A stopped-flow investigation of calcium ion binding by ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid

Smith, Paul D.; Liesegang, Gerald W.; Berger, Robert L.; Czerlinski, George; Podolsky, Richard J.

doi:10.1016/0003-2697(84)90575-x

Cited by 119 publications

(112 citation statements)

References 4 publications

Supporting

Mentioning

103

Contrasting

Unclassified

Order By: Relevance

“…The values of kl that were obtained by Harafuji and Ogawa (1980), 1.0-3.0 • 106 M-is -1 at 20~ are in good agreement with the value obtained by Smith et al (1984), 1.5 • 106 M-as -x at 25~ consistent with the idea that there is little delay between the complexation of Ca by H2EGTA 2-and the dissociation of protons from CaH2EGTA. Second, in the experiments of Smith et al (1984), the change in pH began almost immediately after mixing and showed no detectable delay, also suggesting that protons dissociate from CaH2EGTA almost immediately after Ca is complexed by H~EGTA 2-.…”

Section: At Neutral Ph Ca Z+ Is Complexed By H2egtae-and Two Protonssupporting

confidence: 87%

“…In this case, EGTA is expected to capture about 96% of the Ca released by an action potential. The value of "rc~ is expected to be independent of pH because, in stopped-flow experiments, the value of hi is independent of pH between pH 5.8 and 7.3 (Smith, Liesegang, Berger, Czerlinski, and Podolsky, 1984). Under the conditions of our experiments, the value of 7c~ is estimated to be 22 ~s.…”

Section: The Time Course Of the A [Ca] Signal Elicited By An Action Pmentioning

confidence: 57%

“…This value lies near the upper end of the range of values that have been measured in vitro with 100 mM KC1. Harafuji and Ogawa (1980) obtained values of 1.0-3.0 X 106 M-is -x at 20~ and Smith et al (1984) obtained a value of 0.9 • 106 M-is -1 at 16~ Harafuji and Ogawa (1980) attributed the differences among their values to the three different pH buffers that were used. Our estimate of kl would be smaller than 2.5 • 106 M-is -1 if the value of [EGTA]R were greater than 18.24 mM; for example, the concentration of free EGTA might be essentially the same as that in the end pools, as seems likely after the long periods of equilibration that were used in our experiments, and an additional amount of EGTA might be bound inside the myoplasm and able to react with Ca.…”

Section: (Ms) 500mentioning

confidence: 99%

“…Consequently, pathway (3) can be neglected. Smith et al (1984) carried out stopped-flow measurements of the kinetics of Ca complexation by EGTA and obtained information about the relative importance of pathways (1) and (2) at physiological pH. Between pH 5.8 and 7.3, the value of the apparent forward rate constant, kl, was found to be approximately constant, 1.5 • 106 M-is -1 at 25~ Because the value of [H2EGTA2-]/[EGTA] is also approximately constant at these pH's (~1) and because the value of [HEGTA3-]/[EGTA] is expected to change sixfold (see above), Smith et al (1984) concluded that most of the complexation of Ca by EGTA occurs through pathway 1, with Ca 2+ complexing H2EGTA 2-to form CaH2EGTA.…”

Section: At Neutral Ph Ca Z+ Is Complexed By H2egtae-and Two Protonsmentioning

confidence: 99%

“…First, in the stopped-flow experiments of Smith et al (1984), the rate of Ca complexation by EGTA was determined from measurements of changes in pH whereas, in another series of stopped-flow experiments (Harafuji and Ogawa, 1980), it was determined from measurements of changes in free [Ca]. The values of kl that were obtained by Harafuji and Ogawa (1980), 1.0-3.0 • 106 M-is -1 at 20~ are in good agreement with the value obtained by Smith et al (1984), 1.5 • 106 M-as -x at 25~ consistent with the idea that there is little delay between the complexation of Ca by H2EGTA 2-and the dissociation of protons from CaH2EGTA. Second, in the experiments of Smith et al (1984), the change in pH began almost immediately after mixing and showed no detectable delay, also suggesting that protons dissociate from CaH2EGTA almost immediately after Ca is complexed by H~EGTA 2-.…”

Section: At Neutral Ph Ca Z+ Is Complexed By H2egtae-and Two Protonsmentioning

confidence: 99%

See 4 more Smart Citations

Calcium release and its voltage dependence in frog cut muscle fibers equilibrated with 20 mM EGTA.

