Binding to tubulin of the colchicine analog 2-methoxy-5-(2', 3', 4'-trimethoxyphenyl)tropone. Thermodynamic and kinetic aspects

The thermodynamics and kinetics of the binding to tubulin of the colchicine analog 2-methoxy-5-(2', 3', 4'-trimethoxyphenyl) tropone (termed AC because it lacks the B-ring of colchicine) have been characterized by fluorescence techniques. The fluorescence of AC is weak in aqueous solution and is enhanced 250-fold upon binding to tubulin. The following thermodynamic values were obtained for the interaction at 37 degrees C: K = 3.5 X 10(5) M-1; delta G0 = -7.9 kcal/mol; delta H0 = -6.8 kcal/mol; delta S0 = 3.6 entropy units. The AC-tubulin complex is 1-2 kcal/mol less stable than the colchicine-tubulin complex. The change in fluorescence of AC was employed to measure the kinetics of the association process, and quenching of protein fluorescence was used to measure both association and dissociation. The association process, like that of colchicine, could be resolved into a major fast phase and a minor slow phase. The apparent second order rate constant for the fast phase was found to be 5.2 X 10(4) M-1 S-1 at 37 degrees C, and the activation energy was 13 kcal/mol. This activation energy is 7-11 kcal/mol less than that for the binding of colchicine to tubulin. The difference in activation energies can most easily be rationalized by a mechanism involving a tubulin-induced conformational change in the ligand ( Detrich , H. W., III, Williams, R. C., Jr., Macdonald, T. L., Wilson, L., and Puett , D. (1981) Biochemistry 20, 5999-6005). Such a change would be expected to have a small activation energy in AC because it possesses a freely rotating single bond in place of the B-ring of colchicine.     

AMP interaction sites in glycogen phosphorylase b. A thermodynamic analysis

The binding of AMP to activator site N and to inhibitor site I in glycogen phosphorylase b has been characterized by calorimetry, potentiometry and ultracentrifugation in the pH range 6.5-7.5 at 25 degrees C (mu = 0.1). Calorimetric titration data of phosphorylase b with adenosine 5'-phosphoramidate are also reported at pH 6.9 (T = 25 degrees C, mu = 0.1). Calorimetric curves have been analyzed on the basis of potentiometric and sedimentation velocity results to determine thermodynamic quantities for AMP binding to the enzyme. The comparison of calorimetric titration data of AMP and adenosine 5'-phosphoramidate at pH 6.9 supports the hypothesis previously suggested that the dianionic phosphate form of the nucleotide preferentially binds to the allosteric activator site. The thermodynamic parameters for AMP binding to site N are as follows: delta G0 = -22 kJ mol-1, delta H0 = -34 kJ mol-1 and delta S0 = -40 J mol-1 K-1. The binding of the nucleotide to site I was found to be strongly dependent on the pH. This behaviour may be explained in terms of coupled protonations of three groups having pKa values of 6.0, 6.0 and 6.1 in the unbound form and 7.0, 7.5 and 7.2 in the enzyme-nucleotide complex. The thermodynamic parameters for nucleotide binding to site I for the enzymatic form in which all the modified groups are completely deprotonated or protonated have been calculated to be: delta G0 = -7.7 kJ mol-1, delta H0 = -28 kJ mol-1 and delta S0 = -68 J mol-1 K-1 and delta G0 = -28 kJ mol-1, delta H0H = -10 kJ mol-1 and delta S0H = 61 J mol-1 K-1, respectively. These results suggest that attractive dispersion forces are of primary significance for AMP binding to activator site N, although electrostatic interactions act as a stabilizing factor in the nucleotide binding. The protonation states of those residues of which the pKa values are modified by AMP binding to site I highly influence the thermodynamic parameters for the nucleotide binding to this site.     

Thermodynamics of DNA hairpins: contribution of loop size to hairpin stability and ethidium binding.

