Interaction of effecting ligands with lac repressor and repressor-operator complex.

The equilibrium association constants for the binding of a wide variety of effecting ligands of the lac repressor were measured by equilibrium dialysis. Also, detailed investigations of the apparent rate of dissociation of repressor-operator comples as a function of ligand concentration were carried out for several inducers and anti-inducers. The affinity of repressor-ligand comples for operator DNA was evaluated from the specific rate constants at saturating concentrations of effecting ligand. By fitting the experimental data depicting the functional dependence of the rate of dissociation upon ligand concentrations to calculated curves, assuming simple models of the induction mechanism, the equilibrium association constant for the binding of effecting ligand to repressor-operator comples was determined. Inducers reduce the affinity of lac repressor for operator DNA by a factor of approximately 1000 under standard conditions; the extent of destabilization depends on Mg2+ ion concentration. Anti-inducers increase the affinity of repressor for operator at most a factor of five. Only one neutral ligand, which binds to repressor without altering the stability of repressor-operator comples, was found. No homotropic or heterotropic interactions in the binding of effecting ligands either to repressor or to repressor-operator complex are evident.     

Interaction of ADP and magnesium with the active site of myosin subfragment-1 observed by reactivity changes of the critical thiols and by direct binding methods at low and high ionic strength

Comprehensive binding studies using direct and indirect methods yield stoichiometry and affinities for the binding of Mg X ADP and uncomplexed ADP to the active site of myosin subfragment-1. Additionally, the binding parameters for Mg2+ in the ternary complex protein X Mg X ADP are presented for the first time. The indirect method makes use of reactivity changes of the critical thiol-1 and thiol-2 groups, which occur upon the binding of the ligand at the active site. The affinity constants derived by this method are corroborated by two independent direct methods, equilibrium dialysis and centrifugation transport. For Mg2+, ADP and Mg X ADP just one mole of ligand binds/mole subfragment-1. The affinity of Mg X ADP at low ionic strength is 2.1 X 10(6) M-1 and only five-times lower in the absence of Mg2+. In the ternary complex Mg2+ has a low affinity of 4.1 X 10(4) M-1. At high ionic strength the uncomplexed ADP binds with a 43-times-lower affinity than Mg X ADP, whose affinity is 6.9 X 10(5) M-1. In this case Mg2+ interacts in the ternary complex with the higher affinity of 3.2 X 10(5) M-1, implying that at high salt concentration it plays a more prominent role in anchoring ADP at the active site.     

Initial rate kinetic analysis of the mechanism of initiation complex formation and the role of initiation factor IF-3.

Initial rate kinetics of the formation of ternary complexes of Escherichia coli 30S ribosomal subunits, poly(uridylic acid), and N-acetylphenylalanyl transfer ribonucleic acid in the presence and in the absence of IF-3 are consistent with the hypothesis that the ternary complex is formed through a random order of addition of polynucleotide and aminoacyl-tRNA to separate and independent binding sites on the 30S ribosomes. The transformation of an intermediate into a stable ternary complex which probably entails a rearrangement of the ribosome structure leading to a codon-anticodon interaction represents the rate-limiting step in the formation of the ternary complex. The rate constant of this transformation, as well as the association constants for the formation of the 30S-poly(U) and 30S-N-AcPhe-tRNA binary complexes, are enhanced by the presence of IF-3 which acts as a kinetic effector on reactions which are intrinsic properties of the 30S ribosome. The IF-3-induced modification of these kinetic parameters of the 30S ribosomal subunit can per se explain the effect of IF-3 on protein synthesis without invoking a specific action at the level of the mRNA-ribosome interaction. This seems to be confirmed by the finding that IF-3 can stimulate several-fold the formation of a ternary complex even if one by-passes the ribosome-template binding step by starting with a covalent 30S-polynucleotide binary complex. Furthermore, the above-mentioned changes induced by IF-3 appear to be compatible with the previously proposed idea that the binding of the factor modifies the conformation of the 30S subunit. The random order of addition of substrates determined for the 30S-N-AcPhe-tRNA-poly(U) model system was found to be valid also for the more physiological 30S initiation complex containing poly(A,U.G) and (fMet-tRNA formed at low Mg2+ concentration in the presence of GTP and all three initiation factors.     

Kinetic mechanism of elongation factor Ts-catalyzed nucleotide exchange in elongation factor Tu.

