Thermodynamics of the interaction of daunomycin with DNA

The association constant for the interaction of daunomycin with DNA was determined as a function of temperature (using [³H] daunomycin in conventional equilibrium dialysis cells) and ionic strength (using a spectrophotometric titration method). The association constant varied between 3.1 × 10⁶ M^?1 (4°C) and 3.9 × 10⁵ M^?1 (65°C). The free energy change was ?8.2 to ?8.8 $\text{kcal}\text{mol}$, the enthalpy change ?5.3 $\text{kcal}\text{mol}$ and the entropy change +10 to +11 eu, all values being consistent with that expected of an intercalation process. The apparent number of intercalation sites detected (0.15 to 0.16 per nucleotide) was independent of temperature. The large positive entropy change accompanying the interaction appeals to be due to extensive release of water from the DNA and daunomycin. The apparent number of binding sites increased dramatically with decrease of ionic strength, although the apparent association constant remained largely unaffected by ionic strength.     

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.     

Acid dissociation of cyclohexaamylose and cycloheptaamylose

Acid dissociation constants of aqueous cyclohexaamylose (6-Cy) and cycloheptaamylose (7-Cy) have been determined at 10–47 and 25–55°C, respectively, by pH potentiometry. Standard enthalpies and entropies of dissociation derived from the temperature dependences of these pK_a's are ΔH⁰ = 8.4 ± 0.3 kcal mol^?1, $\text{Δ}\text{S}{}^0\text{= ?28. ± 1}\text{cal mol}{}^{\mathrm{?1}}{}^0\text{K}{}^{\mathrm{?1}}$ for 6-Cy and ΔH⁰ = 10.0 ± 0.1 kcal mol^?1, $\text{Δ}\text{S}{}^0\text{= ?22.4 ±0.3}\text{cal mol}{}^{\mathrm{?1}}{}^0\text{K}{}^{\mathrm{?1}}$ for 7-Cy. Intrinsic ¹³C nmr resonance displacements of anionic 6- and 7-Cy were measured at 30°C in 5% D₂O ($\text{v}\text{v}$). These results indicate that the dissociation of 6- and 7-Cy involves both C2 and C3 2⁰-hydroxyl groups. The thermodynamic and nmr parameters are discussed in terms of interglucosyl hydrogen bonding.     

Dissociation of pig liver carboxylesterase measured by quantitative micro-complement fixation

Rate and apparent equilibrium constants for the dissociation of pig liver carboxylesterase into three subunit molecules have been determined by complement fixation. The dependence of the dissociation equilibria on pH are consistent with dissociation reactions involving the addition of two protons per subunit, a pH-independent dissociation, and a dissociation upon the loss of one proton per subunit. The rate constants for dissociation are consistent with terms first order in hydrogen and hydroxide ions and a pH-independent path. The equilibrium constants in the range 3–35 °C at pH 7.2 exhibit no dependence on temperature; the association reaction is entropy driven with $\text{ΔS = 68}\text{cal mol}{}^{\mathrm{?1}}{}^{\mathrm{°}}\text{K}{}^{\mathrm{?1}}$. The rate constants for the pH-independent dissociation follow ΔH^≠ ? 6 kcal mol^?1. The order of effectiveness of concentrated salts in promoting denaturation is correlated with their effect on the activity coefficient of acetyltetraglycine ethyl ester and suggests that peptide groups become more exposed upon dissociation. The increased dissociation in the presence of urea derivatives containing alkyl substituents suggests exposure of hydrophobic regions upon dissociation; this is also consistent with ΔH = 0 for dissociation. It is likely that hydrophobic interactions contribute to the stability of the trimeric whole molecule.     

