A calorimetric investigation of the heats of binding of Mg ++ to poly A, to poly U, and to their complexes

The heats of binding of Mg⁺⁺ ions to poly A, poly U, and to their complexes, in the presence of Na⁺ ions, have been measurd calorimetrically. In all cases the heat, ΔH(θ), exhibitis a distinct dependence on the extent of binding, θ, and in the cases of poly A and poly U also on the Na⁺ concentration. The values of ΔH(θ) range from +2 to +3 kcal/mole of Mg⁺⁺ bound at θ = 0 to 1.3 kcal/mole at θ = 0.5 except in poly A where at θ = 0 ΔH(θ) = ?2 to ?3 kcal/mole. This is interpreted as being due to a facilitation of base stacking by the binding of Mg⁺⁺. The extent of facilitation is consistent with current estimates of base stacking. A similar effect but of much smaller magnitude is believed to obtain in poly A poly U. An interpretation of the dependence of ΔH(θ) on θ in terms of simple electrostatic interactions, but neglecting solvent effects, was attempted and found to be inadequate.     

The interaction of Na ions with synthetic polynucleotides

The interaction of Na⁺ with poly A, poly U, poly A·poly U, and Poly A·2 poly U has been investigated by means of potentiometry, by means of potentiometry, by means of a linked-function analysis of its effect on the binding of Mg⁺⁺ ions, and of K⁺ by means of an analysis of its effect on the sedimentation coefficients of the polymers. The last method was found to be inapplicable. The results of the other two methods were found to be consistent, except in the case of poly A where the existence of base stacking, influenced by the binding of Mg⁺⁺, significantly affects the linked-function analysis. The results are also consistent with the effects of the concentration of Na⁺ ions on the thermally induced conformational transitions of poly A·poly U and poly A·2 poly U, and with the extents of “binding” of Na⁺ to DNA measured by equilibrium and by transport methods. The interaction of Na⁺ with polynucleotides appears to be physically quite specific, although its thermodynamic basis is not clear. The extent of binding of Na⁺, Ψ, was found to be independent of the total Na⁺ concentration but a quadratic function of the extent of Mg⁺⁺ binding, θ. In the absence of Mg⁺⁺, Ψ = 0.35–0.38 for poly U, 0.40 for poly A, 0.59 for poly A·poly U, and 0.66 for poly A·2 poly U.     

Molecular properties and sensitivity to cations of β-Galactosidase from Streptococcus thermophilus with four enzyme substrates

Summary -Galactosidase (E.C. 3.2.1.23) from an autolytic strain of Streptococcus thermophilus was purified to near homogeneity (466 U/mg protein). The quaternary structure of the -galactosidase was complex, the enzyme apparently existing in three forms in solution (as determined by HPLC ion-exchange or gel permeation chromatography). One form of the enzyme was stable but a second form dissociated with time in a temperature- and concentration-dependent manner to give the stable form. A single subunit was identified with a molecular weight of 116 000.Activation of enzyme activity by cations (Mg²⁺, K⁺ and Na⁺) was complex and varied markedly according to whether o-nitrophenyl--d-galactopyranoside (ONPG), d-nitrophenyl--d-galactopyranoside (PNPG), 4-methylumbelliferyl--d-galactopyranoside (4MeUmG) or lactose was the enzyme substrate. With all substrates there was synergistic activation with either Mg⁺⁺ and K⁺ or Mg⁺⁺ and Na⁺. Na⁺ was the better activator with either ONPG or 4MeUmG as the substrate while K⁺ was the better activator of lactose and PNPG hydrolysis. With either ONPG or 4MeUmG as substrate, and in the presence of both Mg²⁺ and K⁺, Na⁺ further enhanced activity. In contrast, Na⁺ was a competitive inhibitor of the Mg²⁺ and K⁺ activated reaction with either lactose or PNPG as the substrate. Analysis of the effect of the cations on the kinetics of lactose hydrolysis showed they all acted by increasing the binding of lactose of the enzyme as well as by increasing the maximum activity. Weak competitive inhibition of activity (with lactose) by galactose was found with a K_i of 350 mM galactose.     

