Regulation of translation in rabbit reticulocytes and mouse L-cells; comparison of the effects of temperature

Various parameters of protein synthesis were analyzed in rabbit reticulocytes exposed to various temperatures for up to five hours. Between 10°C and 40°C total protein synthesis exhibited two different apparent activation energies (36 kcal/mole, 10–24°C; 22 kcal/mole, 24–40°C), as did protein elongation and release (35 kcal/mole, 10–25°C; 12 kcal/mole, 25–40°C). However, the level of polysomes remained essentially unchanged between 0°C and 42°C which implies that the activation energy for polypeptide initiation is quite similar to that for elongation and is also biphasic. This situation is different from that in cultured mouse L-cells where the polysome level is dependent on temperatures. Nevertheless, reticulocytes and L-cells appear to be similar in their temperature dependence of initiation and in their rate of elongation (5–6 amino acids/second at 36°C).     

Temperature dependence of ultrasound-induced cell killing: The role of membrane fluidity

Chinese hamster cells in suspension were exposed to 20 kHz ultrasound (US) at 54 W/cm² and various temperatures between 2 and 44 °C. Activation energies were 2.6 and 24 kcal/mole below and above 35 °C, respectively. Procaine, a local anaesthetic drug known to increase membrane fluidity, enhanced cellular inactivation by US above 41 °C, increasing the activation energy to 62 kcal/mole. The inactivation of the bacterium Salmonella typhimurium by US was also dependent on the exposure temperature, with an activation energy of 2.9 kcal/mole between 2 and 44 °C. These data are most simply explained by the hypothesis that membranes are a major target for cellular inactivation by US and that the fluidity of the membranes is important in this respect.     

Calorimetric determination of polymerization enthalpy for bacterial flagellin (Salmonella)

The polymerization of bacterial flagellin protein (Salmonella strain SJ814) into flagellar filaments has been found by direct calorimetric measurement to be $\text{exothermic}$ at 25° in .15M KCl, pH 6.8 with a ΔH of ?12.7 ± 0.6 kcal per mole of monomer polymerized. The calorimetric result at 25° contrasts sharply with the endothermic ΔH of +38 kcal/mole inferred from temperature dependence of the critical monomer concentration near 40°C. Comparison between these two values implies that unless a different mechanism of polymerization prevails at the two temperatures the heat capacity change for flagellin polymerization may be as large as 3.3 kcal/mole deg.     

Cytochrome c interaction with membranes. The enthalpy of oxidation of cytochromes c, c2, and a1,2.

The enthalpy of oxidation of horse-heart cytochrome c bound to phospholipid vesicles was found to be 14.6 ± 0.3 kcal/mole at 25 °C, pH 7.0, equal to the value for oxidation of the free form of the cytochrome. The affinity constants for binding of the reduced and oxidized forms of cytochrome c were the same at 4 °C and 30 °C, indicating that ΔH ° of binding contributes negligibly to the overall enthalpy of oxidation of the bound cytochrome c. The free energy (ΔG °′) of oxidation of the bound cytochrome c was 1.3 kcal/mole smaller than that for the free form, the difference being due to the change in entropy favoring the oxidized state of the cytochrome in the bound state. Measurement of the ΔH °′ for the oxidation of cytochrome a relative to the ferri/ferrocyanide couple shows it to be the same, within the limits of experimental error to that for the oxidation of cytochrome c.     

The binding of deoxyguanosine to polycytidylic acid: an application of the thermodynamic model of monomer-polymer complex formation.

The thermodynamic model (equation 1) for formation of monomer-polymer complexes developed for better interpretation of the sigmoidal isotherms for the binding of adenosine to polyuridylic acid (2) and chemically modified polyuridylic acids (3) has been successfully applied to reproduce the isotherms for both the duplex binding of deoxyguanosine (d-G) to polycytidylic acid (at pH 6.8) and the triplex binding at pH 4.1. The value for the equilibrium constant, K, of the triplex complex (per unit of C-G-C) is ～2000 at the optimum value of n = 5 (n is the number of d-G units in the smallest complex that can form). The value of K for the duplex complex is 555 and the optimum value of n is 4.The value of ΔG for the triple helical complex is 4.15 kcal/mole, the value of w (the stacking free energy of the d-G units in the complex) is 2.05 kcal/mole. For the double helical complex at pH 6.8, ΔG° is 3.45 kcal/mole, w = 1.55 kcal/mole.It is also shown that equation (1) predicts that the shape and mid-point slope (i.e., w) of a binding isotherm depends only on the value of n; and thus the isotherms for rA-poly U (n = 5) and dG-poly C (n = 5) have the same mid-point slopes, and thus the same values of w. The difference between ΔG° and w is taken as a relative measure of the free energy of hydrogen bonding; values are calculated for the rA-poly U, the dG-poly C triple helix, and the dG-poly C double helical complexes.     