The reversal of the myosin and actomyosin ATPase reactions and the free energy of ATP binding to myosin

Evidence is presented that both myosin and actomyosin in presence of Mg²⁺ and KCl catalyze an incorporation of ³²P_i into ATP. The rate with actomyosin is about $\text{1}\text{500}$ the rate of ATP hydrolysis; the rate with myosin is less than $\text{1}\text{100}$ of that with actomyosin. With myosin, but not with actomyosin, an apparent initial “burst” of ³²P_i incorporation into ATP is observed. Actin binding thus promotes ATP dissociation. The data with myosin allow estimation of both the amount of enzyme-bound [³²P]-ATP present and the rate constant, k_?1, for dissociation of the myosin· ATP. From these results and other data a ?ΔG^o for ATP binding to myosin of 12–13 kcal/mole may be estimated, with a much lower ?ΔG^o for hydrolysis of enzyme-bound ATP. Protein conformational change accompanying ATP binding appears to be the principal means of capture of energy from the overall reaction of ATP cleavage.     

Configurational effects in antibody–antigen interactions studied by microcalorimetry

In this paper we study the binding of two monoclonal antibodies, E3 and E8, to cytochrome c using high-sensitivity isothermal titration calorimetry. We combine the calorimetric results with empirical calculations which relate changes in heat capacity to changes in entropy which arise from the hydrophobic effect. The change in heat capacity for binding E3 is ?350 ± 60 cal K^?1 mol^?1 while for E8 it is ?165 ± 40 cal K^?1 mol^?1. This result indicates that the hydrophobic effect makes a much larger contribution for E3 than for E8. Since the total entropy change at 25°C is very similar for both antibodies, it follows that the configurational entropy cost for binding E3 is much larger than for binding E8 (?77 ± 15 vs. ?34 ± 11 cal K^?1 mol^?1). These results illustrate a case of entropy compensation in which the cost of restricting conformational degrees of freedom is to a large extent compensated by solvent release. We also show that the thermodynamic data can be used to make estimates of the surface area changes that occur upon binding. The results of the present study are consistent with previous hydrogen–deuterium exchange data, detected using 2D NMR, on the two antibody–antigen interactions. The NMR study indicated that protection from exchange is limited to the binding epitope for E8, but extends beyond the epitope for E3. These results were interpreted as suggesting that a larger surface area was buried on cytochrome c upon binding to E3 than to E8, and that larger changes in configurational entropy occur upon binding of E3 than E8. These findings are confirmed by the present study using isothermal titration calorimetry. © 1995 Wiley-Liss, Inc.     

Kinetics and thermodynamics of the P870+Q−A → P870+Q−B reaction in isolated reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides

The temperature dependences of the P870⁺Q^?_A → P870Q_A and P870⁺Q^?_B → P870Q_B recombination reactions were measured in reaction centers from Rhodopseudomonas sphaeroides. The data indicate that the P870⁺Q^?_B state decays by thermal repopulation of the P870⁺Q^?_A state, followed by recombination. ΔG° for the P870⁺Q^?_A → P870⁺Q^?_B reaction is ?6.89 kJ · mol^?1, while ΔH° = ?14.45 kJ · mol^?1 and ?TΔS° = + 7.53 kJ · mol^?1. The activation ethalpy, $\text{H}{}^3$, for the P870⁺Q^?_A Δ P870⁺Q^?_B reaction is +56.9 kJ · mol^?1, while the activation entropy is near zero. The results permit an estimate of the shape of the potential energy curve for the P870⁺Q^?_A → P870⁺Q^?_B electron transfer reaction.     

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.     

Sodium exchange between two sites the binding of sodium to halotolerant bacteria

The longitudinal and transverse relaxation curves of sodium undergoing exchange between two sites are presented. When the two sites are ‘bound’ and ‘free’ sodium respectively, the relaxation curves are, in general, not exponential. It is shown that in some cases only one exponential decay could be detected experimentally though the true decay curve is much more complicated. In such cases where the population of ‘free’ and ‘bound’ sodium are equal, only 40–70% of the total intensity would be detected, depending on the lifetime of sodium in the two sites. It is also shown that the fast exchange approximation, usually applied in the interpretation of sodium relaxation curves, might lead to wrong conclusions.Measurements of sodium relaxation times in halotolerant bacteria show that T₁ and T₂ are different and frequency-dependent. The intensity of the sodium signal is 40% of the tota sodium concentration. It was possible to simulate the relaxation behaviour and intensity measurements by applying the following model. There are three types of sodium: the extracellular sodium (A) which exchanges with part of the intracellular sodium (B) and a fraction (C) which is bound but does not exchange with the extracellular sodium. It was possible to estimate the physical properties of sodium at site B. The quadrupole coupling constant $\text{(e}{}^2\text{qQ/}\text{h}\text{?}\text{)}{}_{\mathrm{<mtext>B</mtext>}}\text{= 9 · 10}{}^6\text{rad/s}$, the correlation time ⊥_cB = 5.5 · 10^?7 s and the lifetime of sodium at site B, ⊥_B = 6 · 10^?4 s.     