Conformational changes of membrane-bound (Na+−K+)-ATPase as revealed by antibody inhibition

Summary As different structural states of the (Na⁺–K⁺)-ATPase (EC 3.6.1.3) may lead to a changed reactivity to antibodies, the influence of Na⁺, K⁺, Mg⁺⁺, P_i and ATP on the reaction between highly purified (Na⁺–K⁺)-ATPase and antibodies directed against the membrane-bound enzyme was measured. The antigen antibody reaction was registered by measuring the antibody inhibition of (Na⁺–K⁺)-ATPase activity.In themembrane-bound but not in thesolubilized enzyme four different degrees of antibody inhibition were obtained at equilibrium of the antigen antibody reaction if different combinations of Na⁺, K⁺, Mg⁺⁺ and ATP were present during the incubation with the antibodies. Corresponding to the different degrees of inhibition, different rates of enzyme inhibition were measured. (a) The smallest degree of enzyme inhibition was obtained when (i) only Mg⁺⁺, (ii) Mg⁺⁺ and Na⁺ or (iii) Mg⁺⁺ and K⁺ were present during the antigen antibody reaction. (b) The enzyme activity was inhibited more strongly if Na⁺, Mg⁺⁺ and ATP were present together. (c) It was inhibited even more if only (i) Na⁺, (ii) K⁺, (iii) ATP or both (iv) ATP and Na⁺, (v) ATP and K⁺, (vi) ATP and Mg⁺⁺, or if (vii) no ATP and activating ions were present. (d) The highest degree of antibody inhibition was obtained if Mg⁺⁺, ATP and K⁺ were present together.In the presence of Mg⁺⁺ plus ADP and in the presence of Mg⁺⁺ plus the ATP analog adenylyl (--methylene) diphosphonate, Na⁺ and K⁺ did not influence the degree of antibody inhibition as they did in the presence of Mg⁺⁺ plus ATP. It was further found that the degree of antibody inhibition in the presence of Mg⁺⁺, ATP and K⁺ was affected by the sequence in which K⁺ and ATP were added to the enzyme prior to the addition of the antibodies.It is suggested that by antibody inhibition different conformations of the (Na⁺–K⁺)-ATPase could be detected. These conformations may possibly not occur in the solubilized enzyme and therefore do not seem to be necessarily linked to the intermediary steps of the ATP hydrolysis of the enzyme. The structural changes which are induced by Na⁺ and K⁺ in the presence of Mg⁺⁺ plus ATP are proposed to occur during the Na⁺–K⁺ transport.     

Interaction of magnesium ions with poly A and poly U

The binding of Mg⁺⁺ to poly A and poly U has been measured quantitatively by using the metallochromic indicator calmagite. The method is described in detail. It is shown that there is electrostatic interaction between the binding sites, viz., the phosphate groups, and the intrinsic association constant, for the specific binding can be determined. After extrapolation to zero ionic strength we find that, for the binding of Mg⁺⁺ to poly A, k_int = 4 × 10⁴ and for that, to poly U, k_int = 3 × 10⁴. The intrinsic enthalpy of association is negative. The effect of Mg⁺⁺ on the secondary structure of poly A and poly U has been studied by measuring the ultraviolet absorbance, optical rotatory dispersion and viscosity as a function of the amount of added Mg⁺⁺ ions. It was found that Mg⁺⁺ promotes the formation of a more ordered secondary structure by neutralizing or screening the negative charges. It is concluded from the absorbance measurements that for poly A at pH ? 7 and for poly U at pH >xs 9 this ordering involves stacking of the bases. Likewise, in solutions of UDP with a pH around 10, base stacking occurs on addition of Mg⁺⁺.     

