Kinetics of monensin complexation with sodium ions by 23Na NMR spectroscopy

The kinetics of the sodium binding to the ionophore monensin (Mon) in methanol has been studied by ²³Na NMR spectroscopy. Fast quadrupole relaxation of the bound sodium affected the relaxation rate of the free sodium through an exchange process between these two species. The exchange was found to be dominated by the reaction: Na⁺ + Mon^? ? MonNa. The dissociation rate constant at 25°C is 63 s^?1, with an activation enthalpy of 10.3 $\text{kcal}\text{mol}$ and activation entropy of ?15.8 $\text{cal}\text{mol}$ deg. These results indicate that the specificity of the binding of sodium ions to monensin is reflected in the relatively slow dissociation process. The entropy changes indicate that the activated monensin-sodium complex undergoes a conformational change, but the existence of a conformational change in monensin anion prior to complexation is excluded.     

Relaxation kinetics of the helix-coil transition of a self-complementary ribo-oligonucleotide: A7U7

Relaxation measurements on the kinetics of the double helix to coil transition for the self-complementary ribo-oligonucleotide A₇U₇ are reported over a concentration range of 6.9 μM to 19.6 μM in single strand in 1 M NaCl. The rate constants for helix formation are about 2 × 10⁶ M^?1 s^?1 and decrease with increasing temperature yielding an activation enthalpy of ?6 $\text{kcal}\text{mole}$. The rate constants for helix dissociation range from 3 to 250 s^?1 and increase with increasing temperature yielding an activation enthalpy of +45 $\text{kcal}\text{mole}$. The kinetic data reported here for 1 M NaCl is compared with previously published results obtained at lower salt concentrations. These data are discussed in terms of the quantitative effect of ionic strength on the kinetics of helix-coil transitions in oligo- and polynucleotides.     

Investigations on purine and pyrimidine bases stacking associations in aqueous solutions by the fluorescence quenching method: III. Intramolecular association of 9,9'-[1,3-propylene]-bis-2-aminopurine

General equations relating fluorescence quantum yield and lifetime of a compound with its intramolecular stacking equilibrium and kinetics were derived. Intramolecular stacking association of 9,9'-[1,3-propylene]-bis-2-aminopurine in aqueous solution was examined within the range of temperatures from 0 to 90°C. A two-state thermodynamic model of the association was verified. The stacking enthalpy and entropy can be taken, with a good approximation, as temperature-independent (δH = ?2.0 $\text{kcal}\text{mol}$, ΔS = ?3.25 e.u.) although the function ΔG = ?0.00886 T² + 8.847 T ?2876 describes more precisely the observed changes of stacking free enthalpy with temperature. The association rate constants were determined. Activation energy of the reaction (2 $\text{kcal}\text{mol}$) is the same as in the case of association between free 2-aminopurine molecules. It confirms a two-step mechanism of the process. The advantages and shortcomings of the fluorescence quenching method are discussed.     

Analysis of the kinetics of electron transfer reactions of hemoglobin and myoglobin with inorganic complexes.

The kinetics of methemoglobin reduction by Fe(EDTA)^2? have been studied and found to follow a second order rate law with k = 29.0 $\text{M}$^?1 s^?1 [25°C, μ = 0.2 $\text{M}$, pH 7.0 (phosphate)], $\text{ΔH}{}^3\text{= 5.5 ± 0.7 kcal/mol}$, and $\text{ΔS}{}^2\text{= ?33 ± 2 e.u.}$. The electrostatics-corrected self-exchange rate constant (k₁₁^corr) for hemoglobin based on the Fe(EDTA)^2? cross-reaction is 2.79×10^?3$\text{M}$^?1 s^?1. This rate constant is compared with others reported for a water-soluble iron porphyrin and calculated from published data for the reactions of myoglobin and hemoglobin with Fe(EDTA)^2? and Fe(CDTA)^2?/?. The k₁₁^corr values for these systems range over ten orders of magnitude with heme ? myoglobin > hemoglobin.     

A simple procedure for resolution of Escherichia coli RNA polymerase holoenzyme from core polymerase.