Thermodynamics and mechanism of high-pressure deactivation and dissociation of porcine lactic dehydrogenase

Lactic dehydrogenase (LDH) from pig heart and pig skeletal muscle can be reversibly dissociated into monomers at high hydrostatic pressure. The reaction can be quantitatively filled by a reversible consecutive dissociation-unfolding mechanism according to Na = 4M ? 4M* (where N is the native letramer, and M and M* two different conformations of the monomer) (K. Müller, et al., Biophys. Chem. 14 (1981) 101). At P ? 1 kbar, the pressure deactivalion of both isoenzymes (H₄ and M₄) is described by the two-state equilibrium N ? 4M. From the respective equilibrium constant and the temperature and pressure dependence of the change in free energy, the thermodynamic parameters of the dissociation/deactivation may be determined, e.g., for LDH-M₄: Δg_Diss = 110 $\text{kJ}\text{mol}$, ΔSDiss = ?860 J/K per mol, ΔH_Diss = ?124 $\text{kJ}\text{mol}$ (enzyme concentration 10 $\text{μg}\text{ml}$, in Tris-HCl buffer, pH 7.6, I = 0.16 M, 293 K, 0.8 kbar); the dissociation volume is found to be ΔV_Diss = ?420 $\text{ml}\text{mol}$ (0.7 < p < 0.9 kbar). Measurements using 8-anilino-1-naphlhalenesulfonic acid (ANS) as extrinsic fluorophore demonstrate that the occurrence of hydrophobic surface area upon dissociation parallels the decrease in reactivation yield after pressurizarion beyond 1 kbar. Within the range of reversible deactivation (p < 1 kbar) no increase in ANS fluorescence is detectable, thus indicating compensatory effects in the process of subunit dissociation. ²H₂O is found to stabilize the enzyme towards pressure dissociation, in accordance with the involvement of hydrophobic interactions in the subunit contact of both isoenzymes of LDH.     

Kinetics and thermodynamics of catalysis and thermal inactivation of a novel α-amylase (Tp-AmyS) from Thermotoga petrophila

Abstract

This research is focussed on kinetic, thermodynamic and thermal inactivation of a novel thermostable recombinant α-amylase (Tp-AmyS) from Thermotoga petrophila. The amylase gene was cloned in pHIS-parallel1 expression vector and overexpressed in Escherichia coli. The steady-state kinetic parameters (V_max, K_m, k_cat and k_cat/K_m) for the hydrolysis of amylose (1.39?mg/min, 0.57?mg, 148.6?s^?1, 260.7), amylopectin (2.3?mg/min, 1.09?mg, 247.1?s^?1, 226.7), soluble starch (2.67?mg/min, 2.98?mg, 284.2?s^?1, 95.4) and raw starch (2.1?mg/min, 3.6?mg, 224.7?s^?1, 61.9) were determined. The activation energy (E_a), free energy (ΔG), enthalpy (ΔH) and entropy of activation (ΔS) at 98?°C were 42.9?kJ mol^?1, 74?kJ mol^?1, 39.9?kJ mol^?1 and ?92.3 J mol^?1 K^?1, respectively, for soluble starch hydrolysis. While ΔG of substrate binding (ΔG_E-S) and ΔG of transition state binding (ΔG_E-T) were 3.38 and ?14.1?kJ mol^?1, respectively. Whereas, EaD, Gibbs free energy (ΔG*), increase in the enthalpy (ΔH*) and activation entropy (ΔS*) for activation of the unfolding of transition state were 108, 107, 105?kJ mol^?1 and ?4.1 J mol^?1 K^?1. The thermodynamics of irreversible thermal inactivation of Tp-AmyS revealed that at high temperature the process involves the aggregation of the protein.     