Studies on (NA + K)-activated ATPase XLV. Magnesium induces two low-affinity non-phosphorylating nucleotide binding sites per molecule

ATP and adenylylimidodiphosphate (AdoPP[NH]P) bind to (Na⁺ + K⁺)-ATPase in the absence of Mg²⁺ (EDTA present) with a homogeneous but 15-fold different affinity, the K_d values being 0.13 μM and 1.9 μM, respectively. The binding capacities of the two nucleotides are nearly equal and amount to 3.9 and 4 nmol/mg protein or 1.7 and 1.8 mol/mol (Na⁺ + K⁺)-ATPase, respectively. The K_d value for ATP is equal to the K_m for phosphorylation by ATP (0.05–0.25 μM) and the binding capacity is equivalent to the phosphorylation capacity of 1.8 mol/mol (Na⁺ + K⁺)-ATPase. Hence, the enzyme contains two high-affinity nucleotide binding and phosphorylating sites per molecule, or one per α-subunit. Additional low-affinity nucleotide binding sites are elicited in the presence of Mg²⁺, as shown by binding studies with the non-phosphorylating (AdoPP[NH]P). The K_d and binding capacity for AdoPP[NH]P at these sites is dependent on the Mg²⁺ concentration. The K_d increases from 0.06 mM at 0.5 mM Mg²⁺ to a maximum of 0.26 mM at 2 mM Mg²⁺ and the binding capacity from 1.5 nmol/mg protein at 0.5 mM Mg²⁺ to 3.3 nmol/mg protein at 4 mM Mg²⁺. Extrapolation of a double reciprocal plot of binding capacity vs. total Mg²⁺ concentration yields a maximal binding capacity at infinite Mg²⁺ concentration of 3.8 nmol/mg protein or 1.7 mol/mol (Na⁺ + K⁺)-ATPase. The K_d for Mg²⁺ at the sites, where it exerts this effect, is 0.8 mM. The K_d for the high-affinity sites increases from 1.5–1.9 μM in the absence of Mg²⁺ to a maximum of 4.2 μM at 2 mM Mg²⁺ concentration. The binding capacity of these sites (1.8 mol/mol enzyme) is independent of the Mg²⁺ concentration. Hence, Mg²⁺ induces two low-affinity non-phosphorylating nucleotide binding sites per molecule (Na⁺ + K⁺)-ATPase in addition to the two high-affinity, phosphorylating nucleotide binding sites.     

Nature of the transport ATPase-digitalis complex. VI. Purification of and ouabain binding to a highly active cardiac Na+, K+-ATPase

A Na⁺,K⁺-ATPase has been isolated from canine heart with a specific activity as high as 200 μmoles of inorganic phosphate/mg protein/hour. Activity is not due to simple detergent activation since specific ouabain binding (i.e., [Mg⁺⁺,Na⁺,ATP] or [Mg⁺⁺,P_i]-ligand dependent) ranged from 200–450 pmoles/mg protein. Specific ouabain binding activities are up to ten times greater than heretofore reported.     

Effect of spermine on peptide-bond formation,catalyzed by ribosomal peptidyltransferase

The effect of spermine on the binding of AcPhe-tRNA to poly(U)-programmed ribosomes (step 1) and on the puromycin reaction (step 2) has been studied in a cell-free system, derived from E. coli.In the absence of ribosomal wash (FWR fraction) and at suboptimal concentration of Mg⁺⁺ (6 mM), spermine stimulated the binding of AcPhe-tRNA at least five fold, while at 10 mM Mg⁺⁺ there was a three fold stimulation. The above stimulatory effect was decreased at 6 mM Mg⁺⁺, or was abolished at 10 mM Mg⁺⁺ by the presence of FWR during the binding. Beside the stimulatory effect, spermine enhanced the stability of initiation complex AcPhe-tRNA-poly(U)-ribosome.In step 2, spermine affected the final degree of puromycin reaction and the activity status of peptidyltransferase. Both stimulatory and inhibitory effects have been observed, depending on the experimental conditions followed during the binding of the donor and during the peptide bond formation.     