A combination of calorimetric and spectroscopic techniques was used to evaluate the thermodynamic behavior of a set of DNA hairpins with the sequence d(GCGCTnGCGC), where n = 3, 5 and 7, and the interaction of each hairpin with ethidium. All three hairpins melt in two-state monomolecular transitions, with tm's ranging from 79.1 degrees C (T3) to 57.5 degrees C (T7), and transition enthalpies of approximately 38.5 kcal mol-1. Standard thermodynamic profiles at 20 degrees C reveal that the lower stability of the T5 and T7 hairpins corresponds to a delta G degree term of +0.5 kcal mol-1 per thymine residue, due to the entropic ordering of the thymine loops and uptake of counterions. Deconvolution of the ethidium-hairpin calorimetric titration curves indicate two sets of binding sites that correspond to one ligand in the stem with binding affinity, Kb, of approximately 1.8 x 10(6) M-1, and two ligands in the loops with Kb of approximately 4.3 x 10(4) M-1. However, the binding enthalpy, delta Hb, ranges from -8.6 (T3) to -11.6 kcal mol-1 (T7) for the stem site, and -6.6 (T3) to -12.7 kcal mol-1 (T7) for the loop site. Relative to the T3 hairpin, we obtained an overall thermodynamic contribution (per dT residue) of delta delta Hb = delta(T delta Sb) = -0.7(5) kcal mol-1 for the stem sites and delta delta Hb = delta(T delta Sb) = -1.5 kcal mol-1 for the loop sites. Therefore, the induced structural perturbations of ethidium binding results in a differential compensation of favorable stacking interactions with the unfavorable ordering of the ligands.     

Hydroxyl-ion-induced subunit dissociation of east cytoplasmic pyruvate decarboxylase. A circular dichroism study

Cytoplasmic pyruvate decarboxylase (EC 4.1.1.1, from Saccharomyces carlsbergensis) exhibits in its circular dichroic spectrum in the 250--320-nm range a multiple two-signal band. This couplet disappears on increasing the pH up to pH 8.5. Two classes of two protons each can be quantified by these spectral changes. The first class dissociates rapidly and the apparent pK is 7.84. The thermodynamic data are delta G = 87.7 kJ mol-1, delta H = + 56.0 kJ mol-1, delta S = - 108 J mol-1 K-1, very characteristic for the deprotonation of an amino-acid side chain. The second class of the protons has the following thermodynamic data: delta G = 88.3 kJ mol-1, delta H = - 64.3 kJ mol-1, delta S = - 520 J mol-1 K-1 which, in conjunction with kinetic reasoning and in view of enzyme stoichiometry and symmetry, suggests a conformational equilibrium exposing the second two protons. Th enzyme dissociates into two dimeric subunits. This dissociation step is considered to be rate-determining for the overall process. The data are kp = 1.4 . 10(-3), delta H not equal to = + 128.3 kJ mol-1, delta S not equal = + 136 J mol-1 K-1. If there is a conformational equilibrium, the rate constant of product formation kp will be modified by a factor beta = kc/(1 + Kc) (0 < beta less than or equal to 1) where Kc is the conformational equilibrium constant. The subunit dissociation appears to be controlled by the enthalpy of activation indicating that a number of interactions, i.e. ionic, hydrogen and hydrophobic bridges, are to be broken. Optimal conditions for the preparation of the apo-enzyme are derived from the data.     

Hydrophobic acridine dyes for fluorescence staining of mitochondria in living cells. 1.Thermodynamic and spectroscopic properties of 10-n-alkylacridine orange chlorides