Microviscosity of the lipid domains of normal and hypercholesterolemic very low density lipoprotein.

Very low density lipoprotein (VLDL) has been isolated from normal (n) and dietary-induced hypercholesterolemic (hc) rabbits. Incorporation of the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene into the lipid domains of both n VLDL and hc VLDL allowed assessment of the fluidity characteristics of these particles, utilizing fluorescence polarization techniques. Over the temperature range of 5° – 45°, the lipid region of n VLDL consists of an invariant phase, characterized by a microviscosity, η, at 30° of 0.6 ± 0.2 poise and a fusion activation energy, ΔE, of 7.6 ± 1.5 kcal/mole. The lipid region of hc VLDL, over the same temperature range, also is invariant and is characterized by a value of η at 30° of 4.6 ± 0.3 poise, and a ΔE of 7.8 ± 1.5 kcal/mole. Thus, large differences in the fluidit of the lipid in n VLDL and hc VLDL are evident, most probably due to the greatly increased content of cholesterol esters in hc VLDL, compared to n VLDL.     

The rate of γ-benzyl-L-glutamate NCA polymerizations

The primary amine initiated homopolymerization of γ-benzyl-L -glutamate NCA in dioxane at 25°C, 35°C, 50°C, and 65°C has been investigated. The reactions were virtually independent of temperature indicating an activation energy of less than 1 kcal/mole. The entropy of activation was estimated to be ?65 entropy units at 300°K. The reaction proceeded in two stages. The first stage was zero-order with respect to monomer, whereas the second was first-order with respect to monomer. Both stages were first-order with respect to initiator. These results were interpreted by assuming that the rate constant for propagation was not independent of the degree of polymerization up to the point where a conformational transition to α-helix occurred.     

Ordered substrate binding and evidence for a thermally induced change in mechanism for E. coli aspartate transcarbamylase

Isotopic exchange kinetics at equilibrium for E. coli native aspartate transcarbamylase at pH 7.8, 30 °C, are consistent with an ordered BiBi substrate binding mechanism. Carbamyl phosphate binds before l-Asp, and carbamyl-aspartate is released before inorganic phosphate. The rate of [¹⁴C]Asp C-Asp exchange is much faster than [³²P]carbamyl phosphate P_i exchange. Phosphate, and perhaps carbamyl phosphate, appears to bind at a separate modifier site and prevent dissociation of active-site bound P_i or carbamyl phosphate. Initial velocity studies in the range of 0–40 °C reveal a biphasic Arrhenius plot for native enzyme: E_a (>15 °C) = 6.3 kcal/ mole and E_a (<15 °C) = 22.1 kcal/mole. Catalytic subunits show a monophasic plot with E_a ? 20.2 kcal/mole. This, with other data, suggests that with native enzyme a conformational change accompanying aspartate association contributes significantly to rate limitation at t > 15 °C, but that catalytic steps become definitively slower below 15 °C. Model kinetics are derived to show that this change in mechanism at low temperature can force an ordered substrate binding system to produce exchange-rate patterns consistent with a random binding system with all exchange rates equal. The nonlinear Arrhenius plot also has important consequences for current theories of catalytic and regulatory mechanisms for this enzyme.     

