Kinetics and mechanism of CO substitution of [η-CpFe(CO)3]PF6 with PPh3 in the presence of substituted pyridine N-oxide

The kinetics of substitution reactions of [η-CpFe(CO)₃]PF₆ with PPh₃ in the presence of R-PyOs have been studied. For all the R-PyOs (R = 4-OMe, 4-Me, 3,4-(CH)₄, 4-Ph, 3-Me, 2,3-(CH)₄, 2,6-Me₂, 2-Me), the reactions yeild the same product [η⁵-CpFe(CO)₂PPh₃]PF₆, according to a second-order rate law that is first order in concentrations of [η⁵-CpFe(CO)₃]PF₆ and of R-PyO but zero order in PPh₃ concentration. These results, along with the dependence of the reaction rate on the nature of R-PyO, are consistent with an associative mechanism. Activation parameters further support the bimmolecular nature of the reactions: ΔH^≠ = 13.4 ± 0.4 kcal mol⁻¹, ΔS^≠ = −19.1 ± 1.3 cal k⁻¹ mol⁻¹ for 4-PhPyO; ΔH^≠ = 12.3 ± 0.3 kcal mol⁻¹, ΔS^≠ = 24.7 ±1.0 cal K⁻¹ mol⁻¹ for 2-MePyO. For the various substituted pyridine N-oxides studied in this paper, the rates of reaction increase with the increasing electron-donating abilities of the substituents on the pyridine ring or N-oxide basicities, but decrease with increasing ¹⁷O chemical shifts of the N-oxides. Electronic and steric factors contributing to the reactivity of pyridine N-oxides have been quantitatively assessed.     

Complexation of lithium(I), sodium(I), silver(I) and thallium(I) by 4,7,13,16-tetraoxa-1,10-diazabicyclo[8.5.5]eicosane and 4, 7, 13-trioxa-1,10-diazabicyclo[8.5.5]eicosane in trimethyl phosphate. A potentiometric titration and Li and Na nuclear magnetic resonance study

Complexation of M⁺=Li⁺, Na⁺, Ag⁺ and TI⁺ by the cryptands 4, 7, 13, 18-tetraoxa-l, 10-diazabicyclo[8.5.5]eicosane (C211) and 4,7,13-trioxa-1,10-diazabicyclo[8.5.5]eicosane (C21C₅) to form the cryptates [M.C211]⁺ and [M.C21C₅]⁺ has been studied in trimethyl phosphate by potentiometric titration and ⁷Li and ²³Na NMR spectroscopy. For [M.C211]⁺ the logarithm of the apparent stability constants, log K (dm³ mol^-1)=6.98±0.05, 5.38±0.05, 9.82±0.02 and 3.95±0.02 for M⁺ =Li⁺, Na⁺, Ag⁺ and TI⁺, respectively; and for [M.C21C₅]⁺ log K (dm³ mol^-1)=2.40±0.10, 1.90±0.05, 6.04±0.02 and 2.42±0.10 for M⁺=Li⁺, Na⁺, Ag⁺ and Tl⁺, respectively. The decomplexation kinetic parameters for [Na.C211]⁺ are: k_d (298.2 K)=6.924±0.50 s^-l, ΔH_d≠=62.2±0.9 kJ mol^-1, and ΔS_d≠= -20.3±2.7 J K^-1 mol^-1; and those for [Li.C21C₅]⁺ are: k_d (298.2 K)=23.3±0.4 s^-1, ΔH_d≠ =61.2±1.1 kJ mol^-1, and ΔS_d≠= -13.6±3.6 J K^-1 mol^-1. Metal ion exchange on [Li.C211]⁺ is in the very slow extreme of the NMR timescale up to 390 K and k_d « 4 s^-1 at 298.2 K, while in contrast exchange on [Na.C21C₅]⁺ is in the fast extreme of the NMR timescale at 298.2 K (k_d≈ 10⁴ s^-1). These data are compared with those obtained in other solvents.     

Dinuclear complexes of transition metals containing carbonate ligands. VIII. Kinetics and mechanism of carbon dioxide uptake by the tri-μ-hydroxo-bis (1,5-diamino-3-aza-pentane)cobalt(III) ion in weakly basic aqueous carbonate solution

The kinetics of formation of the complex ion, μ-carbonato-di-μ-hydroxo-bis((1,5-diamino-3-aza-pentane) cobalt(III), from the tri-μ-hydroxo-bis((1,5-diamino-3-aza-pentane(III)cobalt(III)) ion in aqueous buffered carbonate solution have been studied spectrophotometrically at 295 nm over the ranges 20.0θ°C34.8, 8.03pH9.44, 5 mM [CO₃²⁻35 mM and at an ionic strength of 0.1 M (LiClO₄). On the basis of the kinetic results a mechanism, involving rapid cleavage of an hydroxo bridge followed by carbon dioxide uptake with subsequent bridge formation, has been proposed. At 25 °C, the rate of the carbon dioxide uptake is 0.58 M⁻¹ s⁻¹ with ΔH≠ = (13.2±0.7) kcal mol⁻¹ and ΔS≠ = (−15.1 ± 0.7) cal deg⁻¹ mol⁻¹. The results are composed with those obtained for several mononuclear cobalt(III) and one dinuclear cobalt(III) complexes.     