Alterations in phospholipid-dependent (Na++K+)-ATPase activity due to lipid fluidity: Effects of cholesterol and Mg2+

The (Na⁺+K⁺)-activated, Mg²⁺-dependent ATPase from rabbit kidney outer medulla was prepared in a partially inactivated, soluble from depleted of endogenous phospholipids, using deoxycholate. This preparation was reactivated 10 to 50-fold by sonicated liposomes of phosphatidylserine, but not by non-sonicated phosphatidylserine liposomes or sonicated phosphatidylcholine liposomes. The reconstituted enzyme resembled native membrane preparations of (Na⁺+K⁺)-ATPase in its pH optimum being around 7.0 showing optimal activity at Mg²⁺: ATP mol ratios of approximately 1 and a K_m value for ATP of 0.4 mM.Arrhenius plots of this reactivated activity at a constant pH of 7.0 and an Mg²⁺: ATP mol ratio of 1:1 showed a discontinuity (sharp change of slope) at 17 °C, With activation energy (E_a) values of 13–15 kcal/mol above this temperature and 30–35 kcal below it. A further discontinuity was also found at 8.0 °C and the E_a below this was very high (> 100 kcal/mol).Incresed Mg²⁺ concentrations at Mg²⁺: ATP ratios in excess of 1:1 inhibited the (Na⁺+K⁺)-ATPase activity and also abolished the discontinuities in the Arrhenius plots.The addition of cholesterol to phosphatidylserine at a 1:1 mol ratio partially inhibited (Na⁺+K⁺)-ATPase reactivation. Arrhenius plots under these conditions showed a single discontinuity at 20°C and E_a values of 22 and 68kcal/mol above and below this temperature respectively. The ouabain-insensitive Mg²⁺-ATPase normally showed a linear Arrhenius plot with an E_a of 8 kcal/mol. The cholesterol-phosphatidylserine mixed liposomes stimulated the Mg²⁺-ATPase activity, which now also showed a discontinuity at 20 °C with, however, an increased value of 14 kcal/mol above this temperature and 6 kcal/mol below. Kinetic studies showed that cholesterol had no significant effect on the K_m for ATP.Since both of cholesterol and Mg²⁺ are know to alter the effects of temperature on the fluidity of phospholipids the above result are discussed in this context.     

The binding of adenosine to polyuridylic acid in which uracil residues are chemically altered.

The binding of adenosine-¹⁴C to polyuridylic acid (poly(U)) and several modified poly(U)s has been studied by equilibrium dialysis. The poly(U) was modified by addition of appropriate reagents across the 5,6-double bond of the uracil ring to form the photohydrate, photodimer, dihydrouracil, the HOBr addition product and the HSO₃^? addition product. Modification of the uracil rings decreases the amount of adenosine which can be bound to the poly(U); the decrease in binding is a function of the fraction of uracil rings which have been changed. Using the expression S = S₀(1 ? αr)² to relate the fraction of uracil rings modified (r) to the number of binding “sites” remaining (S), it is found that α is about 1 for all the modifications except photodimer where it is about 2. These observations are taken to mean that the loss of binding capacity of the poly(U) resulting from modifications of the uracil ring is caused by loss of planarity of the uracil rings caused by the modifications, and consequent loss of double helix structure, but that for all modifications except photodimer there is no disruption of the poly(U) double helix on either side of the leison. There does appear to be local melting on either side of the photodimer lesion. The sigmoidal binding isotherms (A_b versus C_a) of modified and unmodified poly(U) can be approximated closely by the following equation: ((1)) (1) where A_b = bound A, C_a = free A, n = minimum number of adjacent A′s in complex, S = concentration of sites on poly(U), and K₁ = (K_m)^1/m for all m ≥ n. The stacking energy of adenosine (w) can be calculated accurately using the following equation, where dθ/d ln C_a is obtained from Eq. (1). ((2)) (2) For unmodified poly(U), w is ?2.0 kcal/mole and ΔG° (?;RT ln K₁) is ?3.2 kcal/mole. The difference (?1.2 kcal/mole) is attributed to hydrogen bonding. Heavily photohydrated poly(U) does not bind guanosine or guanosine-5′-phosphate.     