Characterization of sodium and proton flows in sub-bacterial particles of Halobacterium halobium in terms of nonequilibrium thermodynamics

A thermodynamic characterization of the Na⁺-H⁺ exchange system in Halobacterium halobium was carried out by evaluating the relevant phenomenological parameters derived from potential-jump measurements. The experiments were performed with sub-bacterial particles devoid of the purple membrane, in 1 M NaCl, 2 M KCl, and at pH 6.5–7.0. Jumps in either pH or pNa were brought about in the external medium, at zero electric potential difference across the membrane, and the resulting relaxation kinetics of protons and sodium flows were measured. It was found that the relaxation kinetics of the proton flow caused by a pH-jump follow a single exponential decay, and that the relaxation kinetics of both the proton and the sodium flows caused by a pNa-jump also follow single exponential decay patterns. In addition, it was found that the decay constants for the proton flow caused by a pH-jump and a pNa-jump have the same numerical value. The physical meaning of the decay constants has been elucidated in terms of the phenomenological coefficients (mobilities) and the buffering capacities of the system. The phenomenological coefficients for the Na⁺-H⁺ flows were determined as differential quantities. The value obtained for the total proton permeability through the particle membrane via all available channels, $\text{L}{}_{\mathrm{<mtext>H</mtext>}}\text{= (}\text{?J}{}_{\mathrm{<mtext>H\; +</mtext>}}\text{?Δ}\text{pH}\text{)}{}_{\mathrm{<mtext>\Delta \psi ,\Delta </mtext><mtext>pNa</mtext>}}$, was in the range of 850–1150 nmol H⁺·(mg protein)^?1·h^?1·(pH unit)^?1 for four different preparations; for the total Na⁺ permeability, $\text{L}{}_{\mathrm{<mtext>Na</mtext>}}\text{= (}\text{?J}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{+}}\text{?Δ}\text{pNa}\text{)}{}_{\mathrm{<mtext>\Delta \psi ,\Delta </mtext><mtext>pH</mtext>}}$, it was 1620–2500 nmol Na⁺·(mg protein)^?1·h^?1·(pNa unit)^?1; and for the proton ‘cross-permeability’, $\text{L}{}_{\mathrm{<mtext>HNa</mtext>}}\text{= (}\text{?J}{}_{\mathrm{<mtext>H</mtext>}}{}^{\mathrm{+}}\text{?Δ}\text{pNa}\text{)}{}_{\mathrm{<mtext>\Delta \psi ,\Delta </mtext><mtext>pH</mtext>}}$, it was 220–580 nmol H⁺·(mg protein)^?1·h^?1·(pNa unit)^?1, for different preparations. From the above phenomenological parameters, the following quantities have been calculated: the degree of coupling (q), the maximal efficiency of Na⁺-H⁺ exchange (η_max), the flow and force efficacies (?) of the above exchange, and the admissible range for the values of the molecular stoichiometry parameter (r). We found q ? 0.4; η_max ? 5%; 0.36 ? r ? 2; ?_JNa⁺ ? 1.3 · 10⁵μmol · (RT unit)^?1 at J_Na = 1 μmolNa⁺ · (mgprotein)^?1 · h^?1; and ?_ΔpNa ? 5 · 10⁴ ΔpNa · (mg protein) · h · (RT unit)^?1 at ΔpNa = 1 unit, for different preparations.     