Thermodynamics of equilibria of hemoglobins M Milwaukee-I and Saskatoon and isolated chains of hemoglobin a with carbon monoxide

The thermodynamic parameters of the CO-equilibria of isolated chains of hemoglobin A and of two α-chains in hemoglobins M Milwaukee-I and Saskatoon at 25°, pH 7.0 were determined. The parameters for the binding of the first CO molecule to the hemoglobins M were ΔH′=?17 and ?18 kcal/mole heme and ΔS′=?30 and ?29 e.u. for hemoglobins M Milwaukee-I and Saskatoon, respectively. In contrast to this the characteristics of the second step of the binding were ΔH′=+5.9^· and +4.3 kcal/mole and ΔS′=+51 and +49 e.u. These values for the second step were also significantly different from those of the isolated α-chain (ΔH′=?15 kcal/mole and ΔS′=?11 e.u.).     

The beta-galactosidase-catalyzed hydrolysis of o-nitrophenol-beta-D-galactoside at subzero temperatures: evidence for a galactosyl-enzyme intermediate.

The reaction of β-galactosidase ($\text{E. coli}$ K12) with $\text{o}$-nitrophenyl-β-$\text{D}$-galactoside has been investigated over the temperature range +25° to ?30° using 50% aqueous dimethyl sulfoxide as solvent. At temperatures below ?10° turnover becomes very slow and a burst of $\text{o}$-nitrophenol is observed. Such a burst indicates the existence of a galactosyl-enzyme intermediate whose breakdown is rate-limiting and provides a means of determining the active site normality. The Arrhenius plot for turnover is linear in the ?25 to +25° range with E_a = 26 ± 3 kcal/mole. The presence of the 50% DMSO had no effect on K_m but caused a small decrease in K_cat.     

Death kinetics of yeast in spray drying

The death kinetics of a strain of Saccharomyces cerevisiae were studied in an industrial scale spray drier. In solution studies, the death kinetics of yeast was found to be comparable to pathogen destruction. From the studies in drying of yeast a prediction of a 4 log cycle decrease in viable cells of pathogens could be made for normal processing conditions. This should insure the safety of spray-dried foods unless after contamination occurs. It was found that during drying, although the rate of death is high, the activation energy is greatly decreased over that of death in aqueous solution (reduction from 130 kcal/mole to 5 kcal/mole). The reduction in E_a may be attributed to the thermodynamic compensation phenomenon in which the resulting negative entropy of reaction acts to protect the cells through a water–protein interaction. However, the possibility of a change in death mechanism cannot be precluded. Overall, these results suggest the danger in extrapolating death kinetics to high temperature.     

Further studies on the temperature dependence of the binding of testosterone to human pregnancy plasma proteins

The binding of testosterone by pregnancy plasma proteins has been studied by a new equilibrium dialysis system. The temperature dependence on the association constant has been investigated and the enthalpy change ΔH and entropy change ΔS have been calculated.By a computer optimization program, the binding constant of the high affinity testosterone binding protein has been estimated from Scatchard plots. The binding reactions were carried out at 5°, 25° and 37° C. The corresponding values were 3.1.10 1.2.10⁹ and 7.2.10⁸ liter/mole. The resulting enthalpy and entropy changes were ?2.0 kcal/mole and 35.0 cal/(mole.degree) respectively.It can be concluded that the binding of testosterone to the specific binding protein is an exothermic reaction and is stabilized by hydrophobic binding forces.     

Heat of denaturation of lysozyme

The enthalpy of denaturation of lysozyme was determined by measuring the heat, capacity of an aqueous solution of this protein in the vicinity of the transition temperature, 46 °C at pH 1. Within experimental error the calorimetric, heat (56 ± 8 kcal/mole) was found to agree with the van't Hoff transition enthalpy (63 ± 6 kcal/mole) determined from optical rotation measurements as a function of temperature. This indicates that denaturation of this protein can be interpreted in terms of a two-state model. Successive measurements of the same sample showed, from several lines of evidence, that the transition was about 80% reversible for the particular environmental conditions and thermal history involved in the study.     

Thermal transitions in E. coli +RNA fMet and two of its molecular fragments

Melting curves of tRNA^fMet and two fragments derived from this molecule by limited ribonuclease T1 digestion (i.e., the anticodon arm and loop [K fragment] and the larger fragment representing three-fourths of the tRNA chain from the 3′ terminus including two potential limbs of the cloverleaf structure [L fragment]) are presented. The profiles observed are consistent with the presence of base paired structures in all those molecules. At low salt concentration (0.02M Na⁺) the stabilities of these molecules measured by the apparent midpoints of the denaturation profiles are in the order K > L > tRNA. The relative stabilities approach each other at 0.2M Na⁺ (the tRNA profile being biphasic), while at high salt (2M) the L fragment seems to be more stable than either K or t-RNA ^fMet. Estimation of the enthalpy of denaturing the K structure in 0.02M Na⁺ gives a value of 40 ± 3 kcal/mole corresponding to an enthalpy per effective G.C. base pair disrupted of 10 ± 1 kcal/mole.     