Ouabain receptor interactions in (Na + K)-ATPase preparations. III. On the stability of the ouabain receptor against physical treatment, hydrolases and SH reagents

The action of ATP and its analogs as well as the effects of alkali ions were studied in their action on the ouabain receptor. One single ouabain receptor with a dissociation constant (K_D) of 13 nM was found in the presence of (Mg²⁺ + P_i) and (Na⁺ + Mg²⁺ + ATP). pH changes below pH 7.4 did not affect the ouabain receptor. Ouabain binding required Mg²⁺, where a curved line in the Scatchard plot appeared. The affinity of the receptor for ouabain was decreased by K⁺ and its congeners, by Na⁺ in the presence of (Mg²⁺ + P_i), and by ATP analogs (ADP-C-P, ATP-OCH₃). Ca²⁺ antagonized the action of K⁺ on ouabain binding. It was concluded that the ouabain receptor exists in a low affinity (R_α) and a high affinity conformational state (R_β). The equilibrium between both states is influenced by ligands of (Na⁺ + K⁺)-ATPase. With 3 mM Mg²⁺ a mixture between both conformational states is assumed to exist (curved line in the Scatchard plot).     

Modulation of single cardiac Na+ channels by cytosolic Mg++ ions

Elementary Na⁺ currents were recorded in inside-out patches excised from cultured neonatal rat heart myocytes in order to study the influence of cytosolic Mg⁺⁺ and other bivalent cations present at the cytoplasmic membrane surface on cardiac Na⁺ channel gating. Exposing the cytoplasmic membrane surface to a Mg⁺⁺-free environment shortened the open state of cardiac Na⁺ channels significantly. _open declined to 62±2% of the value obtained at 5 mmol/l Mg_i ⁺⁺. Other channel properties including the tendency to reopen and the elementary current size either changed insignificantly within a 10% range or remained completely unchanged. An almost identical change of _open can be caused by switching from a Mn⁺⁺ (5 mmol/l) containing internal solution to a Mn⁺⁺-free internal solution. But _open failed to significantly respond to a variation in internal Ni⁺⁺ from 5 mmol/l to 0 mmol/l. The same response to internal Mg⁺⁺ withdrawal was obtained with (–)-DPI-modified, non-inactivating Na⁺ channels, indicating that the exit rate from the open state remains as sensitive to cytosolic Mg⁺⁺ variations as in normal Na⁺ channels with operating inactivation. Offprint requests to: M. Kohlhardt     