The interaction of Escherichia coli elongation factor Tu (EF-Tu) with elongation factor Ts (EF-Ts) and guanine nucleotides was studied by the stopped-flow technique, monitoring the fluorescence of tryptophan 184 in EF-Tu or of the mant group attached to the guanine nucleotide. Rate constants of all association and dissociation reactions among EF-Tu, EF-Ts, GDP, and GTP were determined. EF-Ts enhances the dissociation of GDP and GTP from EF-Tu by factors of 6 x 10(4) and 3 x 10(3), respectively. The loss of Mg(2+) alone, without EF-Ts, accounts for a 150-300-fold acceleration of GDP dissociation from EF-Tu.GDP, suggesting that the disruption of the Mg(2+) binding site alone does not explain the EF-Ts effect. Dissociation of EF-Ts from the ternary complexes with EF-Tu and GDP/GTP is 10(3)-10(4) times faster than from the binary complex EF-Tu.EF-Ts, indicating different structures and/or interactions of the factors in the binary and ternary complexes. Rate constants of EF-Ts binding to EF-Tu in the free or nucleotide-bound form or of GDP/GTP binding to the EF-Tu.EF-Ts complex range from 0.6 x 10(7) to 6 x 10(7) M(-1) s(-1). At in vivo concentrations of nucleotides and factors, the overall exchange rate, as calculated from the elemental rate constants, is 30 s(-1), which is compatible with the rate of protein synthesis in the cell.     

Kinetic and equilibrium characterization of the Tet repressor-tetracycline complex by fluorescence measurements. Evidence for divalent metal ion requirement and energy transfer

The interaction of Tet repressor protein with the inducer tetracycline was studied by fluorescence measurements, equilibrium dialysis and nitrocellulose filter binding. The repressor-tetracycline complex was formed from two molecules of tetracycline and one Tet repressor dimer. Formation of the complex requires divalent cations, and results in drastic effects upon the fluorescence spectra of both compounds. The fluorescence of Tet repressor was quenched about 70%, while that of tetracycline was increased between three- and eightfold, depending upon pH. In addition, the emission maximum of the protein was shifted from 330 to 340 nm, and the excitation maximum of tetracycline dropped from 380 to 370 nm. The latter shift is accompanied by a similar change in the absorption spectra. An analogous effect was observed upon changing the environment of the drug by the addition of sodium dodecyl sulphate. These results suggest that tetracycline binds to a hydrophobic region of the protein. A new excitation band in the fluorescence spectrum of the complex is observed. This presumably arises from energy transfer from a tryptophan to the drug. The association rate constant for formation of the complex is 3.3(+/- 0.3) X 10(5) M-1 s-1, and the equilibrium association constant is 2.8(+/- 0.5) X 10(9) M-1. These results are discussed with respect to the biological function of the Tet repressor.     

Dynamics of repressor-operator recognition: the Tn10-encoded tetracycline resistance control

Binding of the Tet repressor to nonspecific and specific DNA leads to quenching of the Tet fluorescence by approximately 22% and approximately 35%, respectively. This effect is used for a direct, quantitative characterization of the binding equilibria and dynamics involved in the recognition of the operator by its repressor. From the dependence of the nonspecific binding constant on the ion concentration, it is concluded that nonspecific binding is almost completely driven by the entropy change resulting from the release of three to four Na+ ions from the double helix upon protein binding. Formation of the specific complex is driven by a higher entropy term resulting from the release of seven to eight Na+ ions and in addition by a free energy term of -33 kJ/mol from nonelectrostatic interactions, which are attributed to the specific contacts. The dynamics of the repressor-operator recognition are resolved by stopped-flow measurements at various salt concentrations and for different DNA chain lengths into two separate steps. The first step follows a second-order mechanism and results in an intermediate complex associated with formation of about three to four electrostatic contacts between protein and DNA; apparently, this complex is equivalent to the nonspecific complex. The existence of an intermediate is also indicated by experiments in mixed Na+-Mg2+ buffers, which can be described with high accuracy by competition of Mg2+ and protein. The intermediate complex is formed at a rate of 3 X 10(8) M-1 s-1 and is converted in the second reaction step to the specific complex with a rate constant of 6 X 10(4) s-1, which is almost independent of the salt concentration. Our interpretation and the parameters obtained from our model are confirmed by competition of nonspecific DNA with operator DNA for repressor binding. The observed maximal rate constant of 3 X 10(8) M-1 s-1 is very close to theoretical predictions for the association without a sliding mechanism. The very small dependence of the observed rate constants on the chain length shows that the Tet repressor is not able to slide over any substantial distance even at low salt concentrations. The question of a potential contribution from sliding under our experimental conditions is critically discussed. The absence of sliding in the case of the Tet repressor under physiological conditions is compared with the high sliding efficiency of the lac repressor and is discussed with respect to possible molecular mechanisms of sliding in relation to biological function.     