Influence of Glu-376 --> Gln mutation on enthalpy and heat capacity changes for the binding of slightly altered ligands to medium chain acyl-CoA dehydrogenase

We showed that the alpha-CH(2) --> NH substitution in octanoyl-CoA alters the ground and transition state energies for the binding of the CoA ligands to medium-chain acyl-CoA dehydrogenase (MCAD), and such an effect is caused by a small electrostatic difference between the ligands. To ascertain the extent that the electrostatic contribution of the ligand structure and/or the enzyme site environment modulates the thermodynamics of the enzyme-ligand interaction, we undertook comparative microcalorimetric studies for the binding of 2-azaoctanoyl-CoA (alpha-CH(2) --> NH substituted octanoyl-CoA) and octenoyl-CoA to the wild-type and Glu-376 --> Gln mutant enzymes. The experimental data revealed that both enthalpy (DeltaH degrees ) and heat capacity changes (DeltaC(p) degrees ) for the binding of 2-azaoctanoyl-CoA (DeltaH degrees (298) = -21.7 +/- 0.8 kcal/mole, DeltaC(p) degrees = -0.627 +/- 0.04 kcal/mole/K) to the wild-type MCAD were more negative than those obtained for the binding of octenoyl-CoA (DeltaH degrees (298) = -17.2 +/- 1.6 kcal/mole, DeltaC(p) degrees = -0.526 +/- 0.03 kcal/mole/K). Of these, the decrease in the magnitude of DeltaC(p) degrees for the binding of 2-azaoctanoyl-CoA (vis-à-vis octenoyl-CoA) to the enzyme was unexpected, because the former ligand could be envisaged to be more polar than the latter. To our further surprise, the ligand-dependent discrimination in the above parameters was completely abolished on Glu-376 --> Gln mutation of the enzyme. Both DeltaH degrees and DeltaC(p) degrees values for the binding of 2-azaoctanoyl-CoA (DeltaH degrees (298) = -13.3 +/- 0.6 kcal/mole, DeltaC(p) degrees = -0.511 +/- 0.03 kcal/mole/K) to the E376Q mutant enzyme were found to be correspondingly identical to those obtained for the binding of octenoyl-CoA (DeltaH degrees (298) = -13.2 +/- 0.6 kcal/mole, DeltaC(p) degrees = -0.520 +/- 0.02 kcal/mole/K). However, in neither case could the experimentally determined DeltaC(p) degrees values be predicted on the basis of the changes in the water accessible surface areas of the enzyme and ligand species. Arguments are presented that the origin of the above thermodynamic differences lies in solvent reorganization and water-mediated electrostatic interaction between ligands and enzyme site groups, and such interactions are intrinsic to the molecular basis of the enzyme-ligand complementarity.     

NMR study of rapid water diffusion across lipid bilayers in dipalmitoyl lecithin vesicles

A method for measurement of rapid diffusional exchange between external and internal water in lecithin vesicles is described. Paramagnetic ions were inserted inside DPL vesicles and the NMR relaxation times for water protons were measured as a function of temperature. It was found that water diffusion rate is described by a single activation energy of 15±1 kcal/mole in the temperature range 16 – 35°C and exhibits a maximum at 44°C. The permeability of DPL vesicles to water was calculated to 16–18 × 10^?4 cm/s at 44°C and 1.7 × 10^?4 cm/s at 20°C.     

Kinetics of the renaturation of yeast tRNA3 leu.