The association constant, K_A, for myosin subfragment-1 binding to actin was measured as a function of ionic strength [KCl, LiCl, and tetramethylammonium chloride (TMAC)]and temperature by the method of time-resolved fluorescence depolarization. The following thermodynamic values were obtained from solutions of 0.20 × 10^?6m S-1, 1.00 × 10^?6m actin in 0.15 m KCl, pH 7.0, at 25 °C: ΔG ° = ?39 ± 1 kJ M^?1, ΔH⁰ = 44 ± 2 kJ M^?1 and $\text{ΔS}{}^0\text{= 0.28 ± 0.01 kJ}\text{M}{}^{\mathrm{?1}}{}^0\text{K}{}^{\mathrm{?1}}$. For measurements in KCl (0.05 to 0.60 m), In $\text{K}{}_{\mathrm{a}}\text{= ?8.36 (KCl)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$. Thus, the binding is endothermic and strongly inhibited by high ionic strength. When KCl was replaced by LiCl or TMAC the ionic effects on the binding were cation specific. The nature of actin-(S-1) binding in the rigor state is discussed in terms of these results.     

Investigations on purine and pyrimidine bases stacking associations in aqueous solutions by the fluorescence quenching method: I. Autoassociation of 2-aminopurine

A general equation was derived, describing fluorescence quantum yield and lifetime of an autoassociating compound in liquid solutions. The autoassociation of 2-aminopurine in aqueous solution was examined within the range from 0 to 90°C. The compound seemed to associate cooperatively. The thermodynamic parameters of polymerization change with temperature, so that its free enthalpy ΔG = ?0.0797 T² + 45.4 T ?7893. The dimerization enthalpy and entropy are approximately temperature-independent (ΔH₂ = ?4.17 $\text{kcal}\text{mol}$, ΔS₂ = ?10.9 e.u.), although the function: ΔG₂ = ?0.0308 T² + 30.3 T - 7213 fits experimental points better. The observed dependences can be explained by the increasing role of the hydrophobic effect with temperature and size of the aggregates. The association rate constants were determined, and a two-step reaction mechanism was demonstrated. The first step is diffusion-controlled. The second is characterized by an activation energy of ~2 $\text{kcal}\text{mol}$ and an encounter distance of ~8.3 Å.     

Kinetic properties of rat hepatic prolactin receptors

Binding of ¹²⁵I-labelled ovine prolactin to female rat liver membranes underequilibrium conditions showed an apparent K_d of 200 pM, and a Hill coefficient of 1.0. The association rate was second order, with a rate constant K₁, of 2.1 × 10⁷, 1.4 × 10⁷, 1.2 × 10⁷ and 4 × 10⁶ M^?1. min^?1 at 37, 30, 24 and 4° respectively. At 24° there were two components to the dissociation; a faster phase with K_?1=1.26 × 10^?2. min^?1 ($\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{=55}\text{minutes}$) and a slower phase with K_?1=1.103 × 10^?3. min^?1. The apparent K_d (from K_?1K₁) was 1.05 nM for the faster phase and 87.5 pM for the slower phase. These data suggest that there is a conformational change following hormone binding which results in an increased receptor affinity, which effectively prevents release of bound hormone.     

A direct measurement of the unzippering rate of a nucleic acid double helix

The rate of double helix unzippering was determined directly by application of a fast temperature jump method to a nucleotide system of partly unzippered helices formed from oligoriboadenylates and oligoribouridylates of equal chain lengths (14 and 18 nucleotide residues). These helices showed a relaxation process in the time range of 0.1 to 0.3 μsec, that is assigned to the unzippering reaction. Measurements at 0.05 M and 0.1 M [Na⁺] demonstrated a rather small dependence upon the ionic strength. Increase of temperature increases the rate of unzippering. Simulation of the unzippering relaxation by a zipper model yielded a rate constant of base pair formation adjacent to a helix sequence of 8 × 10⁶ sec^?1 at 25°C associated with an activation enthalpy of 4 $\text{kcal}\text{mole}$. This elementary rate constant is higher than that obtained from a simulation of the overall recombination and dissociation rates of entire helices. The difference is attributed to reduced electrostatic and steric hindrance effects for base pair equilibration at helix ends.     

Some association properties of bovine SH-k-casein

(1) It is shown ibal ^k-casein association is characterized hv a critical micelle concentration which decreases as the ionic strength is increased. (2) The ^k-casein polymer molecular weight was calculated from the weight-average apparent molecular weight by taking into consideration the monomer concentration and the excluded volume. The degree of polymerization is 30 and does not depend on ionic strength between 0.1 and 1 M. (3) The non-electrical contribution to the standard free energy of association is ?38 $\text{kJ}\text{mol}$ monomer. The electrical part is small: 1–2 $\text{kJ}\text{mol}$ monomer depending on the ionic strength and ^k-casein genetic variant. (4) The limitation of size and the size itself of the ^k-casein polymer can be explained by the theory of self-assembly of virus particles by Caspar and Klug (D.L.D. Caspar and A Klug. Cold Spring Harb. Symp. Quant. Biol. 27 (1962)1). (5) Extending this theory to casein micelle assembly, it is predicted that micelles are distributed preferentially over a restricted number of sizes.     

