Dissociation Constant For Mg2 Research Articles

Tuning Mg(II) Selectivity: Comparative Analysis of the Photophysical Properties of Four Fluorescent Probes with an Alkynyl-Naphthalene Fluorophore.

Four alkynyl-1-naphthyl fluorophores have been synthesised with tri- or pentadentate ligating groups suited to the binding of magnesium. Their photophysical and binding properties for magnesium, calcium and zinc ions have been assessed using absorption, emission and excitation spectroscopy. Each compound has a pKa value between 6.2 and 5.1 and exhibits no significant pH response around pH 7.2. Enhanced selectivity for Mg2+ over Ca2+ was observed with a pentadentate phosphinate substituted system, compared to its carboxylate analogue, due to a 10-fold lowering of affinity for the Ca2+ ion. In each case, the overall dissociation constants for Mg2+ fall in the low mm range.

Blockade of NMDA-activated channels by magnesium in the immature rat hippocampus.

1. N-methyl-D-aspartate (NMDA)-receptor single-channel currents were examined in outside-out patches from thin slices of rat hippocampus to examine possible age dependence in the Mg(2+)-block of NMDA-mediated responses. The kinetics of Mg2+ block, among other channel properties, were compared between CA1 pyramidal cells from neonatal and 2-wk-old animals and dentate gyrus granule cells from neonatal and 4-wk-old animals. 2. Two distinct conductance states were seen consistently in nominally Mg(2+)-free solutions. The main conductance state was 50 and 56 pS in pyramidal cells and granule cells, respectively. The difference in NMDA-receptor channel conductance between the two cell types was statistically significant at all ages examined. Both cell types exhibited a less frequent 35- to 40-pS conductance state. 3. Channel closing rates showed no intrinsic voltage dependence in Mg(2+)-free solutions in any group. Open times became strongly voltage dependent when Mg2+ was added. The rate of block by Mg2+ was similar in all groups. 4. Voltage and Mg2+ influenced primarily the shortest time constant of shut-time distributions. Longer components varied relatively little with voltage or [Mg2+]. The effects of voltage and [Mg2+] suggest that brief shuttings represent closed states in the absence of Mg2+ and primarily a Mg(2+)-blocked state in the presence of Mg2+. 5. The rate of unblocking by Mg2+ was similar in all groups. Thus the dissociation constant for Mg2+ binding (i.e., the ratio of the unblocking and blocking rates) also showed little variation. 6. NMDA-receptor channels in two regions of the hippocampus behaved in a qualitatively similar fashion both in neonates and 2-or 4-wk-old rats. These observations are inconsistent with previous studies obtained using other methods, which suggested that block of NMDA receptor channels by Mg2+ increases substantially with age. Nevertheless, subtle developmental and regional differences in other aspects of NMDA-receptor channel behavior were detected, perhaps reflecting variations in molecular structure tailored to specific functional requirements.

Steady-state kinetics of substrate hydrolysis by vacuolar H(+)-pyrophosphatase. A simple three-state model.

The results of analyses of the steady-state kinetics of the vacuolar H(+)-translocating pyrophosphatase (V-PPase) of native tonoplast vesicles isolated from etiolated hypocotyls of Vigna radiata (mung bean) and purified enzyme from the same source under a wide range of Mg2+, pyrophosphate (PPi) and K+ concentrations are consistent with a minimal reaction scheme in which dimagnesium pyrophosphate is the active substrate species and catalysis is mediated by preformed enzyme-Mg2+ complex. When account is taken of the sensitivity of the V-PPase to ionic strength, additional kinetic interactions are not required to describe the behavior of the enzyme. N-Ethylmaleimide-protection assays show that the dissociation constant for Mg2+ binding in the absence of PPi is an order of magnitude smaller than that estimated from the steady-state kinetics of PPi hydrolysis. Two distinct Mg(2+)-binding sites are therefore invoked. Since the protective action of Mg2+ is independent of the nature of the monovalent cations and Mg2+ and K+ do not compete during substrate hydrolysis, divalent and monovalent cations are concluded to bind at separate sites. The pH dependencies of the kinetic parameters are consistent with the participation of groups of pKa 5.7 and 8.6 in substrate binding and groups of pKa 6.1 and 9.0 in the substrate-conversion step, indicating that at least four ionizable groups are essential for catalysis. These findings are discussed with respect to the reaction mechanism of the V-PPase and the potential regulatory significance of cytosolic free Mg2+ and K+ in vivo.

Adenosine cyclic 3',5'-monophosphate dependent protein kinase: kinetic mechanism for the bovine skeletal muscle catalytic subunit.

The kinetic mechanism for adenosine cyclic 3',5'-monophosphate dependent protein kinase was determined from initial velocity studies in the absence and presence of the product MgADP and dead-end inhibitors. Data are consistent with random addition of MgATP and Ser-peptide and ordered release of phospho-Ser-peptide and MgADP with a dead-end E-MgADP-Ser-peptide complex. In addition to the metal required for the nucleotide, we also characterized the binding of Mg2+ to a second site. Increasing the Mg2+ results in a 5-6-fold decrease in V,,, in the presence or absence of 0.1 M KCl. There is a 5-fold increase in V/KMgATP in the absence of KCl and a 13-fold increase in V/KMdTP at 0.1 M KC1. The effect of increasing free Mg2+ on V,,, and V/K was also obtained with MgITP (20% the V,,, with MgATP) and MgGTP (10% the V,,, with MgATP) as substrates. The dissociation constant for Mg2+ from E-Ser-peptide-Mg2+ and central complexes is 2-3 mM. At low concentrations of free Adenosine cyclic 3',5'-monophosphate dependent protein kinase catalyzes the phorphorylation of a variety of proteins according to

The Mg2+-induced conformational change in rabbit skeletal muscle G-actin.