10-n-Alkyl-acridine-orange-chlorides (alkyl-AOs) are excellent dyes for fluorescence staining of mitochondria in living cells. The thermodynamic and spectroscopic properties of the series alkyl = methyl to nonyl have been investigated. The dyes form dimers in aqueous solution. The dimerisation is mainly a consequence of the hydrophobic interaction. The dissociation constant K respectively association constant K-1 of the dimers describes the hydrophobic interaction and therefore the hydrophobic properties of the dye cations. The dissociation constant K = K0 at the standard temperature T = 298 K has been determined spectroscopically in aqueous solution. It depends on the length of the alkyl residue n-CmH2m + 1 (m = 1 - 9) (Table 2). In addition the standard dissociation enthalpies (energies) delta H0 and dissociation entropies delta S0 have been determined from the temperature dependence of K (Table 2). With increasing chain length m the thermodynamic parameters K0, delta H0, delta S0 decrease. Therefore with growing m the dimers are stabilized. This stabilization is an entropic effect which is diminished by the energetic effect. The change of the thermodynamic parameters with m is in agreement with the concept of hydrophobic interaction and the stabilization of water structure in the surroundings of hydrophobic residues. As one would expect nonyl-AO is the most hydrophobic dye of the series. As an example the spectroscopic properties of nonyl-AO have been determined. We measured the absorption, luminescence and polarization spectra in rigid ethanol at 77 K. Under these conditions alkyl-AOs associate like dyes in Water at room temperature. The spectra depend on the concentration of the solution. In very dilute solution we observe mainly the spectra of the monomers M, in concentrated solution the spectra of the dimers D. The spectra of M and D are characteristically different. The monomers have one long wave length absorption M1 = 20.000 cm-1 with resonance fluorescence. In addition there is a long living phosphorescence at 16.600 cm-1. Its polarization is nearly perpendicular to the plane of the AO residue. The dimers have two long wave length absorption bands D1 = 18.700 and D2 = 21.200 cm-1 with very different intensities. D1 has very low intensity and is forbitten, D2 is allowed. D1 shows fluorescence. Phosphorescence has not been observed. D1, D2 and also M1 are polarized in the plane of the AO residue. At short wave length absorption and polarization spectra are very similar. From the spectra we constructed the energy level diagram of M and D (Fig. 9). The first excited state of M splits in D in two levels. The level splitting and the transition i     

Thermodynamics of porphyrin dimerization in aqueous solutions.

The dimerization equilibrium of deuteroporphyrin IX and of mesoporphyrin IX in aqueous solutions were studied by fluorimetric techniques over the 0.01-1 microM concentration range, where dimerization is the dominant aggregation process. Deuteroporphyrin IX was studied at several temperatures over the range 22-37 degrees C, and mesoporphyrin at 25 and 37 degrees C. The magnitudes determined for the dimerization equilibrium constants (25 degrees C, neutral pH, phosphate-buffered saline) are 2.3 X 10(6)M-1 and 5.4 X 10(6)M-1 for the deutero and meso derivatives respectively. The meso, deutero and haemato species tested show a similar temperature effect, namely dimerization decreasing with increasing temperature, indicating the involvement of a negative enthalpy change. Van''t Hoff isochore of the dimerization constants determined for deuteroporphyrin IX was linear within the temperature range of 22-37 degrees C, allowing the calculation of the thermodynamic parameters. For deuteroporphyrin dimerization, those were found to be delta G0 = -36. 4kJ X mol-1; delta H0 = -46. 0kJ X mol-1 and delta S0 = -32.2J X K-1 X mol-1 (at neutral pH, 25 degrees C, phosphate-buffered saline), showing the process to be enthalpy-driven. Similar trends have been found for porphyrin species other than those studied here. Our data fit with a hypothesis giving a major role to the solvent in driving porphyrins to aggregate in aqueous solution. The magnitudes and directions of the energetic changes fit better with the expectation of the '' solvophobic force'' theory predicting enthalpy-driven association, than with the classic hydrophobic bonding, predicting the association to be entropy-driven.     

Thermodynamic analysis of rat brain opioid mu-receptor-ligand interaction

Opioid mu-receptors are membrane bound receptors. The mechanism by which they transduce their biological effect into the inner compartment of the postsynaptic cell is still not fully understood. The present study was attempted to the measurement of changes of the thermodynamic parameters of the receptor--agonist/antagonist interaction. We have set up the binding assays of a mu-receptor agonist (3H-dihydromorphine) as well as an antagonist (3H-naloxone). The saturation isotherms of both ligands have been assayed at various temperatures and from the resulting KD values the standard changes of Gibbs energy, enthalpy and entropy have been calculated. While the binding of the mu-receptor agonist 3H-dihydromorphine appears to be entropy driven (delta S0 = 230 J mol-1 K-1) and endothermic (delta H0 = 19 kJ mol-1), the binding of the mu-receptor antagonist 3H-naloxone is apparently driven by a decrease of standard enthalpy (delta H0 = -27 kJ mol-1; i.e. the reaction is exothermic) and is also characterized by an increase of standard entropy (delta S0 = 76 J mol-1 K-1). The maximal number of 3H-naloxone binding sites has to be determined by incubation at 0-4 degrees C. The present data to not support the view that opioid mu-receptors transduce their biological signal through the adenylatecyclase system by a mechanism similar to beta-adrenergically stimulated adenylatecyclase.     

