme to retain the appropriate configuration for the subsequent reconstitution.
| S-EPMC1161350 | biostudies-other
Project description:Bivalent metal ions have multiple roles in subunit association and ATPase regulation in scallop adductor-muscle myosin. To help elucidate these functions, the rates of Ca2+ and Mg2+ dissociation from the non-specific high-affinity sites on the regulatory light chains were measured and compared with those of rabbit skeletal-muscle myosin subfragments. Ca2+ dissociation had a rate constant of about 0.7 s-1 in both species, as measured by the time course of the pH change on EDTA addition. Mg2+ dissociation had a rate constant of 0.05 s-1, as monitored by its displacement with the paramagnetic Mn2+ ion. It is concluded that the exchange between Ca2+ and Mg2+ at the non-specific site, on excitation of both skeletal and adductor muscles, is too slow to contribute to the activation itself. The release of bivalent metal ions from the non-specific site is, however, the first step in release of the scallop regulatory light chain (Bennett & Bagshaw (1986) Biochem. J. 233, 179-186). In scallop myosin additional specific sites are present, which can bind Ca2+ rapidly, to effect activation of the ATPase. In the course of this work, Ca2+ dissociation from EGTA was studied as a model system. This gave rates of 1 s-1 and 0.3 s-1 at pH 7.0 and pH 8.0 respectively.
| S-EPMC1153000 | biostudies-other
Project description:The relative activity of a zinc-containing cyclic AMP phosphodiesterase towards the (Sp)- compared with the (Rp)-diastereoisomer of cyclic adensine phosphorothioate varied with the identity of the free bivalent metal ion from more than 35 to 0.074 along the series Mg2+ greater than Mn2+ greater than Co2+ greater than Zn2+ greater than Cd2+, showing that this ion, and not the tightly bound zinc, bonds to the phosphorothioate moiety of the substrate.
| S-EPMC1144795 | biostudies-other
Project description:Correctly calculating the structure of metal coordination sites in a protein during the process of nuclear magnetic resonance (NMR) structure determination and refinement continues to be a challenging task. In this study, we present an accurate and convenient means by which to include metal ions in the NMR structure determination process using molecular dynamics (MD) simulations constrained by NMR-derived data to obtain a realistic and physically viable description of the metal binding site(s). This method provides the framework to accurately portray the metal ions and its binding residues in a pseudo-bond or dummy-cation like approach, and is validated by quantum mechanical/molecular mechanical (QM/MM) MD calculations constrained by NMR-derived data. To illustrate this approach, we refine the zinc coordination complex structure of the zinc sensing transcriptional repressor protein Staphylococcus aureus CzrA, generating over 130 ns of MD and QM/MM MD NMR-data compliant sampling. In addition to refining the first coordination shell structure of the Zn(II) ion, this protocol benefits from being performed in a periodically replicated solvation environment including long-range electrostatics. We determine that unrestrained (not based on NMR data) MD simulations correlated to the NMR data in a time-averaged ensemble. The accurate solution structure ensemble of the metal-bound protein accurately describes the role of conformational sampling in allosteric regulation of DNA binding by zinc and serves to validate our previous unrestrained MD simulations of CzrA. This methodology has potentially broad applicability in the structure determination of metal ion bound proteins, protein folding and metal template protein-design studies.
| S-EPMC3773525 | biostudies-literature
Project description:Triphenylsulphonium ions inhibit mitochondrial oxidative phosphorylation and adenosine triphosphatase activity. The site of action is on the soluble F1 adenosine triphosphatase component. Triphenylsylphonium ions also inhibit electron transfer in the NAD-cytochrome b region of the respiratory chain. In both types of inhibition, triphenylsulphonium ions are effective at low concentrations, half-maximal inhibition being produced by a concentration of about 20-30 muM. These effects resemble the effects of alkylguanidines on mitochondria and are discussed in relation to the effects of alkylguanidines and other lipophilic cations such as ethidium and dibenzyldimethylammonium ions. A modification of the purification procedure for the soluble mitochondrial adenosine triphosphatase [Beechey, Hubbard, Linnett, Mitchell & Munn (1975) Biochem. J. 148, 533-537] IS DESCRIBED, WHICH YIELDS A PREPARATION WITH A HIGHER SPECIFIC ACTIVITY AND SHOWING FEWER BANDS IN GEL ELECTROPHORESIS.
| S-EPMC1163751 | biostudies-other
Project description:Metal ions play essential roles in many aspects of biological chemistry. Detecting their presence and location in proteins and cells is important for understanding biological function. Conventional structural methods such as X-ray crystallography and cryo-transmission electron microscopy can identify metal atoms on protein only if the protein structure is solved to atomic resolution. We demonstrate here the detection of isolated atoms of Zn and Fe on ferritin, using cryogenic annular dark-field scanning transmission electron microscopy (cryo-STEM) coupled with single-particle 3D reconstructions. Zn atoms are found in a pattern that matches precisely their location at the ferroxidase sites determined earlier by X-ray crystallography. By contrast, the Fe distribution is smeared along an arc corresponding to the proposed path from the ferroxidase sites to the mineral nucleation sites along the twofold axes. In this case the single-particle reconstruction is interpreted as a probability distribution function based on the average of individual locations. These results establish conditions for detection of isolated metal atoms in the broader context of electron cryo-microscopy and tomography.
| S-EPMC5651765 | biostudies-literature
Project description:Integrins are cell-surface heterodimeric proteins that mediate cell-cell, cell-matrix, and cell-pathogen interactions. Half of the known integrin alpha subunits contain inserted domains (I domains) that coordinate ligand through a metal ion. Although the importance of conformational changes within isolated I domains in regulating ligand binding has been reported, the relationship between metal ion binding affinity and ligand binding affinity has not been elucidated. Metal and ligand binding by several I domain mutants that are stabilized in different conformations are investigated using isothermal titration calorimetry and surface plasmon resonance studies. This work suggests an inverse relationship between metal ion affinity and ligand binding affinity (i.e. constructs with a high affinity for ligand exhibit a low affinity for metal). This trend is discussed in the context of structural studies to provide an understanding of interplay between metal ion binding and ligand affinities and conformational changes.
| S-EPMC2040231 | biostudies-literature
Project description:1. Titration curves of insulin in the presence and absence of various metal ions are reported. 2. The difference in base consumption with and without the metal ions is compared with calculated curves. 3. These experiments suggest that in dilute solutions Zn(2+) and Cu(2+) ions are bound to alpha-amino groups.
| S-EPMC1198580 | biostudies-other