Differential scanning calorimetry of dipalmitoyl phosphatidylcholine analogues and of their interaction products with basic polypeptides

The thermotropic behaviour of dipalmitoyl phosphatidylcholine analogues with a varying number (n) of CH₂ groups between the phosphate and the quaternary ammonium has been investigated. The temperature (T_m) and the enthalphy (ΔH) of the phase transition are non-monotonous functions of the number of CH₂ groups. T_m oscillates between 40 and 45°C and ΔH between 7 and 13 kcal/mol for a variation of n between 2 and 11.It is concluded that the hydrocarbon chains in the head groups do not penetrate the hydrocarbon region and do not contribute directly to the melting of the acyl chains. It is suggested that their length may affect the critical ballance between the attractive and the repulsive forces within the bidimensional lattice of the head groups.Copolypeptides of lysine with phenylalanine do not appreciably affect the T_m but have a pronounced effect on ΔH of the lipid phase transition, which depends strongly on the ratio of the two amino acids in the polypeptide. The effect of copolypeptide of any defined composition on ΔH is also a non-monotonous function of the number of CH₂ groups in the phosphatidylcholine head group, but it does not parallel completely the oscilations in the T_m and ΔH of the pure lipids.     

α-Synuclein aggregation variable temperature and variable pH kinetic data: A re-analysis using the Finke–Watzky 2-step model of nucleation and autocatalytic growth

The aggregation of proteins is believed to be intimately connected to many neurodegenerative disorders. We recently reported an “Ockham's razor”/minimalistic approach to analyze the kinetic data of protein aggregation using the Finke–Watzky (F–W) 2-step model of nucleation (A → B, rate constant k₁) and autocatalytic growth (A + B → 2B, rate constant k₂). With that kinetic model we have analyzed 41 representative protein aggregation data sets in two recent publications, including amyloid β, α-synuclein, polyglutamine, and prion proteins (Morris, A. M., et al. (2008) Biochemistry 47, 2413-2427; Watzky, M. A., et al. (2008) Biochemistry 47, 10790–10800). Herein we use the F–W model to reanalyze protein aggregation kinetic data obtained under the experimental conditions of variable temperature or pH 2.0 to 8.5. We provide the average nucleation (k₁) and growth (k₂) rate constants and correlations with variable temperature or varying pH for the protein α-synuclein. From the variable temperature data, activation parameters ΔG^‡, ΔH^‡, and ΔS^‡ are provided for nucleation and growth, and those values are compared to the available parameters reported in the previous literature determined using an empirical method. Our activation parameters suggest that nucleation and growth are energetically similar for α-synuclein aggregation (ΔG^‡_nucleation = 23(3) kcal/mol; ΔG^‡_growth = 22(1) kcal/mol at 37 °C). From the variable pH data, the F–W analyses show a maximal k₁ value at pH ~ 3, as well as minimal k₁ near the isoelectric point (pI) of α-synuclein. Since solubility and net charge are minimized at the pI, either or both of these factors may be important in determining the kinetics of the nucleation step. On the other hand, the k₂ values increase with decreasing pH (i.e., do not appear to have a minimum or maximum near the pI) which, when combined with the k₁ vs. pH (and pI) data, suggest that solubility and charge are less important factors for growth, and that charge is important in the k₁, nucleation step of α-synuclein. The chemically well-defined nucleation (k₁) rate constants obtained from the F–W analysis are, as expected, different than the 1/lag-time empirical constants previously obtained. However, k₂ × [A]₀ (where k₂ is the rate constant for autocatalytic growth and [A]₀ is the initial protein concentration) is related to the empirical constant, k_app obtained previously. Overall, the average nucleation and average growth rate constants for α-synuclein aggregation as a function of pH and variable temperature have been quantitated. Those values support the previously suggested formation of a partially folded intermediate that promotes aggregation under high temperature or acidic conditions.     