Yeast tRNA₃^Leu is one of several tRNA molecules which can adopt a stable, biologically inactive, denatured conformation. The circular dichroism of the native and denatured conformers differs, providing the basis for the present study of the mechanism for the renaturation process. Conversion of the denatured structure to the native takes place in two steps: a rapid change occurring immediately on addition of Mg⁺⁺, followed by a slower, strongly temperature-dependent step which returns the molecule to its biologically active state. Optimal kinetic data for the second step could be obtained at 285 nm. Analysis of the time dependence of Δε₂₈₅ by the Guggenheim method demonstrated that this step follows first-order kinetics. The temperature dependence of the rate constants over the range 32–41°C yielded the following parameters for the rate-limiting step: E_a = 69 kcal/mole, ΔH^? = 69 kcal/mole, and ΔS^? = 146 cal/mole deg. Values of this magnitude are typical of order—order transitions in nucleic acids.     

Experimental thermodynamics of the helix--random coil transition. 3. Determination of the transition enthalpies of the helical complexes poly(I+C) and poly I in solution

The enthalpies of the helix-coil transitions of the ordered polynucleotide systems of poly(inosinic acid)–poly(cytidylic acid) [poly(I + C)], (helical duplex), and of poly (inosinic acid) [poly(I + I + I)], (proposed secondary structure: a triple-stranded helical complex), were determined by using an adiabatic twin-vessel differential calorimeter. Measuring the temperature course of the heat capacity of the aqueous polymer solutions, the enthalpy values for the dissociation of the helical duplex poly (I + C) and the three-stranded helical complex poly(I + 1 + 1), respectively, were obtained by evaluating the additional heat capacity involved in the conformational change of the polynucleotide system in the transition range. The ΔH values of the helix-coil transition of poly (I + C) resulting from the analysis of the calorimetric measurements vary between the limits 6.5 ± 0.4 kcal/mole (I + C) and 8.4 ± 0.4 kcal/mole (I + C). depending on the variation of the cation concentration ranging from 0.063 mole cations kg H₂O to 1.003 mole cations/kg H₂O. The calorimetric investigation of an aqueous poly I solution (cation concentration 1.0 mole/kg H₂O) yielded the enthalpy value ΔH = 1.9 ± 0.4 kcal/mole (I), a result which has been interpreted qualitatively following current models of inter- and intramolecular forces of biologically significant macromolecules. Additional information on the transition behavior of poly(I+ C)Was obtained by ultraviolet and infrared absorption measurements.     

Cooperative and thermodynamic parameters for oligoinosinate-polycytidylate complexes

The cooperative nature of the binding between polycytidylate and the oligoinosinates I(pI)_5–10 has been determined. Using the data of Tazawa, Tazawa, and Ts'o, it is shown that knowledge of the slope of the adsorption isothern allows one to determine the oligomer-polymer binidng constant, the oligomer–oligomer interaction constant, and the average degree of association (cooperative clustering) of the oligomers on the polymer. Knowledge of the above equilibrium constants as a function of temperature yields the respective thermodynamic parameters; no assumptions need to be made about the nature of the equilibrium constants or the thermodynamic parameters. For very long chains of polycytidylate, simple, explicit relations are given for the determination of the equilibrium constants involved. For finite chains of polycytidylate, the calculation of a single graph for each oligomer and polymer size allows the equilibrium constants to be determined for all experimental conditions of temperature and concentration. We find that the enthalpy and entropy of binding an oligomer n, bases to be δH_n = ±13.7 ? n(6.65) and δS_n = +32.5 ? n(18.8) given, respectively, in kcal/mole and e.u.; these parameters predict a melting temperature of 81°C for the poly(I)·poly(C) complex compared with the experimental value of 75°C. If the enthalpy is interpreted as arising from a sum of hydrogen bonding and stacking interactions, then the enthalpy of stacking is ?13.7 kcal/mole while the enthalpy of hydrogen bonding is +7 ± 4 kcal/mole; the positive enthalpy of hydrogen bonding presumably is a result of the fact that in the inosine-cytosine base pair, only two of the three sites on cytosine can hydrogen bond, the third being blocked from hydrogen bonding with water. The enthalpy of interaction between neighboring bound oligomers is found to be ?10.4 kcal/mole while the corresponding entropy is ?26.1 e.u. The binding is bound to be cooperative, though the extent of clustering varies markedly with temperature; the average number of oligomers in a cluster on the polymer is found to about five at a melting temperature of 25°C. The approach and equations given have generally applicability to oligomer-polymer associations.