Study of salt effects on adenosine–polyuridylic acid interaction

Complex formation between poly(U) and adenosine in solutions of salts that stabilize (Na₂SO₄), destabilize (NaClO₄), or have little effect on the water structure (NaCl), as well as the poly(U)·poly(A) interaction in NaClO₄, was studied by equilibrium dialysis and uv spectroscopy. At 3°C and neutral pH, Ado·2 poly(U) is formed in 1M NaCl and 0.33M Na₂SO₄. In NaClO₄ solutions under the same conditions, an Ado·poly(U) was found over the whole range of salt concentration investigated (10 mM?1M), which has not been previously observed under any conditions. The Ado-poly(U) was also found in a NaCl/NaClO₄ mixture, the transition from the triple- to the double-helical complex occurring within a narrow range of concentration of added NaClO₄. In the presence of 1M NaCl this transition is observed on adding as little as 10 mM NaClO₄, i.e., at a [ClO]/[Cl^?] ratio of about 1:100. However, when NaClO₄ is added to a 1M solution of the stabilizing salt Na₂SO₄, no transition occurs even at a [ClO]/[SO] ratio of 1:4. Investigation of melting curves and uv spectra has shown that in an equimolar mixture of the polynucleotides, only a double-helical poly(U)·poly(A) exists in 1M NaClO₄ at low temperatures; this also holds for 1M NaCl. This changes to a triple-helical 2 poly(U)·poly(A) and then dissociates as the temperature increases. At low temperatures and the poly(U)/poly(A) concentration ratio of 2:1, a mixture of 2 poly(U)·poly(A) and poly(U)·poly(A) was observed in 1M NaClO₄, in contrast to the case of 1M NaCl. Thus, sodium perchlorate, a strong destabilizer of water structure, promotes formation of double-helical complexes both in the polynucleotide–monomer and the polynucleotide–polynucleotide systems. Beginning with a sufficiently high ionic strength (μ ? 0.9), a further increase in the salt molarity results in an increase of the poly(U)·adenosine melting temperature in both stabilizing and neutral salts and a decrease in the destabilizing salt. In Na₂SO₄ concentrations higher than 1.2M Ado·2 poly(U) precipitates at room temperature. Analysis of the binding isotherms and melting profiles of the complexes between poly(U) and adenosine according to Hill's model shows that the cooperativity of binding, due to adenosine stacking on poly(U), increases in the order NaClO₄ < NaCl < Na₂SO₄. The free energy of adenosine stacking on the template is similar to that of hydrogen bonding between adenosine and poly(U) and ranges from ?1 to ?2 kcal/mol. The values of ΔH_t [the effective enthalpy of adenosine binding to poly(U) next to an occupied site, obtained from the relationship between complex melting temperature and free monomer concentration at the midpoint of the transition] are ?14.2, ?18.3, and ?16.8 kcal/mol for 1M solutions of NaClO₄, NaCl, and Na₂SO₄, respectively. The results indicate that the effects of anions of the salts studied are related to water structure alterations rather than to their direct interaction with the complexes between poly(U) and adenosine.     

Ionic Requirements for the Specific Binding of [3H]GBR 12783 to a Site Associated with the Dopamine Uptake Carrier

At 0°C, when Na⁺ was the only cation present in the incubation medium, increasing the Na⁺ concentration from 3 to 10 mM enhanced the affinity of [³H]l-[2-(di-phenylmethoxy)ethyl]-4-(3-phenyl-2-propenyl)piperazine ([³H]GBR 12783) for the specific binding site present in rat striatal membranes without affecting the 5_max. For higher Na⁺ concentrations, specific binding values plateaued and then slightly decreased at 130 mM Na⁺. In a 10 mM Na⁺ medium, the K_D and the B_max were, respectively, 0.23 nM and 12.9 pmol/mg of protein. In the presence of 0.4 nM [³H]GBR 12783, the half-maximal specific binding occurred at 5 mM Na⁺. A similar Na⁺ dependence was observed at 20°C. Scatchard plots indicated that K⁺, Ca²⁺, Mg²⁺, and Tris⁺ acted like competitive inhibitors of the specific binding of [³H]GBR 12783. The inhibitory potency of various cations (K⁺, Ca²⁺, Mg²⁺, Tris⁺, Li⁺ and choline) was enhanced when the Na⁺ concentration was decreased from 130 to 10 mM. In a 10 mM Na⁺ medium, the rank order of inhibitory potency was Ca²⁺ (0.13 mM) > Mg²⁺ > Tris⁺ > K⁺ (15 mM). The requirement for Na⁺ was rather specific, because none of the other cations acted as a substitute for Na⁺. No anionic requirement was found: Cl^-, Br^-, and F^- were equipotent. These results suggest that low Na⁺ concentrations are required for maximal binding; higher Na⁺ concentrations protect the specific binding site against the inhibitory effect of other cations.     