NMR studies of glycerol permeability in lipid vesicles, erythrocytes and the alga Dunaliella

Glycerol diffusional permeabilities through the cytoplasmic cell membrane of Dunaliella salina, the cell envelope of pig erythrocyte and egg phosphattidylcholine vesicles were measured by NMR spectroscopy employing the spin-echo method and nuclear T₁ relaxation. The following permeability coefficients (P) and corresponding enthalpies of activation (ΔH^‡) were determined for glycerol at 25°C: for phosphatidylcholine vesicles 5·10⁻⁶ cm/s and 11±2 kcal/mol; for pig erythrocytes 7·10⁻⁸ cm/s and 18±3 kcal/mol, respectively; for the cytoplasmic membrane of D. salina the permeability at 17°C was found to be exceptionally low and only a lower limit (P<5·10⁻¹¹cm/s) could be calculated. At temperatures above 50°C a change in membrane permeability occurred leading to rapid leakage of glycerol accompanied by cell death. The data reinforce the notion that the cytoplasmic membrane of Dunaliella represents a genuine anomaly in its exceptional low permeability to glycerol.     

Thermodynamic and kinetic analysis of the interaction between hepatitis B surface antibody and antigen on a gold electrode modified with cysteamine and colloidal gold via electrochemistry

Hepatitis B surface antibody (HBsAb) was immobilized to the surface of a gold electrode modified with cysteamine and colloidal gold as matrices to detect hepatitis B surface antigen (HBsAg). Differential pulse voltammetry (DPV) method was used for the investigation of the specific interaction between the immobilized HBsAb and HBsAg in solution, which was followed as a change of peak current in DPV with time. With the modified gold electrode, the differences in affinity of HBsAb with HBsAg at the temperatures of 37 and 40 °C were easily distinguished and the kinetic rate constants (k_ass and k_diss) and kinetic affinity constant K were determined from the curves of current versus time. In addition, the thermodynamic constants, ΔG, ΔH and ΔS, of the interaction at 37 °C were calculated, which were −56.65, −64.54 and −25.45 kJ mol⁻¹, respectively.     

3-(1′,1′-Dimethylbutyl)-1-deoxy-Δ-THC and related compounds: synthesis of selective ligands for the CB2 receptor

The synthesis and pharmacology of 15 1-deoxy-Δ⁸-THC analogues, several of which have high affinity for the CB₂ receptor, are described. The deoxy cannabinoids include 1-deoxy-11-hydroxy-Δ⁸-THC (5), 1-deoxy-Δ⁸-THC (6), 1-deoxy-3-butyl-Δ⁸-THC (7), 1-deoxy-3-hexyl-Δ⁸-THC (8) and a series of 3-(1′,1′-dimethylalkyl)-1-deoxy-Δ⁸-THC analogues (2, n=0–4, 6, 7, where n=the number of carbon atoms in the side chain−2). Three derivatives (17–19) of deoxynabilone (16) were also prepared. The affinities of each compound for the CB₁ and CB₂ receptors were determined employing previously described procedures. Five of the 3-(1′,1′-dimethylalkyl)-1-deoxy-Δ⁸-THC analogues (2, n=1–5) have high affinity (K_i=<20 nM) for the CB₂ receptor. Four of them (2, n=1–4) also have little affinity for the CB₁ receptor (K_i=>295 nM). 3-(1′,1′-Dimethylbutyl)-1-deoxy-Δ⁸-THC (2, n=2) has very high affinity for the CB₂ receptor (K_i=3.4±1.0 nM) and little affinity for the CB₁ receptor (K_i=677±132 nM).

Scheme 3. (a) (C₆H₅)₃PCH₃⁺ Br⁻, n-BuLi/THF, 65°C; (b) LiAlH₄/THF, 25°C; (c) KBH(sec-Bu)₃/THF, −78 to 25°C then H₂O₂/NaOH.     

Temperature effects on kinetic properties of plasma membrane ATPase from the yeast Saccharomyces cerevisiae

The reaction of plasma membrane ATPase from yeast with Mg²⁺ and Mg · ATP was studied in a temperature range of 10 – 30°C. The random mechanism of activation by Mg²⁺ and the pseudocompetitive inhibition at higher concentrations was not altered when the temperature was varied, nor were the kinetic constants representing substrate binding. However, at low temperature, the affinity of the enzyme for Mg²⁺ is greatly reduced. The Arrhenius plot of log V vs. 1/T shows straight lines with an inflection point at 24°C, which disappears in the presence of detergent. Calorimetric studies of the plasma membranes show a transition point at the same temperature. From these findings we suppose that Mg²⁺ is bound at a regulatory site of the ATPase, which is influenced by the surrounding phospholipids.     

