scholarly journals Effect of calcium ions on the irreversible denaturation of a recombinant Bacillus halmapalus alpha-amylase: a calorimetric investigation

2003 ◽  
Vol 373 (2) ◽  
pp. 337-343 ◽  
Author(s):  
Anders D. NIELSEN ◽  
Claus C. FUGLSANG ◽  
Peter WESTH
Keyword(s):  
Calcium Ions ◽  
Calcium Binding ◽  
Thermal Inactivation ◽  
Binding Constants ◽  
Ion Concentration ◽  
Effect Of Temperature ◽  
Titration Calorimetry ◽  
Free Calcium ◽  
Enthalpy Changes ◽  
The Difference

The effect of temperature and calcium ions on the denaturation of a recombinant α-amylase from Bacillus halmapalus α-amylase (BHA) has been studied using calorimetry. It was found that thermal inactivation of BHA is irreversible and that calcium ions have a significant effect on stability. Thus an apparent denaturation temperature (Td) of 83 °C in the presence of excess calcium ions was observed, whereas Td decreased to 48 °C when calcium was removed. The difference in thermal stability with and without calcium ions has been used to develop an isothermal titration calorimetric (ITC) procedure that allows simultaneous determination of kinetic parameters and enthalpy changes of the denaturation of calcium-depleted BHA. An activation energy EA of 101 kJ/mol was found for the denaturation of calcium-depleted BHA. The results support a kinetic denaturation mechanism where the calcium-depleted amylase denatures irreversibly at low temperature and if calcium ions are in excess, the amylase denatures irreversibly at high temperatures. The two denaturation reactions are coupled with the calcium-binding equilibrium between calcium-bound and -depleted amylase. A combination of the kinetic denaturation results and calcium-binding constants, determined by isothermal titration calorimetry, has been used to estimate kinetic stability, expressed in terms of the half-life of BHA as a function of temperature and free-calcium-ion concentration. Thus it is estimated that the apparent EA can be increased to approx. 123 kJ/mol by increasing the free-calcium concentration.

Binding of calcium ions to bovine αsl-casein and precipitability of the protein–calcium ion complexes

1980 ◽  
Vol 47 (1) ◽  
pp. 113-122 ◽  
Author(s):  
Douglas G. Dalgleish ◽  
Thomas G. Parker
Keyword(s):  
Ionic Strength ◽  
Calcium Ions ◽  
Calcium Ion ◽  
Calcium Binding ◽  
Calcium Concentration ◽  
Binding Constants ◽  
Substitution Effects ◽  
Binding Isotherms ◽  
Decreasing Ph ◽  
Increasing Temperature

SummaryBinding isotherms for the calcium ion–αsl-casein system have been measured, as functions of ionic strength, temperature, and pH, and the isotherms have been analysed in terms of binding constants modified by substitution effects. The results demonstrate that the strength of binding is increased with increasing temperature and decreased by increasing ionic strength or decreasing pH, all of which may be explained semi-quantitatively. Parallel studies on the precipitability of the αsl-casein–Ca2+ complexes showed that there is considerable variation in the extent of calcium binding required to initiate precipitation of the protein, and in the calcium concentration necessary to achieve the required extent of ligand binding.

scholarly journals A mysterious family of calcium-binding proteins from parasitic worms

2016 ◽  
Vol 44 (4) ◽  
pp. 1005-1010 ◽  
Author(s):  
Charlotte M. Thomas ◽  
David J. Timson
Keyword(s):  
Calcium Ions ◽  
Calcium Binding ◽  
Cell Biology ◽  
Fasciola Hepatica ◽  
Family Members ◽  
Ion Concentration ◽  
Pharmacological Significance ◽  
Ef Hand ◽  
Parasitic Worms

There is a family of proteins from parasitic worms which combine N-terminal EF-hand domains with C-terminal dynein light chain-like domains. Data are accumulating on the biochemistry and cell biology of these proteins. However, little is known about their functions in vivo. Schistosoma mansoni expresses 13 family members (SmTAL1–SmTAL13). Three of these (SmTAL1, SmTAL2 and SmTAL3) have been subjected to biochemical analysis which demonstrated that they have different molecular properties. Although their overall folds are predicted to be similar, small changes in the EF-hand domains result in differences in their ion binding properties. Whereas SmTAL1 and SmTAL2 are able to bind calcium (and some other) ions, SmTAL3 appears to be unable to bind any divalent cations. Similar biochemical diversity has been seen in the CaBP proteins from Fasciola hepatica. Four family members are known (FhCaBP1–4). All of these bind to calcium ions. However, FhCaBP4 dimerizes in the presence of calcium ions, FhCaBP3 dimerizes in the absence of calcium ions and FhCaBP2 dimerizes regardless of the prevailing calcium ion concentration. In both the SmTAL and FhCaBP families, the proteins also differ in their ability to bind calmodulin antagonists and related drugs. Interestingly, SmTAL1 interacts with praziquantel (the drug of choice for treating schistosomiasis). The pharmacological significance (if any) of this finding is unknown.