Interaction of anthracycline antibiotics with biopolymers: 9. Comparative study of the interaction kinetics of daunomycin,adriamycin and iremycin with DNA

The interaction kinetics of the three anthracycline antibiotics, daunomycin, adriamycin and iremycin, with calf thymus DNA has been investigated using the temperature-jump technique. Experimental data obtained at high binding ratio have been fitted by a kinetic theory which, for the binding of large ligands to a linear polymer chain, takes into account both nearest-neighbour ligand interaction and the overlap of potential binding sites. The kinetics of such cooperative binding according to a single-step mechanism can be described completely by two independent microscopic parameters, namely one rate constant and a kinetic cooperativity parameter. Both these parameters have been determined for the three anthracyclcine antibiotics, making use of the known equilibrium binding parameters. The association rate constant in the singly contiguous case turns out to be almost the same for all three antibiotics ($\text{7 × 10}{}^6\text{to 8 × 10}{}^6\text{1 mol}{}^{\mathrm{?1}}\text{s}{}^{\mathrm{?1}}$), while the corresponding dissociation rate constant ranges from 3.5 s^?1 for adriamycin to 10 s^?1 for daunomycin and about 35 s^?1 for iremycin. The different equilibrium binding constants thus correspond to different mean attachment times of the antibiotics at the polymer chain, which positively correlate with the inhibitory action of these drugs on in vitro DNA synthesis. Nearest-neighbour interaction in the case of adriamycin-DNA binding kinetics implies that adriamycin molecules dissociate from an isolated binding site nine times more frequently than from a site between two adjacent ligands.     

Selective antilipolytic effect of bacitracin in the isolated fat cell

Bacitracin, an antibiotic which decreases extracellular degradation, has been used to study peptide hormone degradation $\text{in}\text{vitro}$. The biologic effectiveness of these hormones in the presence of bacitracin has received minimal attention. This study demonstrates inhibition of lipolysis induced by both epinephrine and glucagon in the isolated fat cell (IFC). IFC from epididymal tissue were incubated with 0.5 μM epinephrine and increasing concentrations of bacitracin. Lipolysis was inhibited in a dose-dependent fashion, with a concentration of 5.7 × 10^?4$\text{M}$ bacitracin suppressing lipolysis 50%. Increasing the concentration of epinephrine in the presence of a constant dose of bacitracin overcame the antilipolytic effect. Bacitracin did not increase oxidation of glucose-U-C¹⁴ over basal. In the perifusion system, acute exposure to 5.7 × 10^?4$\text{M}$ bacitracin plus 5 × 10^?9$\text{M}$ glucagon suppressed lipolysis below unstimulated basal levels. Constant bacitracin perifusion produced no change in basal lipolysis but blunted the response to glucagon. ¹²⁵I-glucagon degradation was decreased in the presence of bacitracin. Additional studies with dibutyryl cyclic AMP demonstrated that the antilipolytic effect of bacitracin is exerted at a step beyon the second messenger. Bacitracin exerts a direct antilipolytic effect in isolated fat cells without stimulating glucose uptake and may afford a means of studying antilipolysis in the absence of other insulin-like effects.     

Mechaism of Arthrobacter sialophilus neuraminidase: The binding of substrates and transition-state analogs

2-Deoxy-2,3-dehydro-N-acetylneuraminic acid and its methyl ester are competitive inhibitors of Arthrobacter sialophilus neuraminidase with K_i = 1.4 × 10^?6$\text{M}$ and 4.8 × 10^?5$\text{M}$, respectively. The K_m for the substrate, N-acetylneuraminlactose, is 1.0 × 10^?3$\text{M}$. These data, taken together with the conformation of these compounds, indicate that these compounds are transition-state analogs of the enzyme. These results also suggest that the substrate upon binding to neuraminidase is distorted to a conformation approaching that of a half-chair.     

The dimer-monomer equilibrium constant for [125I]β nerve growth factor

The equilibrium constant for $\text{[}{}^{125}\text{I]β}$ nerve growth factor was determined using polyacrylamide gel electrophoresis to separate the monomer and dimer. Various concentrations of the radiolabelled nerve growth factor were incubated for 24 and 48 hours. The equilibrium constants obtained for both incubation periods were the same, 3.2 ± 1.4 × 10^?11$\text{M}$ and 2.6 ± 1.6 × 10^?11$\text{M}$, respectively. Thus, at physiological concentrations the β nerve growth factor is in the dimeric form almost exculsively.     