Multispectroscopic studies of the interaction of folic acid with glycated human serum albumin

Abstract

The interaction between glycated human serum albumin (gHSA) and folic acid (FA) was investigated by various spectroscopic techniques, such as fluorescence, circular dichroism, UV–vis absorption spectroscopy and electrophoretic light scattering technique. These methods characterize the binding properties of an albumin–folic acid system. The binding constants values (K_a) at 300 and 310 K are about 10⁴ M^?1. The standard enthalpy change (ΔH) and the standard entropy change (ΔS) were calculated to be ～?20?kJ mol^?1 and ～16 J mol^?1 K^?1, respectively, which indicate characteristic electrostatic interactions between gHSA and folic acid. The CD studies showed that there are no significant conformational changes in the secondary structure of the protein. Moreover, the zeta potential measurements proved that under physiological conditions the gHSA–folic acid complex shows instability. No significant changes in the secondary structure of the protein and reversible drug binding are the desirable effect from pharmacological point of view.

Communicated by Ramaswamy H. Sarma     

Rate enhancement by catalytic groups in enzymes. Imidazole catalysis of the hydrolysis of N,O-diacetylserinamide as a model for general base catalysis in chymotrypsin

An attempt to estimate the importance of general acid-base catalysis in enzymic catalysis has been made, using the hydrolysis of the ester group of N,O-diacetylserinamide as a model for the deacylation of acyl-chymotrypsins. General base catalysis of this reaction by imidazole is estimated to reduce the activation energy by at least 31 kJ mol^?1. The rate of reaction, however, is not greatly enhanced because of an unfavourable change in the entropy of activation from ?132 to ?197 JK^?1 mol^?1. At about 300 K, a typical temperature for enzyme-catalysed reactions, the reduction in activation energy would cause a rate enhancement of about 3 × 10⁵-fold if the unfavourable entropy change did not occur. For specific acyl-chymotrypsins the entropy of activation for deacylation is about ?89 J K^?1 mol^?1, allowing the full effect of general base catalysis by imidazole to be realized. It is, therefore, postulated that in the active site of an enzyme, a properly oriented imidazole side chain may catalyse the rate of a reaction 10⁵-fold by general base catalysis.     

Imipramine binding site Temperature dependence of the binding of 3H-labeled imipramine and 3H-labeled paroxetine to human platelet membrane

The characteristics of ³H-labeled imipramine and ³H-labeled paroxetine binding to human platelet membranes were determined at various temperatures between 0 and 37°C. Both paroxetine and imipramine probably bind to the same molecular complex in the platelet membrane, but the binding characteristics are different for the two molecules. The dissociation constant ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}$) for imipramine increases from 0.3 nM to 7.0 nM with increasing incubation temperature in a continuous way, whereas $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ for paroxetine is almost constant, about 0.05 nM, between 0 and 19°C, and first begins to increase from 0.06 nM to 0.16 nM between 20 and 37°C. This suggests that the binding of paroxetine to the binding site induces a conformational change in the molecular complex of the binding site, whereas the binding of imipramine takes place without conformational changes in the binding site.     

Errata

Optimal conditions for activation of adenylate cyclase in membrane particles were studied. Enzyme activation with serotonin (5-hydroxytryptamine), NaF, and guanosine $\text{5′-(3-O-}\text{thio}\text{)-}\text{triphosphate}$ (GTPγS) was time- and temperature-dependent. Mg²⁺ was required for enzyme activation. Adenylate cyclase that was activated by NaF or GTPγS was gradually inhibited by $\text{N-}\text{methylmaleimide}$ while enzyme activated with serotonin and GTP responded faster to inhibition by the same sulfhydryl reagent. The enzyme responded in a similar fashion to a spin-labeled $\text{N-}\text{methylmaleimide}$ analog 3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrolidinyloxyl (i.e., $\text{N-}\text{methylmaleimide}$ nitroxide). Binding of the spin label was enhanced following enzyme activation by serotonin, NaF, or GTPγS in the presence of Mg²⁺. Activation of the enzyme was accompanied by an increase in the strong immobilization peaks in the EPR spectra. Both effects, the increase in binding and in the strong immobilization peaks, can be induced by Mg²⁺ alone. The results indicate that a general conformational change induced by Mg²⁺ may be essential for adenylate cyclase activation.     

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.     

Evaluation of proximity inhibition of DNA synthesis in 3T3 cells.