Cyanide Thermodynamics. III. Enthalpies and Entropies of Ionization of Water and Hydrogen Cyanide

1996 ◽  
Vol 49 (6) ◽  
pp. 651 ◽  
Author(s):  
JS Solis ◽  
PM May ◽  
G Hefter
Keyword(s):  
Aqueous Solution ◽  
Ionic Strength ◽  
Stability Constant ◽  
Hydrogen Cyanide ◽  
Infinite Dilution ◽  
Medium Effect ◽  
Titration Calorimetry ◽  
Enthalpy Changes ◽  
The Difference ◽  
Ionization Of Water

The heats (enthalpy changes) associated with the ionization of water and of hydrogen cyanide have been determined by titration calorimetry at 25�C as a function of ionic strength up to 5 M in both NaCl and NaClO4 media. The enthalpy changes for both reactions exhibit a 'medium effect' with ?H being more positive in NaCl than in NaClO4 and with the difference becoming more pronounced with increasing ionic strength. This is attributed to the greater solvation of Cl- cf. CN- in aqueous solution. The present ?H values are similar to previous published results at high ionic strengths, and are in excellent agreement with the well established literature values at infinite dilution. The present ?H values were combined with literature stability constant data to calculate the corresponding entropies for the ionization of H2O and HCN as a function of ionic strength.

The effect of temperature on some calcium-binding properties of troponin C and calmodulin

1980 ◽  
Vol 58 (9) ◽  
pp. 683-691 ◽  
Author(s):  
William D. McCubbin ◽  
Maxwell T. Hincke ◽  
Cyril M. Kay
Keyword(s):  
Calcium Binding ◽  
Heat Stability ◽  
Troponin C ◽  
Effect Of Temperature ◽  
Spectroscopic Techniques ◽  
Binding Properties ◽  
Tyrosine Residues ◽  
Temperature Range ◽  
Skeletal Protein ◽  
The Difference

Some calcium-binding properties of skeletal and cardiac troponin C (TnC) have been measured as functions of temperature employing several physical and spectroscopic techniques. The degree of exposure of the tyrosine residues in brain calmodulin has also been determined by a new approach. Circular dichroism thermal unfolding profiles have been established for the three cases: metal-free protein, high-affinity sites filled, and fully saturated. In addition some thermodynamic parameters have been calculated for these reversible melting processes. It was found that the calcium-binding parameters n and K, where n is the fraction of the total conformational change and K is the apparent association constant, for both skeletal and cardiac TnC, did not vary significantly over the temperature range 10–38 °C, but at 50 °C differences became quite apparent, dramatically so in the case of the skeletal protein. The technique of thermal perturbation difference spectroscopy was applied to determine the degree of exposure of aromatic chromophores for the TnC(s) and calmodulin in the absence and presence of calcium. For skeletal TnC and calmodulin the results were in good agreement with previous observations, but the reduced degree of exposure of the tyrosine residues in cardiac TnC, in the absence of Ca2+, was contrary to the earlier work. Calcium-induced difference absorption spectra have been measured for the TnC(s) over the temperature range 10–70 °C. Cardiac TnC showed greater heat stability than its skeletal counterpart, in terms of the rate and the amount of change of the difference spectral maxima.

Effect of Minerals on Casein Micelle Stability of Cows' Milk

2007 ◽  
Vol 74 (2) ◽  
pp. 167-173 ◽  
Author(s):  
Alexandros Tsioulpas ◽  
Michael J Lewis ◽  
Alistair S Grandison
Keyword(s):  
Linear Relationship ◽  
Calcium Ions ◽  
Calcium Ion ◽  
Ion Concentration ◽  
Free Calcium ◽  
Casein Micelle ◽  
Clotting Time ◽  
Rennet Coagulation ◽  
Calcium Ion Concentration ◽  
Ethanol Stability

The effects of minerals on casein micelle stability of individual cows' milk, throughout a complete lactation, were investigated. Calcium and calcium ions, magnesium, phosphorus, sodium, potassium and citrate contents were analysed, together with the following physical properties of milk; pH, ethanol stability, rennet clotting time and coagulum firmness. There was an inverse non-linear relationship between free calcium ion concentration and ethanol stability (ES; r=0·84). Rennet coagulation time showed a weaker relationship with free calcium ion concentration (r=0·44) but a stronger relationship with pH (r=0·66). In addition, samples containing higher amounts of free calcium ions produced a firmer gel. Citrate in natural samples acts as a stabilizing factor, as it slightly improves milk stability. Potassium, on the other hand, exhibited a negative correlation, but only with rennet clotting time (r=−0·52). Throughout lactation the average values were; free Ca2+ concentration 1·88 mM, pH 6·63, ES 83·2% and clotting time 13·6 min. The equilibrium relationship between pH and free Ca2+ concentration was investigated by adjusting milk pH from 5·9 to 7·1, using acid and alkali. There was a good inverse linear relationship between pH and log (free Ca2+) for individual milk samples, with a gradient of −0·62 and a standard deviation of 0·042.