Leucine aminopeptidase (bovine lens). Effect of pH on the relative binding of Zn2+ and Mg2+ to and on activation of the enzyme.

Incubation of leucine aminopeptidase (bovine lens) (EC 3.4.1.1) with various concentrations of Mg2+ at various pH values in 1 M KCl and 0.155 M trimethylamine-HCl at 37 degrees confirms that Mg2+ competes with Zn2+ for binding only 1 site per 54,000-dalton subunit. The ratio of the apparent association constants (1KZn:1KMg = 1KZn/Mg) at this site (site 1) was estimated to be 20,720 at pH 8.16, 10,570 at pH 8.44, 3,590 at pH 8.78, and 660 AT PH 9.14. The decrease in values of 1KZn/Mg with increasing pH in the activation of leucine aminopeptidase by Mg2+ is attributed to the lowering of the free Zn2+ concentration relative to that of free Mg2+ caused by the formation of ZnOH+ and Zn(OH)2 complexes with increasing OH- concentration. When corrections are made for the binding of Zn2+ by OH- ions, the pH-independent ratio of association constants (1KZn:1KMg = 1KZn/Mg) for the relative binding of Zn2+ and Mg2+ at site 1 of leucine aminopeptidase in 29,800. From the effect of pH on the relative binding constant, a value (beta2) for the product of the two stepwise association constants for the formation of Zn(OH)2 from Zn2+ and OH- (Zn2+ + OH- in equilibrium ZnOH+; ZnOH+ + OH- in equilibrium Zn(OH)2) was estimated to be 4.42 X 10(10) M-2 at 37 degrees. Values of Km at pH 7.5 AND 30 degrees with L-leucine p-nitroanilide as substrate in the presence of 0.01 M NaHCO3 are 4.13 and 2.01 mM for the zinc-zinc and magnesium-zinc enzymes, respectively. Values for Vmax are 0.2 and 2.49 mumol/min/mg, respectively.     

Kinetics of dissociation of parvalbumin complexes with calcium and magnesium ions

Dissociation kinetics of parvalbumin complexes with calcium and magnesium ions were studied by means of stopped-flow method employing intrinsic protein fluorescence registration. In the temperature range from 10 to 30 degrees C the kinetic curves of Ca2+ and Mg2+ dissociation are best fitted with a sum of two exponential terms, each term is ascribed to a dissociation process in one of two bindings sites of parvalbumin. Dissociation rate constants in this temperature range increase from 0.03 to 0.8 s-1 and from 0.18 to 5 s-1 for Ca2+, and from 0.9 to 4.5 s-1 and from 4 to 33 s-1 for Mg2+. Parvalbumin equilibrium binding constants of Ca2+ and Mg2+ were also measured in the same temperature range. It makes possible to estimate the rate constants of association of Ca2+ and Mg2+. In the case of Ca2+ the rate of association approaches the diffusion controlled limit.     

Equilibrium and kinetic studies on the interaction of tetracyclines with calcium and magnesium

The interaction of Ca2+ and Mg2+ with three Tetracycline antibiotics (tetracycline, chlorotetracycline, and oxytetracycline) has been investigated. Spectrophotometric measurements have been used to determine the apparent association constant for this interaction as a function of pH. It is shown that the results are consistent with a model in which the metal ion can form complexes with both the fully-deprotonated and mono-protonated forms of the Tetracycline. The temperature-jump relaxation method has been used to measure the kinetics of formation of the complexes of Mg2+ with the Tetracyclines. The results are compared with those of previous studies of Mg2+ complex formation reactions and it is shown that the data is consistent with the normal dissociative model. A possible role for metal ion chelation in the mechanism of antibacterial action of the Tetracyclines is discussed.     