Binding of cations by microsomes from rabbit skeletal muscle

Fragmented sarcoplasmic reticulum and transverse tubular system, as isolated in the microsomal fraction from rabbit skeletal muscle, bind H⁺, Na⁺, K⁺, Ca⁺⁺, Mg⁺⁺, and Zn⁺⁺. The binding depends on a cation exchange type of interaction between these cations and the chemical components of the membranous systems of the muscle cell. The monovalent and divalent cations exchange quantitatively for each other at the binding sites on an equivalent basis. Scatchard plots of the H⁺ binding data indicate that the binding groups can be resolved into two major components in terms of their pK values. Component 1 has a pK value of 6.6 and a capacity for H⁺ binding of 2.2 meq/g N . The second component has a much higher H⁺ binding capacity (7–8 meq/g N ), but its pK value, 3.4, is non-physiological. The binding of cations other than H⁺ at a neutral pH occurs at the binding sites making up component 1. The order of affinity of the cations for the microsome binding sites is H⁺ » Zn⁺⁺ > Ca⁺⁺ > Mg⁺⁺ » Na⁺ = K⁺ as reflected by the apparent respective pK_M values: 6.6, 5.2, 4.7, 4.2, 1.3, 1.3. Caffeine, which causes contracture and potentiates the twitch of skeletal muscle, does not interfere with the binding of Ca⁺⁺ by the microsomes at neutral pH.     

Early changes of 'leak flux' and the cation content of lymphocytes by concanavalin A.

The leak fluxes of Na⁺, K⁺, Mg⁺⁺ and Ca⁺⁺ in mouse thymocytes are increased by Concavaline A (Con A), within minutes after mitogen addition. The intracellular Mg⁺⁺ and K⁺ concentrations were decreased and the Na⁺ and Ca⁺⁺ contents were increased by Con A in mouse thymocytes and spleen cells.     

On the selective adsorption of cations in the cell wall of the green algaValonia utricularis

The selective adsorption of the cations Na⁺, K⁺, Mg⁺⁺ and Ca⁺⁺ by the cell wall of the Mediterranean algaValonia utricularis (Siphonocladales, Chlorophyceae) from sea water of 40 %. S was investigated by extraction of cell-wall preparations, eluted before in 1.1 mol methanol (adjusted to pH 8) with 0.1 n formic acid in a Soxhlet apparatus. Na⁺ and K⁺ were determined by flame photometry, Mg⁺⁺ and Ca⁺⁺ by complexometric titration with EDTA. From calculation of the dry weight:fresh weight ratios and the chloride determinations in the eluates, the Donnan free-space fraction of the total cell-wall volume was calculated to about 35 %, and the analytical results of the cation concentrations in the extracts expressed asVal cm^–3 DFS. This calculation is based on the assumption that the acidic groups of the noncellulosic matrix material, carrying negative charges by dissociation at the reaction of sea water (ph about 8) are responsible for the adsorption of cations by exhibition of a Donnan effect. The results obtained show clearly that besides the divalent cations Mg⁺⁺ and Ca⁺⁺, which according to the physico-chemical laws of the Donnan distribution must be relatively accumulated to the second power of the monovalent ones, potassium is also enriched by selective adsorption, and the K⁺:Na⁺ ratio increased significantly compared with that in sea water. This seems to indicate that the strength of attraction between the cations and the negative sites is dependent on the radii of the ions and the state of hydration and/or polarisation of the ions and binding sites.     

Thermodynamic and kinetic parameters of ion condensation to polynucleotides: Outer sphere complex formed by Mg++ ions

