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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mitochondrial ATPase from rat liver mitochondria contains multiple nucleotide binding sites. At low concentrations ADP binds with high affinity (1 mole/mole ATPase, KD = 1-2 muM). At high concentrations, ADP inhibits ATP hydrolysis presumably by competing with ATP for the active site (KI = 240-300 muM). As isolated, mitochondrial ATPase contains between 0.6 and 2.5 moles ATP/mole ATPase. This "tightly bound" ATP can be removed by repeated precipitations with ammonium sulfate without altering hydrolytic activity of the enzyme. However, the ATP-depleted enzyme must be redissolved in high concentrations of phosphate to retain activity. AMP-PNP (adenylyl imidodiphosphate) replaces tightly bound ATP removed from the enzyme and inhibits ATP hydrolysis. AMP-PNP has little effect on high affinity binding of ADP. Kinetics studies of ATP hydrolysis reveal hyperbolic velocity vs. ATP plots, provided assays are done in bicarbonate buffer or buffers containing high concentrations of phosphate. Taken together, these studies indicate that sites on the enzyme not directly associated with ATP hydrolysis bind ATP or ADP, and that in the absence of bound nucleotide, Pi can maintain the active form of the enzyme.
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PMID:Interaction of homogeneous mitochondrial ATPase from rat liver with adenine nucleotides and inorganic phosphate. 12 85

Tightly bound adenine nucleotides are removed from multiple binding sites on beef heart mitochondrial ATPase (F1) by chromatography on columns of Sephadex equilibrated with 50% glycerol. Release of nucleotides from the enzyme is associated with large decreases in sedimentation velocity (from 11.9 S to 8.4 S) which may be observed in concentrated solutions of polyols. Polyol-induced conformational changes are reversed when the enzyme is returned to dilute buffers. The nucleotide-depleted enzyme restores oxidative phosphorylation in F1-deficient submitochondrial particles. Reconstitution of nucleotide-depleted F1 with the ATP analog (adenylyl-imidodiphosphate (AMP-PNP), almost 5 moles of AMP-PNP per mole of enzyme, results in preparations with substantially inhibited ATPase activity which nevertheless restores oxidative phosphorylation and the 32Pi-ATP exchange reaction in F1-deficient submitochondrial particles. Incubation of the analog-labeled enzyme with ATP and Mg++ results in partial displacement of the analog and a time-dependent recovery of ATPase activity.
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PMID:Physical and enzymatic properties of nucleotide-depleted beef heart mitochondrial adenosine triphosphatase. 12 61

F1-ATPase of rat liver was examined for its capacity to interact with both metal ions and nucleotides and for the effect of covalent ATPase inhibitors on these interactions. As isolated, rat liver F1 contains about 2 mol of Mg2+/mol of F1, 1 mol of which can be removed or exchanged. The remaining mole of Mg2+ per mole of F1 remains very tightly associated with F1 and is recovered in the alpha gamma fraction after cold denaturation. Rat liver F1 also contains as isolated a nearly equivalent amount of nucleotide (approximately 1.7 mol/mol of F1) which is readily removed by incubation at room temperature followed by column centrifugation. The "2 Mg2+ enzyme" binds almost 3 mol of 5'-adenylyl imidodiphosphate (AMP-PNP)/mol of F1 in the presence or absence of added divalent cation. When divalent cation is present as Co2+, an equivalent activator to Mg2+ in the ATPase reaction, 1 mol of F1 binds 3 mol of both AMP-PNP and Co2+. under these conditions, the very tight Mg2+ site remains loaded, the exchangeable Mg2+ site is replaced with AMP-PNPCo, and two additional AMP-PNPCo sites are filled. At this point, ADP can be loaded onto the enzyme as a fourth nucleotide at a site separate and distinct from the AMP-PNP sites. Significantly, rat liver F1 contains only a single readily detectable ADP binding site in the presence or absence of divalent cation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Ligand binding studies of the F1 moiety of rat liver ATP synthase: implications about the enzyme's structure and mechanism. 288 76

ATP hydrolysis by the Escherichia coli F1 ATPase (ECF1) induces a conformational change in the gamma subunit. This change can be monitored by fluorescence changes in N-[4-[7-(diethylamino)-4-methyl]coumarin-3-yl)]maleimide (CM) bound at a cysteine introduced by site-directed mutagenesis into the gamma subunit at position 106 [Turina, P., & Capaldi, R. A. (1994) J. Biol. Chem. 269, 13465-13471]. In studies reported here, the magnitude of the fluorescence change has been determined with the noncleavable nucleotide analogue AMP-PNP and by rapid measurements using the slowly cleavable ATP gamma S. The data indicate that maximal fluorescence change occurs with binding of 1 mol of nucleotide triphosphate per mole of ECF1. During unisite catalysis, ATP binding causes a fluorescence enhancement from CM bound at position 106, which is then followed by fluorescence quenching. The kinetics of these fluorescence changes have been measured using both ATP and ATP gamma S as substrate. With ATP gamma S, these kinetics can be simulated using rate constants similar to those for ATP except for an approximately 30-fold slower rate of the bond cleavage and resynthesis steps, i.e., k+2 and k-2. The observed rates and amplitudes of the fluorescence changes on hydrolysis of ATP and ATP gamma S were analyzed by simulations in which the bond cleavage or the Pi release step was responsible for fluorescence quenching. The results indicate that ATP or ATP gamma S binding causes the fluorescence enhancement of CM bound to the gamma subunit and that this conformational change is reversed upon bond cleavage to yield ADP.Pi or ADP.PiS in catalytic sites.
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PMID:ATP binding causes a conformational change in the gamma subunit of the Escherichia coli F1ATPase which is reversed on bond cleavage. 794 38