Effects of pH, ionic strength, and temperature on activation by calmodulin an catalytic activity of myosin light chain kinase

The reversible association of Ca42+-calmodulin with the inactive catalytic subunit of myosin light chain kinase results in the formation of the catalytically active holoenzyme complex [Blumenthal, D. K., & Stull, J. T. (1980) Biochemistry 19, 5608--5614]. The present study was undertaken in order to determine the effects of pH, temperature, and ionic strength on the processes of activation and catalysis. The catalytic activity of myosin light chain kinase, when fully activated by calmodulin, exhibited a broad pH optimum (greater than 90% of maximal activity from pH 6.5 to pH 9.0), showed only a slight inhibition by moderate ionic strengths (less than 20% inhibition at mu = 0.22), and displayed a marked temperature dependence (Q10 congruent to 2; Ea = 10.4 kcal mol-1). Thermodynamic parameters calculated from Arrhenius plots indicate that the Gibb's energy barrier associated with the rate-limiting step of catalysis is primarily enthalpic. The process of kinase activation by calmodulin had a narrower pH optimum (pH 6.0--7.5) than did catalytic activity, was markedly inhibited by increasing ionic strength (greater than 70% inhibition at mu = 0.22), and exhibited nonlinear van't Hoff plots. Between 10 and 20 degrees C, activation was primarily entropically driven (delta S degrees congruent to 40 cal mol-1 deg-1; delta H degrees = -900 cal mol-1), but between 20 and 30 degrees C, enthalpic factors predominated in driving the activation process (delta S degrees congruent to 10 cal mol-1 deg-1; delta H degrees = -9980 cal mol-1). The apparent change in heat capacity (delta Cp) accompanying activation was estimated to be -910 cal mol-1 deg-1. On the basis of these data we propose that although hydrophobic interactions between calmodulin and the kinase are necessary for the activation of the enzyme, other types of interactions such as hydrogen bonding, ionic, and van der Waals interactions also make significant and probably obligatory contributions to the activation process.     

The thermodynamics of protein stability. Cold destabilization as a general phenomenon

A theoretical analysis of the temperature/stability profiles of proteins shows that, where a two-state model represents the denaturation, and where the free energy of denaturation delta G(T) shows a strong temperature dependence, then the protein becomes subject to both high- and low-temperature destabilization. In the simplest case delta G(T) is parabolic, therefore the high temperature TH, where delta (G(TH) = 0, is complemented by a low temperature TL, where delta G(TL) = 0. It is generally stated that the partial molal heat capacity change delta C accompanying the heat denaturation is positive and independent of the temperature. This implies that heating the protein through TL results in a negative delta C which seems physically unsatisfactory. The constant delta C model is explored and a physically more realistic model is advanced which allows for a temperature-dependent delta C which changes sign at some temperature within the range of stability of the native protein; delta G(T) then has the form of a skewed parabola. Experimental heat capacity data for native lysozyme and for a flexible polymer lend support to this model. The molecular basis of cold inactivation of proteins is discussed in the light of the thermodynamic analysis.     

Thermodynamics of hydrolysis of disaccharides. Cellobiose, gentiobiose, isomaltose, and maltose

The thermodynamics of the enzymatic hydrolysis of cellobiose, gentiobiose, isomaltose, and maltose have been studied using both high pressure liquid chromatography and microcalorimetry. The hydrolysis reactions were carried out in aqueous sodium acetate buffer at a pH of 5.65 and over the temperature range of 286 to 316 K using the enzymes beta-glucosidase, isomaltase, and maltase. The thermodynamic parameters obtained for the hydrolysis reactions, disaccharide(aq) + H2O(liq) = 2 glucose(aq), at 298.15 K are: K greater than or equal to 155, delta G0 less than or equal to -12.5 kJ mol-1, and delta H0 = -2.43 +/- 0.31 kJ mol-1 for cellobiose; K = 17.9 +/- 0.7, delta G0 = -7.15 +/- 0.10 kJ mol-1 and delta H0 = 2.26 +/- 0.48 kJ mol-1 for gentiobiose; K = 17.25 +/- 0.7, delta G0 = -7.06 +/- 0.10 kJ mol-1, and delta H0 = 5.86 +/- 0.54 kJ mol-1 for isomaltose; and K greater than or equal to 513, delta G0 less than or equal to -15.5 kJ mol-1, and delta H0 = -4.02 +/- 0.15 kJ mol-1 for maltose. The standard state is the hypothetical ideal solution of unit molality. Due to enzymatic inhibition by glucose, it was not possible to obtain reliable values for the equilibrium constants for the hydrolysis of either cellobiose or maltose. The entropy changes for the hydrolysis reactions are in the range 32 to 43 J mol-1 K-1; the heat capacity changes are approximately equal to zero J mol-1 K-1. Additional pathways for calculating thermodynamic parameters for these hydrolysis reactions are discussed.     

