The intracellular ionic strength of red cells and the influence of complex formation

Mouat, Michael F.; Manchester, K.L.

doi:10.1007/bf02628107

Cited by 37 publications

(24 citation statements)

References 17 publications

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“…The addition of 2 mM Mg 2þ , which is above mammalian cellular levels of 0.2-0.6 mM (22)(23)(24), to the effective physiological salt concentration buffer (211 mM I) did not affect the binding of strongly binding REs but did weaken the binding of a weak binder (Table 1) so that AcK120p53 reached its full selectivity, which was still much higher than that for nonacetylated p53, measured under the same Mg 2þ concentrations. Substitution of KCl for NaCl had a negligible effect on binding (Table 1).…”

Section: Resultsmentioning

confidence: 92%

“…Allowance for formation of complexes between ions reduces the value to 200 mM (23). An effective physiological ionic strength of 215 mM I is also calculated (30).…”

Section: Discussionmentioning

confidence: 99%

“…Mg 2þ ions can affect DNA-protein interactions by competing for DNA sites (31,32). The concentration of Mg 2þ in mammalian cells is about 0.2-0.6 mM (22)(23)(24). The addition of 2 mM Mg 2þ to the effective physiological salt buffer (Table 1) had little effect on the binding of either AcK120p53 or nonacetylated p53 to strongbinding REs, but it weakened the binding to weak-binding REs.…”

Section: Binomial Distribution Experimentalmentioning

confidence: 99%

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Acetylation of lysine 120 of p53 endows DNA-binding specificity at effective physiological salt concentration

Arbely

Natan

Brandt

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Lys120 in the DNA-binding domain (DBD) of p53 becomes acetylated in response to DNA damage. But, the role and effects of acetylation are obscure. We prepared p53 specifically acetylated at Lys120, AcK120p53, by in vivo incorporation of acetylated lysine to study biophysical and structural consequences of acetylation that may shed light on its biological role. Acetylation had no affect on the overall crystal structure of the DBD at 1.9-Å resolution, but significantly altered the effects of salt concentration on specificity of DNA binding. p53 binds DNA randomly in vitro at effective physiological salt concentration and does not bind specifically to DNA or distinguish among its different response elements until higher salt concentrations. But, on acetylation, AcK120p53 exhibited specific DNA binding and discriminated among response elements at effective physiological salt concentration. AcK120p53 and p53 had the highest affinity to the same DNA sequence, although acetylation reduced the importance of the consensus C and G at positions 4 and 7, respectively. Mass spectrometry of p53 and AcK120p53 DBDs bound to DNA showed they preferentially segregated into complexes that were either DNAðp53DBDÞ 4 or DNA ðAcK120DBDÞ 4 , indicating that the different DBDs prefer different quaternary structures. These results are consistent with electron microscopy observations that p53 binds to nonspecific DNA in different, relaxed, quaternary states from those bound to specific sequences. Evidence is accumulating that p53 can be sequestered by random DNA, and target search requires acetylation of Lys120 and/or interaction with other factors to impose specificity of binding via modulating changes in quaternary structure.

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Section: Resultsmentioning

confidence: 92%

“…Allowance for formation of complexes between ions reduces the value to 200 mM (23). An effective physiological ionic strength of 215 mM I is also calculated (30).…”

Section: Discussionmentioning

confidence: 99%

Section: Binomial Distribution Experimentalmentioning

confidence: 99%

See 1 more Smart Citation

Acetylation of lysine 120 of p53 endows DNA-binding specificity at effective physiological salt concentration

Arbely

Natan

Brandt

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Figure 5(a) shows a drastic impact of pH variation in pH sensing capability. The extra- and intra-cellular ionic strength range normally from 0.08 to 0.2 M [47] which is shown in the labelled box. Below or above this range, it is not recommended to measure pH with currently proposed probe, otherwise poor sensitivity and large error would be expected.…”

Section: Resultsmentioning

confidence: 99%

Reflection-mode micro-spherical fiber-optic probes for in vitro real-time and single-cell level pH sensing