We have utilized DSC and high pressure FTIR spectroscopy to study the specificity and mechanism by which ATP protects actin against heat and pressure denaturation. Analysis of the thermograms shows that ATP raises the transition temperature Tm for actin from 69.6 to 75.8 degrees C, and the calorimetric enthalpy, deltaH, from 680 to 990 kJ/mole. Moreover, the peak becomes sharper indicating a more cooperative process. Among the other nucleotide triphosphates, only UTP increases the Tm by 2.5 degrees C, whereas GTP and CTP have negligable effects; ADP and AMP are less active, increasing the Tm by 2.1 and 1.6 degrees C, respectively. Therefore, gamma phosphate plays a key role in this protection, but its hydrolysis is not implicated since the nonhydrolysable analogue of ATP, ATP-PNP have the same activity as ATP. FTIR spectroscopy demonstrates that ATP also protects actin against high pressure denaturation. Analysis of the amide I band during the increase in pressure clearly illustrates that ATP protects particularly a region rich in beta-sheets of the actin molecule.
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PMID:Effects of nucleotides on the denaturation of F actin: a differential scanning calorimetry and FTIR spectroscopy study. 924 36

Ionic channels bathed in mixed solutions of two permeant electrolytes often conduct less current than channels bathed in pure solutions of either. For many years, this anomalous mole fraction effect (AMFE) has been thought to occur only in single-file pores containing two or more ions at a time. Most thinking about channels incorporates this view. We show here that the AMFE arises naturally, as an electrostatic consequence of localized ion specific binding, if the average current through a channel is described by a theory (Poisson-Nernst-Planck, PNP) that computes the average electric field from the average concentration of charges in and near the channel. The theory contains only those ion-ion interactions mediated by the mean field, and it does not enforce single filing. The AMFE is predicted by PNP over a wide range of mean concentrations of ions in the channel; for example, it is predicted when (on the average) less, or much less, than one ion is found in the channel's pore. In this treatment, the AMFE arises, in large measure, from a depletion layer produced near a region of ion-specific binding. The small excess concentration of ions in the binding region repels all nearby ions of like charge, thereby creating a depletion layer. The overall conductance of the channel arises in effect from resistors in series, one from the binding region, one from the depletion zone, and one from the unbinding region. The highest value resistor (which occurs in the depletion zone) limits the overall series conductance. Here the AMFE is not the result of single filing or multiple occupancy, and so previous views of permeation need to be revised: the presence of an AMFE does not imply that ions permeate single file through a multiply occupied pore.
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PMID:Anomalous mole fraction effect, electrostatics, and binding in ionic channels. 959 60

L-type Ca channels contain a cluster of four charged glutamate residues (EEEE locus), which seem essential for high Ca specificity. To understand how this highly charged structure might produce the currents and selectivity observed in this channel, a theory is needed that relates charge to current. We use an extended Poisson-Nernst-Planck (PNP2) theory to compute (mean) Coulombic interactions and thus to examine the role of the mean field electrostatic interactions in producing current and selectivity. The pore was modeled as a central cylinder with tapered atria; the cylinder (i.e., "pore proper") contained a uniform volume density of fixed charge equivalent to that of one to four carboxyl groups. The pore proper was assigned ion-specific, but spatially uniform, diffusion coefficients and excess chemical potentials. Thus electrostatic selection by valency was computed self-consistently, and selection by other features was also allowed. The five external parameters needed for a system of four ionic species (Na, Ca, Cl, and H) were determined analytically from published measurements of thre limiting conductances and two critical ion concentrations, while treating the pore as a macroscopic ion-exchange system in equilibrium with a uniform bath solution. The extended PNP equations were solved with these parameters, and the predictions were compared to currents measured in a variety of solutions over a range of transmembrane voltages. The extended PNP theory accurately predicted current-voltage relations, anomalous mole fraction effects in the observed current, saturation effects of varied Ca and Na concentrations, and block by protons. Pore geometry, dielectric permittivity, and the number of carboxyl groups had only weak effects. The successful prediction of Ca fluxes in this paper demonstrates that ad hoc electrostatic parameters, multiple discrete binding sites, and logistic assumptions of single-file movement are all unnecessary for the prediction of permeation in Ca channels over a wide range of conditions. Further work is needed, however, to understand the atomic origin of the fixed charge, excess chemical potentials, and diffusion coefficients of the channel. The Appendix uses PNP2 theory to predict ionic currents for published "barrier-and-well" energy profiles of this channel.
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PMID:Ion permeation and glutamate residues linked by Poisson-Nernst-Planck theory in L-type calcium channels. 972 31