The divalent cation Mg2+ binds specifically and tightly to a specific site on rabbit muscle G-actin, and it has been shown previously that such binding involves a conformational change in the monomeric actin as measured by a time-dependent change in the fluorescence of G-actin labeled with the fluorescent probe N-iodoacetyl N'-(5-sulfo-1-naphthyl)ethylenediamine (Frieden, C., Lieberman, D., and Gilbert, H. R. (1980) J. Biol. Chem. 255, 8991-8993). The characteristics of specific Mg2+-induced conformational changes using this labeled G-actin have been examined. The data are consistent with an initial poor binding of Mg2+ followed by an isomerization process resulting in tight binding. Mg2+ and Ca2+ compete for this site and the isomerization induced by Mg2+ can be reversed with high Ca2+ concentrations. The overall dissociation constant for Mg2+ binding is highly pH-sensitive, becoming larger with decreasing pH, implying that ionizable groups may control Mg2+ and Ca2+ binding and that these cations bind preferentially to the unprotonated form. Mg2+ binding to this site increases the off rate constant of the tightly bound ATP relative to Ca2+ by about 6-fold. It is suggested that Mg2+ binding to this site may be related to hydrolysis of the ATP.

Factors that influence the translocation of the N-carboxybiotin moiety between the two sub-sites of pyruvate carboxylase.

The active site of pyruvate carboxylase, like those of all biotin-dependent carboxylases, is believed to consist of two spatially distinct sub-sites with biotin acting as a mobile carboxy-group carrier oscillating between the two sub-sites. Some of the factors that influence the location and rate of movement of the N-carboxybiotin were studied. The rate of carboxylation of the alternative substrate, 2-oxobutyrate, was measured at 0 degrees C in an assay system where the isolated enzyme--[14C]carboxybiotin was the carboxy-group donor. The results are consistent with the hypothesis that the location of the carboxybiotin in the active site is determined by the presence of Mg2+, acetyl-CoA and the oxo acid substrate. The presence of Mg2+ favours the holding of the complex at the first sub-site, whereas alpha-oxo acids induce the complex to move to the second sub-site. At low concentrations pyruvate induces this movement but does not efficiently act as a carboxy-group acceptor; hydroxypyruvate, glyoxylate and oxamate, though not carboxylated, still induce the movement. The allosteric activator acetyl-CoA exerts only a slight stimulation on the rate of translocation to the second sub-site, and this stimulation arises from an increase in the dissociation constant for Mg2+.

Interaction of divalent cations with beta-galactosidase (Escherichia coli).

Although the addition of various divalent metals to beta-galactosidase resulted in apparent activation, only Mg2+ and Mn2+ actually did activate. The apparent activation by the other divalent metals was shown to be due to Mg2+ impurities. Calcium did not activate, but experiments suggested that it did bind. Other divalent metals which were studied failed to bind. The dissociation constants for Mg2+ and Mn2+ were 2.8 X 10(-7) and 1.1 X 10(-8) M, respectively, and in each case one ion bound per monomer. These constants corresponded very closely to apparent values which were obtained from activation studies. The apparent binding constant for Ca2+, obtained from competition studies, was 1.5 X 10(-5) M. Data were obtained which showed that Mg2+, Mn2+, and Ca2+ all compete for binding at a single site. Of interest and of possible molecular biological importance was the observation that, while Mg2+ bound noncooperatively (n = 1.0), Mn2+ did so in a highly cooperative manner (n = 3.4). The binding of Mn2+ (as compared to Mg2+) resulted in a twofold drop in the Vmax for the hydrolysis and transgalactosylis reactions of lactose but had little effect on the Vmax of hydrolysis of allolactose, p-nitrophenyl beta-D-galactopyranoside (PNPG), or o-nitrophenyl beta-D-galactopyranoside (ONPG); Km values were not effected differently for any of the substrates by Mn2+ as compared to Mg2+. When very low levels of divalent metal ions were present (0.01 M EDTA added) or when Ca2+ was bound with lactose as the substrate, a greater decrease was observed in the rate of the transgalactosylic reaction than in the rate of the hydrolytic reaction, and the Km values for lactose and ONPG were increased. Of the three divalent metal ions which bound to beta-galactosidase, only Mn2+ had significant stabilizing effects toward denaturing urea and heat conditions.

A fluorescence study of the effects of pH and Mg2+ on the conformation of fructose 1,6-diphosphatase from spinach chloroplasts.

2-p-Toluidino-naphthalene-6-sulfonate is a sensitive fluorescent reporter group which can be used for the detection of the conformation of fructose 1,6-diphosphatase from spinach chloroplasts. When fructose 1,6-diphosphatase was added to a dilute solution of 2-p-toluidino-naphthalene-6-sulfonate at pH 9.0, the fluorescence intensity gradually increased. At this pH, the enzyme activity decreased at the same rate. However, at neutral pH (7.5), this time-dependent fluorescence change was not observed. In the presence of Mg2+, which is an activator of the enzyme, the fluorescence intensity was increased instantly and did not change for 30 min in the pH range 8.0--9.0. From the concentration dependence of the fluorescence intensity, the dissociation constant for Mg2+ was determined, Kdis = 3 mM. The effects of pH and Mg2+ on the conformation and activity of chloroplast fructose 1,6-diphosphatase are discussed.