The coupling of ion binding to the single strand helix—coil transition in poly (A) and poly(C) is used to obtain information about both processes by ion titration and field-jump relaxation methods. Characterisation of the field-jump relaxation in poly(C) at various concentrations of monovalent ions leads to the evaluation of a stability constant K = 71 M^?1 for the ion binding to the polymer. The rate constant of helix formation is found to be 1.3 × 10⁷ s^?1, whereas the dissociation rate is 1.0 × 10⁶ s^?1. Similar data are presented for poly (A) and poly (dA).The interaction of Mg⁺⁺ and Ca⁺⁺ with poly (A) and poly (C) is measured by a titration method using the polymer absorbance for the indication of binding. The data can be represented by a model with independent binding “sites”. The stability constants increase with decreasing salt concentration from 2.7 × 10⁴ M^?1 at medium ionic strengths up to 2.7 × 10⁷ M^?1 at low ionic strength. The number of ions bound per nucleotide residue is in the range 0.2 to 0.3. Relaxation time constants associated with Mg⁺⁺ binding are characterised over a broad range of Mg⁺⁺ concentrations from 5 μM to 500 μM. The observed concentration dependence supports the conclusion on the number of binding places inferred from equilibrium titrations. The rate of Mg⁺⁺ and Ca⁺⁺ association to the polymer is close to the limit of diffusion control (k_R = 1 × 10¹⁰ to 2 × 10¹⁰ M^?1 s^?1). This high rate demonstrates that Mg⁺⁺ and Ca⁺⁺ ions do not form inner-sphere complexes with the polynucleotides. Apparently the distance between two adjacent phosphates is too large for a simultaneous site binding of Mg⁺⁺ or Ca⁺⁺, and inner sphere complexation at a single phosphate seems to be too weak. The data support the view that the ions like Mg⁺⁺ and Ca⁺⁺ surround the polynucleotides in the form of a mobile ion cloud without site binding.     

Effect of magnesium ions on the high-affinity binding of eosin to the (Na+ + K+)-ATPase

(1) The fluorescence of eosin Y in the presence of (Na⁺ + K⁺)-ATPase is enhanced by Mg²⁺. The enhancement by Mg²⁺ is larger than that obtained with Na⁺ (Skou, J.C. and Esmann, M. (1981) Biochim. Biophys. Acta 647, 232–240). Mg²⁺ shifts the excitation maximum from 518 to 524 nm, the emission maximum from 538 to 542 nm. Also a shoulder appears at about 490 nm on the excitation curve, as was also observed with Na⁺. (2) The Mg²⁺-dependent enhancement of fluorescence can be reversed by K⁺ as well as by ATP. In the presence of Mg²⁺ + P_i (i.e. under conditions of phosphorylation), the fluorescence enhancement can be reversed by ouabain. With Mg²⁺ and a low concentation of K⁺ (i.e. conditions for vanadate binding), the enhancement of fluorescence can be reversed by vanadate. (3) There is a low-affinity binding of eosin which increases with the Mg²⁺ concentration. This is observed as a slight increase in the fluorescence when the excitation wavelength is above 520 nm. The low-affinity binding is K⁺-, ATP-, ouabain- and vanadate-insensitive. (4) Scatchard analysis of the binding experiments suggests that there are two high-affinity eosin-binding sites per ³²P-labelling site in the presence of 5 mM Mg²⁺ both of which are ouabain-, vanadate- and ATP-sensitive. With 5 M Mg²⁺ + 0.25 P_i, the K_d values are 0.14 μM and 1.3 μM, respectively. With 5 mM Mg²⁺, 150 mM Na⁺, the K_d values are 0.45 μM and 3.2 μM, respectively. With 5 mM Mg²⁺, the addition of K⁺ gives a pronounced decrease in affinity but does not decrease the number of binding sites (which remains at two per ³²P-labelling site). With 5 mM Mg²⁺ + 150 mM K⁺, the affinities of the two binding sites become identical, at a K_d of 17 μM. (5) The rate of conformational transitions was measured using the stopped-flow method. The rate of the transition from the Mg²⁺-form to the K⁺-form is high. Oligomycin has only a small (if any) effect on the rate. Addition of Na⁺ in the presence of Mg²⁺ does not appreciably change the rate of conversion to the K⁺-form, giving a rate constant of about 110 s^?. However, the addition of oligomycin in the presence of Mg²⁺ + Na⁺ had a profound effect: the rate of conversion to the K⁺-form was decreased by a factor of 2000 to about 0.063 s^?1. This suggests that the conformation with Mg²⁺ alone is different from the conformation with Na⁺ alone. (6) The effects of K⁺, ouabain, vanadate and ATP on the high-affinity binding of eosin suggest that the two eosin molecules bound per ³²P-labelling site are bound to ATP sites.