Thermodynamic and kinetic studies on saccharide binding to soya-bean agglutinin.

The fluorescence of N-dansylgalactosamine [N-(5-dimethylaminonaphthalene-1-sulphonyl)galactosamine] was enhanced 11-fold with a 25 nm blue-shift in the emission maximum upon binding to soya-bean agglutinin (SBA). This change was used to determine the association constants and thermodynamic parameters for this interaction. The association constant of 1.51 X 10(6) M-1 at 20 degrees C indicated a very strong binding, which is mainly due to a relatively small entropy value, as revealed by the thermodynamic parameters: delta G = -34.7 kJ X mol-1, delta H = -37.9 kJ X mol-1 and delta S = -10.9 J X mol-1 X K-1. The specific binding of this sugar to SBA shows that the lectin can accommodate a large hydrophobic substituent on the C-2 of galactose. Binding of non-fluorescent ligands, studied by monitoring the fluorescence changes when they are added to a mixture of SBA and N-dansylgalactosamine, indicates that a hydrophobic substituent at the anomeric position increases the affinity of the interaction. The C-6 hydroxy group also stabilizes the binding considerably. Kinetics of binding of N-dansylgalactosamine to SBA studied by stopped-flow spectrofluorimetry are consistent with a single-step mechanism and yielded k+1 = 2.4 X 10(5) M-1 X s-1 and k-1 = 0.2 s-1 at 20 degrees C. The activation parameters indicate an enthalpicly controlled association process.     

Thermodynamics of glucagon aggregation.

Heats of dilution of concentrated glucagon solutions have been measured calorimetrically at 10 and 25 degrees C in 0.2 M potassium phosphate buffer of pH 10.6. Analysis of the data in terms of a monomer-trimer equilibrium gives the following thermodynamic parameters for the association reaction at 25 degrees C: delta G degrees = 7.34 kcal/mol of trimer, delta H degrees = -31.2 kcal/mol, deltaS degrees = -80 cal/(K mol), deltaCp = 430 cal/(K mol). The sensitivity of heat of dilution data to the association constant and stoichiometry of the reaction is discussed.     

Thermodynamics of the quenching of tyrosyl fluorescence by dithiothreitol

Tyrosyl fluorescence quenching by oxidized dithiothreitol (DTTo) in N-acetyl-L-tyrosine N'-methylamide, and native bovine pancreatic ribonuclease A and its reduced, S-methylated form, in aqueous solution is studied at pH 3.0. From the temperature dependence of the fluorescence quenching, it is demonstrated that the mechanism of the quenching process is probably static (formation of a complex), and not dynamic (collisional), in origin. Although other quenching mechanisms cannot be ruled out, our proposition that the quenching of tyrosyl fluorescence in these molecules is due to the formation of a complex between the tyrosyl moieties and DTTo is consistent with previously reported evidence indicating a strong tendency for aromatics to complex with various disulfide-containing compounds. The strength of binding is approximately the same for these three tyrosine-containing compounds, indicating that the microenvironments of their tyrosyl residues may be similar. With 1 M as the reference standard state, the following average thermodynamic parameters are established for the complexation (at 298 K): delta G0 = -3.32 kcal/mol, delta H0 = -1.1 kcal/mol, and delta S0 = 7.4 eu. The large positive value of delta S0 suggests that hydrophobic interactions may play an important role in the stabilization of such tyrosyl-disulfide complexes; the negative value of delta H0 suggests that polar interactions may also contribute to the formation of these complexes. Some possible implications with regard to protein-folding studies are discussed.     

Thermodynamic studies of the reversible association of Escherichia coli ribosomal subunits.