Pape

Jong

Chandler

1995

The Journal of General Physiology

129

295

View full text Add to dashboard Cite

Sarcoplasmic reticulum (SR) Ca release was studied at 13-16~ in cut fibers (sarcomere length, 3.4-3.9 I~m) mounted in a double Vaseline-gap chamber. The amplitude and duration of the action-potential stimulated free [Ca] transient were reduced by equilibration with end-pool solutions that contained 20 mM EGTA with 1.76 mM Ca and 0.63 mM phenol red, a maneuver that appeared to markedly reduce the amount of Ca complexed by troponin. A theoretical analysis shows that, under these conditions, the increase in myoplasmic free [Ca] is expected to be restricted to within a few hundred nanometers of the SR Ca release sites and to have a time course that essentially matches that of release. Furthermore, almost all of the Ca that is released from the SR is expected to be rapidly bound by EGTA and exchanged for protons with a 1:2 stoichiometry. Consequently, the time course of SR Ca release can be estimated by scaling the ApH signal measured with phenol red by -13/2. The value of 13, the buffering power of myoplasm, was determined in fibers equilibrated with a combination of EGTA, phenol red, and fura-2; its mean value was 22 mM/pH unit. The Ca content of the SR (expressed as myoplasmic concentration) was estimated from the total amount of Ca released by either a train of action potentials or a depleting voltage step; its mean value was 2,685 I~M in the action-potential experiments and 2,544 IxM in the voltage-clamp experiments. An action potential released, on average, 0.14 of the SR Ca content with a peak rate of release of,'~5 %/ms. A second action potential, elicited 20 ms later, released only 0.6 times as much Ca (expressed as a fraction of the SR content), probably because Ca inactivation of Ca release was produced by the first action potential. During a depolarizing voltage step to 60 mV, the rate of Ca release rapidly increased to a peak value of~3 %/ms and then decreased to a quasi-steady level that was only 0.6 times as large; this decrease was also probably due to Ca inactivation of Ca release. SR Ca release was studied with small step depolarizations that open no more than one SR Ca channel in 7

show abstract

Section: At Neutral Ph Ca Z+ Is Complexed By H2egtae-and Two Protonssupporting

confidence: 87%

Section: The Time Course Of the A [Ca] Signal Elicited By An Action Pmentioning

confidence: 57%

Section: (Ms) 500mentioning

confidence: 99%

Section: At Neutral Ph Ca Z+ Is Complexed By H2egtae-and Two Protonsmentioning

confidence: 99%

Section: At Neutral Ph Ca Z+ Is Complexed By H2egtae-and Two Protonsmentioning

confidence: 99%

See 3 more Smart Citations

Calcium release and its voltage dependence in frog cut muscle fibers equilibrated with 20 mM EGTA.

Pape

Jong

Chandler

1995

The Journal of General Physiology

129

295

View full text Add to dashboard Cite

show abstract

Thapsigargin-inducedgrp78 expression is mediated by the increase of cytosolic free calcium in 9L rat brain tumor cells

