Determination of the intracellular dissociation constant, KD, of the Fluo-3 � Ca2+ complex in mouse sperm for use in estimating intracellular Ca2+ concentrations

1999 ◽  
Vol 54 (4) ◽  
pp. 418-428 ◽  
Author(s):  
Pamela L. Rockwell ◽  
Bayard T. Storey
Keyword(s):  
Dissociation Constant ◽  
Mouse Sperm ◽  
Dissociation Constant Kd

Free cytosolic Ca2+ measured with Ca(2+)-selective electrodes and fura 2 in rat mesenteric resistance arteries

1993 ◽  
Vol 265 (2) ◽  
pp. H741-H746 ◽  
Author(s):  
P. E. Jensen ◽  
M. J. Mulvany ◽  
C. Aalkjaer ◽  
H. Nilsson ◽  
H. Yamaguchi
Keyword(s):  
Dissociation Constant ◽  
Isometric Force ◽  
Force Measurements ◽  
Resistance Arteries ◽  
Fura 2 ◽  
Good Accordance ◽  
Dissociation Constant Kd

Free cytosolic Ca2+ was measured with sub-micrometer-tip, double-barrelled, Ca(2+)-selective electrodes and fura 2 in rat mesenteric resistance arteries. The purpose was to establish intracellular free Ca2+ concentration ([Ca2+]i) values in resting and stimulated vessels. Isolated vessels were mounted for isometric force measurements. Measured with electrodes, mean [Ca2+]i was 115 and 708 nM under resting and norepinephrine-activated conditions, respectively. Fura 2 was calibrated intracellularly including determination of the intracellular dissociation constant (Kd) of the fura 2:Ca2+ complex. The intracellular Kd was 342 nM. With this value of Kd, fura 2 measurements of mean [Ca2+]i were 129 and 537 nM under resting and norepinephrine-activated conditions, respectively. The values measured with the two techniques were thus in good accordance.

scholarly journals Binding of NAD+ by cholera toxin

1987 ◽  
Vol 244 (1) ◽  
pp. 225-230 ◽  
Author(s):  
T S Galloway ◽  
S van Heyningen
Keyword(s):  
Binding Site ◽  
Dissociation Constant ◽  
Cholera Toxin ◽  
Equilibrium Dialysis ◽  
Accurate Determination ◽  
Polyacrylamide Gel ◽  
Nad Glycohydrolase ◽  
Competitive Inhibitors ◽  
Dissociation Constant Kd

1. The Km for NAD+ of cholera toxin working as an NAD+ glycohydrolase is 4 mM, and this is increased to about 50 mM in the presence of low-Mr ADP-ribose acceptors. Only molecules having both the adenine and nicotinamide moieties of NAD+ with minor alterations in the nicotinamide ring can be competitive inhibitors of this reaction. 2. This high Km for NAD+ is also reflected in the dissociation constant, Kd, which was determined by a variety of methods. 3. Results from equilibrium dialysis were subject to high error, but showed one binding site and a Kd of about 3 mM. 4. The A1 peptide of the toxin is digested by trypsin, and this digestion is completely prevented by concentrations of NAD+ above 50 mM. Measurement (by densitometric scanning of polyacrylamide-gel electrophoretograms) of the rate of tryptic digestion at different concentrations of NAD+ allowed a more accurate determination of Kd = 4.0 +/- 0.4 mM. Some analogues of NAD+ that are competitive inhibitors of the glycohydrolase reaction also prevented digestion.

Synthesis and characterization of 19F NMR chelators for measurement of cytosolic free Ca

1987 ◽  
Vol 252 (4) ◽  
pp. C441-C449 ◽  
Author(s):  
L. A. Levy ◽  
E. Murphy ◽  
R. E. London
Keyword(s):  
Chemical Shifts ◽  
Cell Types ◽  
Free Calcium ◽  
Tetraacetic Acid ◽  
Nmr Studies ◽  
Cytosolic Free Calcium ◽  
Fluorine Nmr ◽  
Dissociation Constant Kd

Fluorine 19 nuclear magnetic resonance (NMR) studies of intracellular fluorinated calcium chelators provide a useful strategy for the determination of cytosolic free calcium levels in cells and perfused organs. However, the fluorinated chelator with the highest affinity for calcium ions which has been described to date. 1,2-bis-(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA), exhibits a dissociation constant (Kd) value 5- to 10-fold greater than the intracellular calcium concentration levels in most cell types, thus limiting the ability of fluorine NMR to report these concentrations reliably. We have consequently designed and synthesized several fluorinated calcium chelators with higher affinity for calcium. The best of these, 2-(2-amino-4-methyl-5-fluorophenoxy)-methyl-8 aminoquinidine-N,N,N',N'-tetraacetic acid (quinMF), has a Kd value approximately 10 times lower than that of 5FBAPTA. Several of the newly synthesized indicators have different chemical shifts for the calcium complexed and uncomplexed chelators to allow the simultaneous use of two indicators. In addition to providing information about the level of cytosolic free calcium, chelators containing a quinoline ring exhibit considerable sensitivity to magnesium levels and hence have potential application for the determination of cytosolic-magnesium concentrations. Application of these chelators is illustrated by determination of the cytosolic-free calcium level in erythrocytes. Use of quinMF, the chelator with the lowest Kd value, gives a calcium value of 25-30 nM.

Determination of dissociation constant of phosphinate group in phosphinic pseudopeptides by capillary zone electrophoresis

2002 ◽  
Vol 770 (1-2) ◽  
pp. 145-154 ◽  
Author(s):  
Dušan Koval ◽  
Václav Kašička ◽  
Jiřı́ Jiráček ◽  
Michaela Collinsová ◽  
Timothy A. Garrow
Keyword(s):  
Dissociation Constant ◽  
Capillary Zone Electrophoresis ◽  
Zone Electrophoresis ◽  
Capillary Zone

scholarly journals Determination of ionized magnesium in platelets and correlation with selected variables

1996 ◽  
Vol 42 (5) ◽  
pp. 744-748 ◽  
Author(s):  
J E Niemela ◽  
B M Snader ◽  
R J Elin
Keyword(s):  
Total Cholesterol ◽  
Inverse Correlation ◽  
Reference Interval ◽  
Cholesterol Concentration ◽  
Magnesium Concentration ◽  
Serum Electrolytes ◽  
Ionized Magnesium ◽  
And Gender ◽  
Dissociation Constant Kd

Abstract We describe a method for determining the intracellular ionized magnesium concentration ([Mg2+]i) in platelets by using the fluorescent probe FURAPTRA. We determined the dissociation constant (KD) of FURAPTRA for Mg2+ (2.26 +/- 0.29 mmol/L), within-day assay variability (CV = 6.8%), among-day intraindividual variability (CV = 11.0%), variability after a 4-h delay in processing the blood specimen (t = 1.2, P >0.2; F = 6.2, P <0.02), and the reference interval (0.23-0.59 mmol/L) for this assay. We also evaluated the correlation between platelet [Mg2+]i and concentrations of selected serum electrolytes, proteins, and total cholesterol; age; body mass index; and gender. Only the inverse correlation between platelet [Mg2+]i and serum total cholesterol concentration in men was significant (r=-0.66, P <0.005).

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