scholarly journals The equilibrium constant of the phosphoglyceromutase reaction

1974 ◽  
Vol 139 (3) ◽  
pp. 491-497 ◽  
Author(s):  
John B. Clarke ◽  
Michael Birch ◽  
Hubert G. Britton
Keyword(s):  
Ionic Strength ◽  
Equilibrium Constant ◽  
Dissociation Constants ◽  
Ph Dependence ◽  
Binding Constants ◽  
Acid Dissociation ◽  
Effect Of Temperature ◽  
Acid Dissociation Constants ◽  
The Difference ◽  
Low Ionic Strength

The equilibrium constant of the phosphoglyceromutase reaction was determined over a range of pH (5.4–7.9), in solutions of different ionic strength (0.06–0.3) and in the presence of Mg2+, at 30°C and at 20°C. The values obtained (8.65–11.65) differ substantially from previously published values. The third acid dissociation constants were redetermined for 2- and 3-phosphoglycerate, and in contrast with previous reports the pK values (7.03 and 6.97 respectively at zero ionic strength) were closely similar. The Mg2+-binding constants were measured spectrophotometrically and the values, 286mm-1 and 255mm-1 for 2- and 3-phosphoglycerate at pH7 and ionic strength 0.02, were also very similar. From the relative lack of effect of temperature, pH and ionic strength it is concluded that the equilibrium constant differs from unity largely because of entropic factors. At low ionic strength, in the neutral region, the pH-dependence can be attributed to the small difference in the acid dissociation constants, but the difference in dissociation constants does not explain the pH-dependence in the acid region or at high ionic strength. Within physiological ranges of pH, Mg2+ concentration and ionic strength there will be little variation in equilibrium constant.

Mitochondrial matrix calcium ions regulate energy production in myocardium: A cytochemical study

1990 ◽  
Vol 48 (2) ◽  
pp. 166-167
Author(s):  
W.A. Jacob ◽  
R. Hertsens ◽  
A. Van Bogaert ◽  
M. De Smet
Keyword(s):  
Energy Metabolism ◽  
Calcium Ions ◽  
Energy Production ◽  
Regulation Of Respiration ◽  
Free Calcium ◽  
Mitochondrial Matrix ◽  
Cytochemical Study ◽  
Nmr Techniques

In the past most studies of the control of energy metabolism focus on the role of the phosphorylation potential ATP/ADP.Pi on the regulation of respiration. Studies using NMR techniques have demonstrated that the concentrations of these compounds for oxidation phosphorylation do not change appreciably throughout the cardiac cycle and during increases in cardiac work. Hence regulation of energy production by calcium ions, present in the mitochondrial matrix, has been the object of a number of recent studies.Three exclusively intramitochondnal dehydrogenases are key enzymes for the regulation of oxidative metabolism. They are activated by calcium ions in the low micromolar range. Since, however, earlier estimates of the intramitochondnal calcium, based on equilibrium thermodynamic considerations, were in the millimolar range, a physiological correlation was not evident. The introduction of calcium-sensitive probes fura-2 and indo-1 made monitoring of free calcium during changing energy metabolism possible. These studies were performed on isolated mitochondria and extrapolation to the in vivo situation is more or less speculative.

Confocal imaging of calcium in intact ventricular muscle provides a new view of excitation-contraction coupling

1996 ◽  
Vol 54 ◽  
pp. 738-739
Author(s):  
W.G. Wier
Keyword(s):  
Local Control ◽  
Cardiac Tissue ◽  
Cell Function ◽  
Isolated Heart ◽  
Cardiac Cells ◽  
Confocal Imaging ◽  
Ion Concentration ◽  
Heart Cell ◽  
Free Calcium ◽  
Heart Cells

A fundamentally new understanding of cardiac excitation-contraction (E-C) coupling is being developed from recent experimental work using confocal microscopy of single isolated heart cells. In particular, the transient change in intracellular free calcium ion concentration ([Ca2+]i transient) that activates muscle contraction is now viewed as resulting from the spatial and temporal summation of small (∼ 8 μm3), subcellular, stereotyped ‘local [Ca2+]i-transients' or, as they have been called, ‘calcium sparks'. This new understanding may be called ‘local control of E-C coupling'. The relevance to normal heart cell function of ‘local control, theory and the recent confocal data on spontaneous Ca2+ ‘sparks', and on electrically evoked local [Ca2+]i-transients has been unknown however, because the previous studies were all conducted on slack, internally perfused, single, enzymatically dissociated cardiac cells, at room temperature, usually with Cs+ replacing K+, and often in the presence of Ca2-channel blockers. The present work was undertaken to establish whether or not the concepts derived from these studies are in fact relevant to normal cardiac tissue under physiological conditions, by attempting to record local [Ca2+]i-transients, sparks (and Ca2+ waves) in intact, multi-cellular cardiac tissue.

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