The kinetics of association of Escherichia coli 30S and 50S ribosomal subunits have been carried out as a function of temperature after a magnesium jump from 1.5 to 3 mM. Turbidimetric recordings combined with a stopped-flow apparatus were used to follow the kinetics. The data show that the rates of formation and dissociation of the 70S particles at 3 mM Mg2+ and +25 degrees C were, respectively: k2 = 10(5) M-1 s-1, k1 = 4,5 X 10(-3) s-1; lowering the temperature decreases the rate constants with activation energies equal to E2 = 7.5 kcal/mol, E1 = 26.5 kcal/mol and enhances the association equilibrium towards the 70S species with an enthalpy change (delta H degrees assoc = -19.9 kcal/mol) dominant over the entropy change (delta S degrees assoc = -33 cal/(deg mol)). These thermodynamic parameters were compared to those obtained from studies on the interactions of codon-anticodon in yeast phenylalanine transfer RNA as well as of ribooligonucleotides. The kinetic and thermodynamic data are shown to be consistent with 16S-23S RNA interaction.     

Mechanism of lectin-cell interactions: thermodynamic and kinetic analysis of the binding of winged bean acidic lectin (WBA II) to human erythrocytes.

In order to identify the forces involved in the binding and to understand the mechanism involved, equilibrium and kinetic studies were performed on the binding of the winged bean acidic lectin to human erythrocytes. The magnitudes of delta S and delta H were positive and negative respectively, an observation differing markedly from the lectin-simple sugar interactions where delta S and delta H are generally negative. Analysis of the sign and magnitudes of these values indicate that ionic and hydrogen bonded interactions prevail over hydrophobic interactions resulting in net -ve delta H (-37.12 kJ.mol-1) and +ve delta S (14.4 J.mole-1 K-1 at 20 degrees C), thereby suggesting that this entropy driven reaction also reflects conformational changes in the lectin and/or the receptor. Presence of two kinds of receptors for WBA II on erythrocytes, as observed by equilibrium studies, is consistent with the biexponential dissociation rate constants (at 20 degrees C K1 = 1.67 x 10(-3) M-1 sec-1 and K2 = 11.1 x 10(-3) M-1 sec-1). These two rate constants differed by an order of magnitude accounting for the difference in the association constants of the two receptors of WBA II. However, the association process remains monoexponential suggesting no observable difference in the association rates of the lectin molecule with both the receptors, under the experimental conditions studied. The thermodynamic parameters calculated from kinetic data correlate well with those observed by equilibrium. A two-step binding mechanism is proposed based on the kinetic parameters for WBA II-receptor interaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

NADPH binding induced proton ionization as a cause of nonlinear heat capacity changes in glutamate dehydrogenase

Functional group interactions involved in the formation of the glutamate dehydrogenase-NADPH binary complex have been studied by three independent but complementary approaches: the pH dependence of the overall dissociation constant measured by an improved differential spectroscopic technique; the pH dependence of the enthalpy of complex formation measured by flow calorimetry; and the pH dependence of the number of protons released to, or taken up from, the solvent in the complex formation reaction, measured by titration. We conclude that the coenzyme binds to the enzyme through three distinguishable interactions: a pH-independent process involving the binding of the reduced nicotinamide ring; a relatively weak "proton-stabilizing" process, occurring at low pH involving the shift at a pK of 6.3 in the free enzyme to 7.0 in the enzyme-NADPH complex; and a stronger "proton-destabilizing" process, occurring at a higher pH involving a shift of a pK of 8.5 in the enzyme down to 6.9 in the enzyme-NADPH complex. The proton ionization of the free enzyme involved in this third interaction exhibits some unusual thermodynamic parameters, having delta Go = +11.5 +/- 0.1 kcal mol-1, delta Ho = +19 +/- 1 kcal mol-1, and delta So = +23 eu. We show here that this proton ionization step is directly related to and indeed constitutes the "implicit" shift in enzyme macrostates which we have shown to be responsible for the existence of large highly nonlinear delta Cpo effects in the formation of this complex [Fisher, H. F., Colen, A. H., & Medary, R. T. (1981) Nature (London) 292, 271-272].     