Yang

Wang

Lan

et al. 2015

Sensors and Actuators B: Chemical

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pH sensing at the single-cell level without negatively affecting living cells is very important but still a remaining issue in the biomedical studies. A 70 μm reflection-mode fiber-optic micro-pH sensor was designed and fabricated by dip-coating thin layer of organically modified aerogel onto a tapered spherical probe head. A pH sensitive fluorescent dye 2′, 7′-Bis (2-carbonylethyl)-5(6)-carboxyfluorescein (BCECF) was employed and covalently bonded within the aerogel networks. By tuning the alkoxide mixing ratio and adjusting hexamethyldisilazane (HMDS) priming procedure, the sensor can be optimized to have high stability and pH sensing ability. The in vitro real-time sensing capability was then demonstrated in a simple spectroscopic way, and showed linear measurement responses with a pH resolution up to an average of 0.049 pH unit within a narrow, but biological meaningful pH range of 6.12–7.81. Its novel characterizations of high spatial resolution, reflection mode operation, fast response and high stability, great linear response within biological meaningful pH range and high pH resolutions, make this novel pH probe a very cost-effective tool for chemical/biological sensing, especially within the single cell level research field.

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“…The reported cytosolic free magnesium concentration for different mammalian cells expressed as pMg is between 2.6 and 3.7 (Gunther, 2006). Physiological ionic strength of erythrocyte cytosol is around 0.2-0.25 M (Mouat and Manchester, 1998), and of a dogfish muscle fiber at least 0.3 M (West et al, 2005). However, unicellular organisms grow in media of changing composition and preserve their turgor by absorbing K þ ions thus increasing the intracellular osmotic pressure and protecting the cell from deplasmolysis.…”

Section: Data For Physiological Conditions In the Cytosolmentioning

confidence: 99%

Influence of uncertainties in pH, pMg, activity coefficients, metabolite concentrations, and other factors on the analysis of the thermodynamic feasibility of metabolic pathways

Vojinović

Stockar

2009

Biotech & Bioengineering

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Thermodynamic feasibility analysis (TFA) has been used as a tool capable of providing additional constraints to the mass balance-based methods of analysis of metabolic networks (e.g., flux balance analysis). Several publications have recently appeared in which TFA of different metabolic pathways from relatively simple to the genome-scale networks was described as a means of detecting the possible metabolic control steps. However, in order to perform TFA, many simplifying assumptions were necessary. On the other hand, it has been shown by applying TFA to the well-known pathway of glycolysis that erroneous simplifying assumptions may seriously bias the results of the analysis. A quantitative analysis of the influence of non-ideality of the biochemical system, pH, temperature, and complexation of the metabolites with Mg(2+) ions as well as a number of other factors on the TFA is reported. It is shown that the feasibility of glycolysis is very seriously limited by the reaction of oxidative phosphorylation of glyceraldehyde phosphate, and that the intracellular concentration of the main product of this reaction, biphosphoglycerate, must be anywhere from 10 to 100 times lower than published values. In addition, the driving force for this reaction, and consequently the feasibility of the entire pathway depend strongly on the intracellular pH and ionic strength and to a lesser extent on pMg and temperature. The analysis may also be influenced by uncertainties of the dissociation and magnesium complexation constants of glyceraldehyde phosphate. The analysis demonstrates the crucial importance of taking such factors into account when performing TFA. It also suggests an urgent need for experimental determinations of such factors as a prerequisite for sensible thermodynamic analysis of metabolism on a genome-wide scale.

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The intracellular ionic strength of red cells and the influence of complex formation

Cited by 37 publications

References 17 publications

Acetylation of lysine 120 of p53 endows DNA-binding specificity at effective physiological salt concentration

Acetylation of lysine 120 of p53 endows DNA-binding specificity at effective physiological salt concentration

Reflection-mode micro-spherical fiber-optic probes for in vitro real-time and single-cell level pH sensing

Influence of uncertainties in pH, pMg, activity coefficients, metabolite concentrations, and other factors on the analysis of the thermodynamic feasibility of metabolic pathways

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