Lon (La) proteases are multimeric enzymes that are activated by ATP and Mg(2+) ions and stimulated by unfolded proteins such as alpha-casein. The peptidase activity of the Lon protease from Mycobacterium smegmatis (Ms-Lon) is dependent upon both its concentration and that of Mg(2+). Addition of alpha-casein partially substitutes for Mg(2+) in activating the enzyme. In chemical dissociation experiments, higher concentrations of urea were required to inhibit Ms-Lon's catalytic activities after an addition of alpha-casein. Analytical ultracentrifugation was used to directly probe the effect of activators of peptidase activity on Ms-Lon self-association. Sedimentation velocity experiments reveal that Ms-Lon monomers are in a reversible equilibrium with oligomeric forms of the protein and that the self-association reaction is facilitated by Mg(2+) ions but not by AMP-PNP or ATP gamma S. NaCl at 100 mM facilitates oligomerization and stimulates peptidase activity at suboptimal concentrations of MgCl(2). Sedimentation equilibrium analysis shows that Ms-Lon associates to a hexamer at 50 mM Tris and 10 mM MgCl(2), at pH 8.0 and 20 degrees C, and that the assembly reaction is Mg(2+) dependent; the mole fraction of hexamer decreases with decreasing MgCl(2) to undetectable levels in 10 mM EDTA. The analysis of experiments conducted at a series of initial protein and MgCl(2) concentrations yields two assembly models: dimer <--> tetramer <--> hexamer and timer <--> hexamer, equally consistent with the data. Limited trypsin digestion, CD, and tryptophan fluorescence suggest only minor changes in secondary and tertiary structure upon Mg(2+)-linked oligomerization. These results show that activation of Ms-Lon peptidase activity requires oligomerization and that Ms-Lon self-association reaction is facilitated by its activator, Mg(2+), and stimulator, unfolded protein.
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PMID:Mg2+-linked oligomerization modulates the catalytic activity of the Lon (La) protease from Mycobacterium smegmatis. 1147 99

Anomalies in the permeation properties of the cardiac RyR channel reconstituted into bilayer lipid membranes were investigated systematically. We tested the presence of the anomalous mole fraction effect (AMFE) for the ion conductance and the reversal potential with varying mole fractions of two permeant ions, while the total ion concentration was lower, as in previous studies, to avoid the masking effect of the channel pore saturation with ions. Mixtures of Ba(2+) with other divalents (Ca(2+), Sr(2+)), of Ca(2+) with monovalents (Li(+), Cs(+)), and of Na(+) with other monovalents (Cs(+), Li(+)) were used. We revealed a clear anomaly only for the ion conductance measured in the Na(+)-Cs(+) and Ca(2+)-Li(+) mixtures as computed by a Poisson-Nernst-Planck/density functional theory (PNP/DFT) model. Furthermore, we found a significant minimum in the concentration dependence of the reversal potential determined under Li(+)/Ca(2+) bi-ionic conditions. Our study led to new observations that may have important implications for understanding the mechanisms involved in ion handling in the RyR channel pore; furthermore our results could be useful for further validation of ion permeation models developed for the RyR channel.
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PMID:The cardiac ryanodine receptor: looking for anomalies in permeation properties. 1871 44

This study reports the preparation of chitosan-glutaraldehyde (Chi-Glu) copolymers at modified reaction conditions such as the temperature prior to gelation, pH, and reagent ratios. The chitosan copolymers were characterized using infrared spectroscopy (FT-IR), CHN elemental analysis, and thermal gravimetric analysis (TGA). Evidence of self-polymerized glutaraldehyde was supported by CHN and TGA results. The sorption properties of Chi-Glu copolymers were evaluated in aqueous solutions containing p-nitrophenol at variable pH (4.6, 6.6, and 9.0). The sorption properties of the copolymers correlated with the level of the accessibility of the sorption sites in accordance with the relative cross-linker content. The relative sorption capacity of the Chi-Glu copolymers increases as the level of cross-linking increases. Chitosan displays the lowest sorptive uptake while an optimal sorption capacity was concluded at the 4:1 glutaraldehyde:chitosan monomer mole ratio, in close agreement with the three reactive sites (i.e. OH/NH) per glucosamine monomer. The PNP dye probe was determined to bind to chitosan through an electrostatic interaction due to the increased sorption capacity of the phenolate anion, as evidenced by the change in pH from 4.6 to 9.0.
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PMID:Chitosan-glutaraldehyde copolymers and their sorption properties. 2481 6


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