Kinetic parameters for the binding of p-nitrophenyl alpha-d-mannopyranoside to concanavalin A.

Binding of the chromogenic ligand p-nitrophenyl α-d-mannopyranoside to concanavalin A was studied in a stopped-flow spectrometer. Formation of the protein-ligand complex could be represented as a simple one-step process. No kinetic evidence could be obtained for a ligand-induced change in the conformation of concanavalin A, although the existence of such a conformational change was not excluded. The entire change in absorbance produced on ligand binding occurred in the monophasic process monitored in the stopped-flow spectrometer. The value of the apparent second-order rate constant (k_a) for complex formation (k_a = 54,000 s^?1m^? at 25 °C, pH 5.0, Γ/2 0.5) was independent of the protein concentration when the protein was in the range of 233–831 μm in combining sites and in excess of the ligand. The apparent first-order rate constant (k_?a) for dissociation of the complex was obtained from the rate constant for the decomposition of the complex upon the addition of excess methyl α-d-mannopyranoside (k_?a = 6.2 s^?1 at 25 °C, pH 5.0, Γ/2 0.5). The ratio $\text{k}{}_{\mathrm{<mtext>a</mtext>}}{}_{\mathrm{<mtext>?</mtext><mtext>a</mtext>}}$ (0.9 × 10₄m^?1) was in reasonable agreement with value of 1.1 ± 0.1 × 10⁴m^?1 determined for the equilibrium constant for complex formation by ultraviolet difference spectrometry. Plots of ln($\text{k}{}_{\mathrm{<mtext>a</mtext>}}\text{T}$) and ln($\text{k}{}_{\mathrm{<mtext>a</mtext>}}\text{T}$) vs $\text{1}\text{T}$ were linear (T is temperature) and were used to evaluate activation parameters. The enthalpies of activation for formation and dissociation of the complex are 9.5 ± 0.3 and 16.8 ± 0.2 kcal/mol, respectively. The unitary entropies of activation for formation and dissociation of the complex are 2.8 ± 1.1 and 1.3 ± 0.7 entropy units, respectively. These entropy changes are much less than those usually associated with substantial changes in the conformation of proteins.     

Thermodynamic and kinetic parameters of an oligonucleotide hairpin helix

The thermodynamics of the hairpin helix-single strand transition of A₆C₆U₆ has been analyzed by a staggering zipper model with consideration of single strand stacking. This analysis yields an enthalpy change of +11 kcal/mole for the formation of a first, isolated base pair. The stability constant of a first (intramolecular) base pair in A₆C₆U₆ is around 2 × 1O^?5 at 25°C, whereas a first (intermoleciilar) base pair in an A₆ · U₆ helix is characterised by a stability constant of about 4 × 10^?3M^?1 (25°C, extrapolated from A_n · V_n oligomer measurements). These data indicate a destabilizing effect of the C₆ loop.The rate constant of hairpin helix formation is 2 to 3 × 10⁴ sec^?1 associated with an activation enthalpy of +2.5 kcal/mote. The rate of helix dissociation of the A₆C₆U₆ hairpin is in the range of 10³ to lO⁵ sec^?1 with an activation enthalpy of 21 $\text{kcal}\text{mole}$. A comparison with the kinetic parameters obtained for A · U oligomer helices shows a specific influence of the C₆ loop due to the stacking tendency of the cytosine residues. This intluence is preferentially reflected in the relatively low value of the rate constant of helix formation.     

Kinetics of reduction of ferrioxamine B by chromium(II), vanadium(II), and dithionite

Kinetic studies of the reduction of ferrioxamine B (Fe(Hdesf)⁺) by Cr(H₂O)₆²⁺, V(H₂O)₆²⁺, and dithionite have been performed. For Cr(H₂O)₆²⁺ and V(H₂O)₆²⁺, the rate is ?d[Fe(Hdesf)⁺]/dt = k[Fe(Hdesf)⁺][M²⁺]. For Cr(H₂O)₆²⁺, k = 1.19 × 10⁴ M^?1 sec^?1 at 25°C and μ = 0.4 M, and k is independent of pH from 2.6 to 3.5. For V(H₂O)₆²⁺, k = 6.30 × 10² M^?1 sec^?1 at 25°C, μ = 1.0 M, and pH = 2.2. The rate is nearly independent of pH from 2.2 to 4.0. For Cr(H₂O)₆²⁺ and V(H₂O)₆²⁺, the activation parameters are ΔH^≠ = 8.2 kcal mol^?1, ΔS^≠ ?12 eu and ΔH^≠ = 1.7 kcal mol^?1, ΔS^≠ = ?40 eu (at pH 2.2) respectively. Reduction by Cr(H₂O)₆²⁺ is inner-sphere, while reduction by V(H₂O)₆²⁺ is outer-sphere. Reduction by dithionite follows the rate law ?d[Fe(Hdesf)⁺]/dt =kK^{$\text{1}\text{2}$}[Fe(Hdesf)⁺][S₂O₄^2?]^{$\text{1}\text{2}$} where K is the equilibrium constant for dissociation of S₂O₄^2? into SO₂^? radicals. The value of k at 25°C and μ = 0.5 is 2.7 × 10³ M^?1 sec^?1 at pH 5.8, 3.5 × 10³ M^?1 sec^?1 at pH 6.8, and 4.6 × 10³ M^?1 sec^?1 at pH 7.8, and ΔH^≠ = 6.8 kcal mol^?1 and ΔS^≠ = ?19 eu at pH 7.8.     