Thermodynamic analysis of the lactose repressor-operator DNA interaction

Kinetic and equilibrium constants for lactose repressor-operator DNA interaction have been examined as a function of salt concentration, size and sequence context of the operator DNA, and temperature. Significant salt effects were observed on kinetic and equilibrium parameters for pLA 322-8, an operator-containing derivative of pBR 322, and pIQ, an operator and pseudooperator-containing derivative of pBR 322. The association rate constant and equilibrium constant for the 40 base pair operator fragment were also salt dependent. Data for all the DNAs were consistent with a sliding mechanism for repressor-operator association/dissociation [Berg, O. G., & Blomberg, C. (1978) Biophys. Chem. 8, 271-280]. Calculation of the number of ionic interactions based on salt dependence yielded a value of approximately 8 for repressor binding to pIQ and pLA 322-8 vs. approximately 6 for the repressor-40 base pair fragment. These data and the differences in binding parameters for the plasmids vs. the 40 base pair operator are consistent with the formation of an intramolecular ternary complex in the plasmid DNAs. Unusual biphasic temperature dependence was observed in the equilibrium and dissociation rate constants for pLA 322-8, pIQ, and the 40 base pair fragment. These observations coupled with a discontinuity found in the inducer association rate constant as a function of temperature suggest a structural change in the protein. The large positive entropy contributions associated with repressor binding to all the DNAs examined provide the significant driving force for the reaction and are consistent with involvement of ionic and apolar interactions in complex formation.     

Protein synthesis in rabbit reticulocytes. A study of the mechanism of Co-eIF-2 action

The characteristics of component activities in Co-eIF-2 (where eIF is eukaryotic initiation factor) protein complex have been studied. (i) At limiting concentrations, Co-eIF-2 promoted rapid GDP binding to eIF-2 and also GDP displacement from eIF-2 X GDP during ternary complex formation in the presence of GTP and Mg2+ (Co-eIF-2C activity) but did not significantly stimulate ternary complex formation by eIF-2. (ii) At higher concentrations, Co-eIF-2 significantly enhanced ternary complex formation by eIF-2 and also rendered the complex stable to aurintricarboxylic acid presumably as Co-eIF-2 became physically bound to the ternary complex (Co-eIF-2A activity). (iii) Ternary complex preformed in the presence of Co-eIF-2 and without Mg2+ dissociated upon subsequent addition of Mg2+ (Co-eIF-2B activity). This dissociation reaction was presumably due to loss of interaction of the Co-eIF-2A component in Co-eIF-2 with the ternary complex (reversal of Co-eIF-2A activity) as the complex became increasingly sensitive to aurintricarboxylic acid with increasing Mg2+ concentration. In another study, purified eIF-2 was freed of bound GDP by treatment with alkaline phosphatase and the characteristics of native and GDP-free eIF-2 were compared. (i) One mM Mg2+ inhibited (60%) ternary complex formation by native eIF-2 but not by GDP-free eIF-2. Addition of exogenous GDP rendered GDP-free eIF-2 sensitive to Mg2+ indicating that Mg2+ inhibition is due to eIF-2-bound GDP. (ii) In the presence of Mg2+, Co-eIF-2 stimulated similarly ternary and Met-tRNAf X 40 S X AUG complex formation by both native and GDP-free eIF-2. Such stimulatory activity in each case was strongly inhibited by prior phosphorylation of eIF-2 alpha subunit by heme-regulated translational inhibitor. (iii) Ternary complexes preformed using either native and GDP-free eIF-2 and excess Co-eIF-2A80 in the absence of Mg2+ did not form Met-tRNAf X 40 S X AUG complex. They required trace amounts of Co-eIF-2 for such activity.     

Lateral ligand-receptor interactions on membranes probed by simultaneous fluorescence-interference detection

We describe an experimental approach for studying ligand-receptor interactions in the plane of the membrane. The extracellular domains of the type I interferon receptor subunits ifnar1-EC and ifnar2-EC were tethered in an oriented fashion onto solid-supported, fluid lipid bilayers, thus mimicking membrane anchoring and lateral diffusion of the receptor. Ligand-induced receptor assembling was investigated by simultaneous total internal reflection fluorescence spectroscopy and reflectance interferometry (RIf). Based on a rigorous characterization of the interactions of fluorescence-labeled IFNalpha2 with each of the receptor subunits, the dynamics of the ternary complex formation on the fluid lipid bilayer was addressed in further detail making use of the features of the simultaneous detection. All these measurements supported the formation of a ternary complex in two steps, i.e., association of the ligand to ifnar2-EC and subsequent recruitment of ifnar1-EC on the surface of the membrane. Based on the ability to control and quantify the receptor surface concentrations, equilibrium, and rate constants of the interaction in the plane of the membrane were determined by monitoring ligand dissociation at different receptor surface concentrations. Using mutants of IFNalpha2 binding to ifnar2-EC with different association rate constants, the key role of the association rate constants for the assembling mechanism was demonstrated.     