Binding of basic pancreatic trypsin inhibitor and related isoinhibitors to leukocytic elastase. Determination of thermodynamic parameters

The effect of temperature, ionic strength and solvation power of mono- and divalent cations on the interaction of BPTI-like inhibitors with human leukocytic elastase has been determined. The binding process is characterized by a non-linear dependence of the equilibrium association constant on 1/T indicating a thermal transition at temperature values ranging between 20 degrees C and 35 degrees C depending on the solvent. The marked dependence of the thermodynamic parameters (delta H degrees, delta S degrees, delta G degrees) and of the transition temperature on the concentration and nature of the cations present in solution seems to indicate that the transition, probably of conformational nature, is related to removal of water molecules upon enzyme/inhibitor complex formation.     

Chemical model of reaction cascades induced by activated enzymes or catalysts. Two-step cascades in visual transduction.

A dissipative system is approximated by a nonlinear rate equation: Z congruent to K1Z - K2Z3 (K2 greater than 0), in which the right side is derived from -delta G/delta Z of Taylor's series of the thermodynamic potential given by Gibbs' function G(Tc, Pc) (Z) at about the critical point C(Tc, Pc) of the control variables (parameters) T and P. The stability or instability of the system is treated by the changes in the control parameters. In the case that T not equal to P not equal to 0 in the steady state, Z = 0, and T and P pass the point C, K1 becomes negative. By this change, the G function is convex at Z = 0 and each product is created rapidly with concentration or number of the molecules Z = ([K1]/K2)1/2. This dynamic theory is applied to enzyme cascades. Based on cyclic GMP (cGMP) hypothesis in visual transduction, the cascade hydrolysis of cGMP of vertebrates is analyzed by dividing it into two-step reaction cascades: The initial process is that metarhodopsin II catalyzes the exchange of GDP for GTP by transducin (Gtd) and that GTP-Gtd complex is hydrolyzed to GDP-Gtd complex. In the following cascade cGMP is hydrolyzed with amplification of phosphodiesterase (PDE) activated by the removal of the small inhibitory subunit. The quantity of the hydrolysis of cGMP is estimated as approximately 5 x 10(4-5) molecules per photolyzed rhodopsin semiempirically, and this coincides well with experiments.     

Analysis of the Escherichia coli ribosome-ribosomal subunit equilibrium using pressure-induced dissociation

Hydrostatic pressure can be used to perturb the ribosome-ribosomal subunit equilibrium. We have used glutaraldehyde fixation and subsequent sucrose gradient analysis to determine the equilibrium concentrations of Escherichia coli 70 S, 50 S, and 30 S particles at pressures from 1 to 1400 atm. This method is shown to be sufficiently rapid and free of interfering ribosomal aggregation artifacts when performed at Mg2+ concentrations below 8 mM. We show directly that the E. coli ribosome is in equilibrium with its subunits and that the pressure-sensitive reaction is appropriately described by the expression: In Kp = ln K0 + (P delta V/RT), where Kp and K0 are the equilibrium constants at pressure P and 1 atm, respectively, and delta V is the change in molecular volume that occurs during the reaction. The method provides values for K0 under different conditions, and the effects of Mg2+ ion can be readily ascertained. K0 and delta V were also estimated by a method of fitting computer-generated sucrose gradient profiles to experimental profiles. Determination of delta H0, delta S0, and delta V0 at 5 mM Mg2+ are presented. The results are discussed in the context of previous thermodynamic studies of the E. coli ribosome.     

Study of DNA melting in the region of the inversion of relative stability of AT and GC pairs]

Systematic data on the dependence of the melting curve parameters of DNA from different organisms on the concentration of salt (C2H5)5NBr have been obtained. The melting curves were studied by spectrophotometric as well as by microcalorimetric methods. The DNA melting range width is shown to pass through the minimum value delta0T = 0.6 +/- 0.1 degrees at the point of inversion of relative stability of AT and GC pairs that corresponds to the concentration of (C2H5)4NBr equal to 2.9 +/- 0.1 M. This concentration, as well as the value of delta0T, are the same for different DNA's of common chemical structure. The T2 and T4 DNA containing hydroxymethylated and glucosylated cytosine residues show an anomalous behaviour. The enthalpy of melting falls very slowly as the salt concentration increases. The possible causes of the observed value of delta0T are discussed. A conclusion is drawn that the main factor which governs the DNA melting process in the region of inversion of the relative stability of AT and GC pairs is the heterogeneity of stacking interaction between different base pairs.