A microphotometric technique that displays rapid length changes of Spirostomum has been used to follow the variation with temperature of three kinetic parameters of myonemal contraction: contraction rate, relaxation rate and stimulus duration at threshold. In each case the exponential form of the relationship indicated that the gross rate constant might be equated with the limiting rate constant, k, of a driving chemical reaction, and from standard expressions of chemical kinetics the change in activation free energy appropriate to this reaction has been computed. The thermal dependence of contraction is described by an activation enthalpy (ΔLH?) of 21.7 kcal mol^?1, and the activation entropy (ΔLS?) of 26.8 e.u. is consistent with a model of contraction requiring neutralization of fixed myonemal charges by divalent cations. The analysis of thermal dependence of relaxation gives a negative activation entropy, a result predicted for a rate-limiting reaction involving dissociation of a neutral molecule. On the other hand, values of ΔLS? and ΔLH? for relaxation fall close to an isokinetic correlation drawn in the literature from analysis of the thermal dependence of ciliary beat frequency in different organisms, and for which breakdown of an ATP-ATPase complex could be the common rate-limiting reaction. ΔLS? for stimulus duration suggests that the rate-limiting step in excitation-contraction coupling is a reaction between ions of like charge, or ion pair formation from a neutral molecule.     

Identification of opiate receptor binding in intact animals.

After intravenous administration of ³H-naloxone to rats, particulate bound radioactivity accumulated in the brain is selectively associated with opiate receptor binding sites, providing a means of labeling the opiate receptor $\text{in vivo}$. The regional distribution of ³H-naloxone bound $\text{in vivo}$ closely parallels regional differences in opiate receptor binding $\text{in vitro}$ with highest levels in the corpus striatum, negligible receptor-associated binding in the cerebellum and intermediate levels in other regions. ³H-Naloxone binding $\text{in vivo}$ is saturable with the same total number of binding sites determined $\text{in vivo}$ as by $\text{in vitro}$ procedures. Nalorphine is markedly more potent than morphine in inhibiting ³H-naloxone binding $\text{in vivo}$ and non-opiates are ineffective. The half-life for dissociation of ³H-naloxone bound to particles $\text{in vivo}$ is the same as its dissociation rate after binding occurs $\text{in vitro}$, and sodium stabilizes ³H-naloxone bound $\text{in vivo}$ from initial rapid dissociation as predicted from the known properties of the opiate receptor $\text{in vitro}$.     

Cytochalasin B inhibition and temperature dependence of 3-O-methylglucose transport in fat cells

The transport of $\text{3-O-}\text{methylglucose}$ in white fat cells was measured under equilibrium exchange conditions at $\text{3-O-}\text{methylglucose}$ concentrations up to 50 mM with a previously described method (Vinten, J., Gliemann, J. and Østerlind, K. (1976) J. Biol. Chem. 251, 794–800). Under these conditions the main part of the transport was inhibitable by cytochalasin B. The inhibition was found to be of competitive type with an inhibition constant of about 2.5 · 10^?7 M, both in the absence and in the presence of insulin (1μM). The cytochalasin B-insensitive part of the $\text{3-O-}\text{methylglucose}$ permeability was about 2 · 10^?9 cm · s^?1, and was not affected by insulin. As calculated from the maximum transport capacity, the half saturation constant and the volume/ surface ratio, the maximum permeability of the fat cell membrane to $\text{3-O-}\text{methylglucose}$ at 37°C and in the presence of insulin was 4.3 · 10^?6 cm · s^?1. From the temperature dependence of the maximum transport capacity in the interval 18–37°C and in the presence of insulin, an Arrhenius activation energy of $\text{14.8 ± 0.44}\text{kcal/mol}$ was found. The corresponding value was $\text{13.9 ± 0.89}$ in the absence of insulin. The half saturating concentration of $\text{3-O-}\text{methylglucose}$ was about 6 mM in the temperature interval used, and it was not affected by insulin, although this hormone increased the maximum transport capacity about ten-fold to 1.7 mmol · s^?1 per 1 intracellular water at 37°C.