et al. 2000

View full text Add to dashboard Cite

Exposure of 9L rat brain tumor cells to 300 nM thapsigargin (TG), a sarcoendoplasmic Ca(2+)-ATPases inhibitor, leads to an immediate suppression of general protein synthesis followed by an enhanced synthesis of the 78-kDa glucose-regulated protein, GRP78. Synthesis of GRP78 increases significantly and continues to rise after 4 h of treatment, and this process coincides with the accumulation of grp78 mRNA. TG-induced grp78 expression can be suppressed by the cytosolic free calcium ([Ca(2+)](c)) chelator dibromo-1, 2-bis(aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA) in a concentration-dependent manner. Induction of grp78 is completely abolished in the presence of 20 microM BAPTA under which the TG-induced increase of [Ca(2+)](c) is also completely prevented. By adding ethyleneglycol bis(beta-aminoethyl)ether-N,N,N',N' tetraacetic acid in the foregoing experiments, in a condition such that endoplasmic reticulum calcium ([Ca(2+)](ER)) is depleted and calcium influx from outside is prevented, TG-induced grp78 expression is also abolished. These data lead us to conclude that increase in [Ca(2+)](c), together with the depletion of [Ca(2+)](ER), are the major causes of TG-induced grp78 expression in 9L rat brain tumor cells. By using electrophoretic mobility shift assays (EMSA), we found that the nuclear extracts prepared from TG-treated cells exhibit an increase in binding activity toward the extended grp78 promoter as well as the individual cis-acting regulatory elements, CRE and CORE. Moreover, this increase in binding activity is also reduced by BAPTA. By competitory assays using the cis-acting regulatory elements as the competitors as well as the EMSA probes, we further show that all of the tested cis elements-CRE, CORE, and C1-are involved in the basal as well as in the TG-induced expression of grp78 and that the protein factor(s) that binds to the C1 region plays an important role in the formation and maintenance of the transcription complex.

show abstract

Modulation of nuclear rotation in neuronal interphase nuclei by nerve growth factor, by γ‐aminobutyric acid, and by changes in intracellular calcium

Fung

Boni

1988

Cell Motil. Cytoskeleton

View full text Add to dashboard Cite

Nuclear rotation (NR) is typically measured as motion of nucleoli within nuclei of cells in vitro. This occurs in cycling cells. However, its observation in neurons arrested in interphase indicates that mechanisms related to mitosis are not a prerequisite. We have recently shown that NR occurs in three dimensions within the nuclear space, that it occurs within the space delineated by the outer nuclear membrane and that it includes chromatin domains in addition to nucleoli and have postulated that this motion of chromatin domains is related to changes in gene expression. We now show that exposure of dorsal root, sensory neurons in vitro to nerve growth factor (NGF) or to gamma-aminobutyric acid (GABA), agents which alter gene expression, and to agents causing redistribution of calcium, such as EGTA and the calcium ionophore A23187, significantly alters NR. The NGF increased the mean rate of NR and did so at a time after exposure when activity of RNA polymerases have been shown to rise. Exposure to GABA resulted, within minutes, in shifts of the nucleolus within the three-dimensional space of the nucleus, associated in some neurons with significant, sigmoidal increases in the rate of NR. The calcium ionophore A23187 as well as chelation of extracellular calcium with EGTA similarly increased rates. Importantly, excess calcium, with EGTA remaining present, returned NR of all nucleoli to rates not different from controls. This indicates that the increase in NR seen with EGTA is specific to the chelation of calcium and not an nonspecific response to EGTA. It is difficult to link the action of agents which alter gene expression or transmembrane ion balance with changes in NR. Nevertheless, in support of our hypothesis, the results presented here show that agents known to alter gene expression, alter NR in a temporally coincident manner and that they do so, possibly, by calcium-dependent mechanisms.

show abstract

A stopped-flow investigation of calcium ion binding by ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid

Cited by 119 publications

References 4 publications

Calcium release and its voltage dependence in frog cut muscle fibers equilibrated with 20 mM EGTA.

Calcium release and its voltage dependence in frog cut muscle fibers equilibrated with 20 mM EGTA.

Thapsigargin-inducedgrp78 expression is mediated by the increase of cytosolic free calcium in 9L rat brain tumor cells

Modulation of nuclear rotation in neuronal interphase nuclei by nerve growth factor, by γ‐aminobutyric acid, and by changes in intracellular calcium

Contact Info

Product

Resources

About