Protein synthesis in rabbit reticulocytes: Mg2+-inhibition of ternary complex (Met-tRNA(f).eIF-2.GTP) formation by reticulocyte eIF-2

There are conflicting reports regarding Mg2+-inhibition of ternary complex formation by reticulocyte eIF-2. Several laboratories have reported that eIF-2 is isolated as eIF-2.GDP and Mg2+ inhibits ternary complex formation, as in the presence of Mg2+, GDP remains tightly bound to eIF-2 and prevents ternary complex formation. A protein factor, GEF is necessary for GDP displacement and subsequent ternary complex formation. Other laboratories have reported that Mg2+ has no effect on eIF-2 activity and eIF-2 forms near stoichiometric amount of ternary complex in the presence of Mg2+. In this paper, we provide evidence which suggests that the Mg2+-insensitive eIF-2 activity as reported by several laboratories might have been the result of the use of high Met-tRNA(f) concentrations in their assays as the nucleotides in excess tRNA bound Mg2+ in the reaction mixture and there was no free Mg2+ available to inhibit eIF-2 activity. Our data will show that the addition of excess tRNA promotes non-enzymatic GDP displacement from eIF-2.GDP and relieves Mg2+ inhibition.     

The binding of ribosomal protein S1 to S1-depleted 30S and 70S ribosomes. A fluorescence anisotropy study of the effects of Mg2+

We have determined the equilibrium constants for the binding of AEDANS-labelled S1 to S1-depleted 30S and 70S ribosomes. For "tight" ribosomes, the association of S1 increases with the sixth power of Mg2+ concentration, but for 30S subunits and "loose" ribosomes, there is virtually no dependence of the association on Mg2+ over the same concentration range, 2-10 mM in Mg2+. The binding of S1 to 70S ribosomes at 10 mM Mg2+ is stabilized by 2 kcal/mol compared to the binding to 30S subunits. When intact S1 binds to tight ribosomes, the fluorescence anisotrophy is that for virtually complete rotational immobilization. The anisotropies vary considerably with the preparation and treatment of both S1 and ribosomes and these variations are detailed here. The results suggest the linkage of Mg2+-dependent conformational changes in the intact ribosomes, perhaps including rRNA, and the binding of S1.     

Kinetic analysis of the interactions between troponin C (TnC) and troponin I (TnI) binding peptides: evidence for separate binding sites for the 'structural' N-terminus and the 'regulatory' C-terminus of TnI on TnC

The Ca(2+)/Mg(2+)-dependent interactions between TnC and TnI play a critical role in regulating the 'on' and 'off' states of muscle contraction as well as maintaining the structural integrity of the troponin complex in the off state. In the present study, we have investigated the binding interactions between the N-terminus of TnI (residues 1-40 of skeletal TnI) and skeletal TnC in the presence of Ca(2+) ions, Mg(2+) ions and in the presence of the C-terminal regulatory region peptides: TnI(96-115), TnI(96-131) and TnI(96-139). Our results show the N-terminus of TnI can bind to TnC with high affinity in the presence of Ca(2+) or Mg(2+) ions with apparent equilibrium dissociation constants of K(d(Ca(2+) ) ) = 48 nM and K(d(Mg(2+) ) ) = 29 nM. The apparent association and dissociation rate constants for the interactions were, k(on) = 4.8 x 10(5) M (-1) s(-1), 3.4 x 10(5) M (-1) s(-1) and k(off) = 2.3 x 10(-2) s(-1), 1.0 x 10(-2) s(-1) for TnC(Ca(2+)) and TnC(Mg(2+)) states, respectively. Competition studies between each of the TnI regions and TnC showed that both TnI regions can bind simultaneously to TnC while native gel electrophoresis and SEC confirmed the formation of stable ternary complexes between TnI(96-139) (or TnI(96-131)) and TnC-TnI(1-40). Further analysis of the binding interactions in the ternary complex showed the binding of the TnI regulatory region to TnC was critically dependent upon the presence of both TnC binding sites (i.e. TnI(96-115) and TnI(116-131)) and the presence of Ca(2+). Furthermore, the presence of TnI(1-40) slightly weakened the affinity of the regulatory peptides for TnC. Taken together, these results support the model for TnI-TnC interaction where the N-terminus of TnI remains bound to the C-domain of TnC in the presence of high and low Ca(2+) levels while the TnI regulatory region (residues 96-139) switches in its binding interactions between the actin-tropomyosin thin filament and its own sites on the N- and C-domain of TnC at high Ca(2+) levels, thus regulating muscle contraction.     