Investigations on purine and pyrimidine bases stacking associations in aqueous solutions by the fluorescence quenching method: II. Heteroassociation between 2-aminopurine and thymidine

Heteroassociation between A and B compounds in liquid solution was considered. Provided that concentration of A molecules is low, a geneial equation describing fluorescence quantum yield and lifetime of compound A as a function of B molecules concentration was derived. The heteroassociation between 2-aminopurine and thymidine in aqueous solutions was examined within the range of temperatures 0 to 90° C. The equilibrium constants of the first step of association, namely heterodimer formation, were determined and its thermodynamic parameters (ΔH = ?2.76 $\text{kcal}\text{mol}$, ΔS = ?5.9 e.u.) were calculated. The observed changes of the stacking rate constants with temperature confirm the two-step mechanism of the reaction. The activation energy (~2.7 $\text{kcal}\text{mol}$) and the encounter distance (~10.7 A) are only slightly larger than in the case of 2-aminopurine autoassociation, most probably because of a stronger solvation of thymidine molecules.     

Nuclear binding of cortisol in intestinal mucosa and liver of a teleost fish (Salmo gairdnerii irideus)

Nuclear binding of corticosteroids in fish tissues was investigated in two target organs of the trout $\text{Salmo gairdnerii irideus}$: the liver and the intestinal mucosa. Incubation of intact nuclei with [³H]-cortisol or [³H]-dexamethasone failed to demonstrate high-affinity binding of these steroids to proteins. Exchange assay of [³H]-cortisol in high-salt nuclear extracts indicated an association constant Ka = 1.9 × 10⁴ M^?1 for intestinal mucosa and 2.1 × 10⁴ M^?1 for liver. In sea water-adapted trout, the association constant remained the same as in fresh water.These results extend previous observations obtained on the cytosol which showed that no high-affinity receptors could be disclosed in fish tissues using these two corticosteroids.     

ATP-dependent Ca2+ accumulation in intracellular membranes of guinea pig macrophages after saponin treatment

Ca²⁺ transport system in the intracellular membranes was studied by using saponin-treated macrophages of the guinea pig, in which the plasma membranes could be selectively destroyed. Saponin-treated macrophages could accumulate 3.1 nmoles $\text{Ca}{}^{\mathrm{2+}}\text{4 × 10}{}^6$ macrophages in the presence of Mg-ATP and sodium azide with an apparent affinity constant of 6 × 10⁶ M^?1. In the absence of sodium azide, the value of Ca²⁺ uptake of saponin-treated macrophages was 95 nmoles/4 × 10⁶ macrophages, and its affinity constant for Ca²⁺ was 3 × 10⁵ M^?1. Saponin-treated macrophages may be suitable for the studies of Ca²⁺ transport systems in the intracellular membranes.     

In vitro triiodothyronine binding to non-histone proteins from rat liver nuclei

$\text{In vitro}$ incubations of non-histone proteins from rat liver nuclei with labelled L-3, 5, 3′ triiodothyronine demonstrate the existence of high affinity, limited capacity binding sites for the hormone in this protein group; the affinity was found identical for triiodothyroacetic acid and lower for L-thyroxine. Binding ability was highly temperature dependent. At 4°C, the rate constant of association was 0.9 × 10⁷ M^?1 h^?1 and the rate constant of dissociation was 0.015 h^?1. The dissociation constant K_d was calculated from these data or measured by Scatchard analysis and found to be between 1.6 and 5 × 10^?9 M. The maximum binding capacity was 10^?13 moles of L-3, 5, 3′ triiodothyronine per 100 μg non-histone proteins or 6000 hormone molecules per nucleus. Protein binding had a half-life of 20 hours at 4°C, in the absence of hormone, but was found to be very stable in the presence of hormone.