Effects of the mutation glycine-222----aspartic acid on the functions of elongation factor Tu

We have studied the properties of a mutant elongation factor Tu, encoded by tufB (EF-TuBo), in which Gly-222 is replaced by Asp. For its purification from the kirromycin-resistant EF-Tu encoded by tufA (EF-TuAr), a method was developed by exploiting the different affinities to kirromycin of the two factors and the competition between kirromycin and elongation factor Ts (EF-Ts) for binding to EF-Tu. The resulting EF-TuBo kirromycin and EF-TuAr EF-Ts complexes are separated by chromatography on diethylaminoethyl-Sephadex A-50. For the first time we have succeeded in obtaining a tufB product in homogeneous form. Compared with wild-type EF-Tu, EF-TuBo displays essentially the same affinity for GDP and GTP, with only the dissociation rate of EF-Tu GTP being slightly faster. Protection of amino-acyl-tRNA (aa-tRNA) against nonenzymatic deacylation by different EF-Tu species indicates that conformational alterations occur in the ternary complex EF-TuBo GTP aa-tRNA. However, the most dramatic modification is found in the EF-TuBo interaction with the ribosome. Its activity in poly(Phe) synthesis as well as in the GTPase activity associated with the interaction of its ternary complex with the ribosome mRNA complex requires higher Mg2+ concentrations than wild-type EF-Tu (Mg2+ optimum at 10-14 vs. 6 mM), even if EF-TuBo can sustain enzymatic binding of aa-tRNA to ribosomes at low Mg2+. The anomalous behavior of EF-TuBo is reflected in a remarkable increase of the fidelity in poly(Phe) synthesis, especially at high Mg2+ concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium-proton and calcium-magnesium antagonisms in calmodulin: microcalorimetric and potentiometric analyses

Microcalorimetry, pH potentiometry, and direct binding studies by equilibrium dialysis or gel filtration were performed to determine the thermodynamic functions delta Ho, delta Go, and delta So guiding the interactions of Ca2+, Mg2+, and H+ with bovine brain calmodulin. At pH 7.5, Ca2+ and Mg2+ binding are both endothermic with enthalpy changes of 19.5 and 72.8 kJ X (mol of calmodulin)-1, respectively. These enthalpy changes are identical for each of the four ion-binding domains. The affinity constants also are identical with intrinsic values of 10(5) M-1 for Ca2+ and 140 M-1 for Mg2+. Ca2+ and Mg2+ do not compete for the same binding sites: at high concentrations of both ions, a calmodulin-Ca4-Mg4 species is formed with an enthalpy value of 24.4 kJ X mol-1 with respect to calmodulin-Ca4 and -28.8 kJ X mol-1 with respect to calmodulin-Mg4. Moreover, in the presence of high concentrations of Ca2+, the affinity of each of the four ion-binding domains in calmodulin for Mg2+ is decreased by a factor of 4 and vice versa, indicative of negative free-energy coupling between Ca2+ and Mg2+ binding. Protons antagonize Ca2+ and Mg2+ binding in a different manner. Ca2+-H+ antagonism is identical in each of the four Ca2+-binding domains in the pH range 7.5-5.2. Our analyses suggest that three chemical geometries, probably carboxyl-carboxylate interactions, are responsible for this antagonism with ionization constants of 10(6.2) M-1 in the metal-free protein. Mg2+-H+ antagonism also is identical for each of the Mg2+-binding sites but is qualitatively different from Ca2+-H+ antagonism.(ABSTRACT TRUNCATED AT 250 WORDS)     

The interaction of magnesium ions with teichoic acid.

The binding of Mg2+ to the wall teichoic acid of Lactobacillus buchneri N.C.I.B. 8007 was measured by equilibrium dialysis at controlled ionic concentration and pH. In an aqueous solution containing 10mM-NaCl at pH 5.0 one Mg2+ ion was bound for every two phosphate groups of the teichoic acid, with an apparent association constant, Kassoc. = 2.7 x 10(3) M-1. On lowering the pH below the pKa of the phosphate groups the amount of bound Mg2+ decreased concomitantly with decreasing ionization of the phosphate groups. Both the amount of Mg2+ bound to the teichoic acid and the apparent association constants were similar in the presence of 10 mM concentrations of NaCl or KCl but decreased markedly in the presence of 10 mM-CaCl2 because of competition between Ca2+ and Mg2+ for the binding sites. A similar effect was found when the concentration of NaCl was increased from 0 to 50 mM. The results are discussed in relation to the function of teichoic acid in the walls of Gram-positive bacteria.