Evidence of α fluctuations in myoglobin's denaturation in the high temperature region: Average relaxation time from an Adam–Gibbs perspective

In this work, we derive an analytical expression for the relaxation time τ as a function of temperature T for myoglobin protein (Mb, PDB:1MBN) in the high temperature limit (T > T_g = 200 K). The method is based on a modified version of the Adam–Gibbs theory (AG theory) for the glass transition in supercooled liquids and an implementation of differential geometry techniques. This modified version of the AG theory takes into account that the entropic component in protein's denaturation has two major sources: a configurational contribution ΔS_c due to the unfolding of the highly ordered native state N and a hydration contribution ΔS^hyd arising from the exposure of non-polar residues to direct contact with solvent polar molecules. Our results show that the configurational contribution ΔS_c is temperature-independent and one order of magnitude smaller than its hydration counterpart ΔS^hyd in the temperature range considered. The profile obtained for log τ(T) from T = 200 K to T = 300 K exhibits a non-Arrhenius behavior characteristic of α relaxation mechanisms in hydrated proteins and glassy systems. This result is in agreement with recent dielectric spectroscopy data obtained for hydrated myoglobin, where at least two fast relaxation processes in the high temperature limit have been observed. The connection between the relaxation process calculated here and the experimental results is outlined.     

Effects of pressure and pentanol on the phase transition in the membrane of Acholeplasma laidlawii B

The phase transition of a complex, biological membrane was studied in relation to two variables: high pressure and the alcohols, pentanol and benzyl alcohol, in order to determine whether earlier studies of defined phospholipid bilayers may be applied to natural membranes. The isolated membrane of A choleplasma laidlawii was used and three independent methods were chosen to detect the transition; optical transmission (giving T_t, an index of the end-of-melting temperature); fluorescence polarisation (giving T_p, the temperature midway through the change in polarisation which characterises the transition) and differential thermal analysis giving a record of the temperature range occupied by the endothermic process. Pressure increased T_t by 0.017 K·atm⁻¹ and T_p by 0.016 K·atm⁻¹, consistent with dT/dP = T·ΔV/ΔH. Pentanol (and benzyl alcohol) lowered T_t, T_p and the temperature of the endotherm seen with differential thermal analysis. Thus the membrane transition responds to pressure and alcohols in agreement with thermodynamic theory.     

Thermal unfolding of the archaeal DNA and RNA binding protein Ssh10

The reversible thermal unfolding of the archaeal histone-like protein Ssh10b from the extremophile Sulfolobus shibatae was studied using differential scanning calorimetry and circular dichroism spectroscopy. Analytical ultracentrifugation and gel filtration showed that Ssh10b is a stable dimer in the pH range 2.5–7.0. Thermal denaturation data fit into a two-state unfolding model, suggesting that the Ssh10 dimer unfolds as a single cooperative unit with a maximal melting temperature of 99.9 °C and an enthalpy change of 134 kcal/mol at pH 7.0. The heat capacity change upon unfolding determined from linear fits of the temperature dependence of ΔH^cal is 2.55 kcal/(mol K). The low specific heat capacity change of 13 cal/(mol K residue) leads to a considerable flattening of the protein stability curve (ΔG (T)) and results in a maximal ΔG of only 9.5 kcal/mol at 320 K and a ΔG of only 6.0 kcal/mol at the optimal growth temperature of Sulfolobus.     

Thermoregulation of water foraging wasps (Vespula vulgaris and Polistes dominulus)

A comparison of the thermoregulation of water foraging wasps (Vespula vulgaris, Polistes dominulus) under special consideration of ambient temperature and solar radiation was conducted. The body surface temperature of living and dead wasps was measured by infrared thermography under natural conditions in their environment without disturbing the insects’ behaviour. The body temperature of both of them was positively correlated with T_a and solar radiation. At moderate T_a (22–28 °C) the regression lines revealed mean thorax temperatures (T_th) of 35.5–37.5 °C in Vespula, and of 28.6–33.7 °C in Polistes. At high T_a (30–39 °C) T_th was 37.2–40.6 °C in Vespula and 37.0–40.8 °C in Polistes. The thorax temperature excess (T_th–T_a) increased at moderate T_a by 1.9 °C (Vespula) and 4.4 °C (Polistes) per kW⁻¹ m⁻². At high T_a it increased by 4.0 °C per kW⁻¹ m⁻² in both wasps. A comparison of the living water foraging Vespula and Polistes with dead wasps revealed a great difference in their thermoregulatory behaviour. At moderate T_a (22–28 °C) Vespula exhibited distinct endothermy in contrast to Polistes, which showed only a weak endothermic activity. At high T_a (30–39 °C) Vespula reduced their active heat production, and Polistes were always ectothermic. Both species exhibited an increasing cooling effort with increasing insolation and ambient temperature.     

Using species-specific paleotemperature equations with foraminifera: a case study in the Southern California Bight

Species-specific paleotemperature equations were used to reconstruct a record of temperature from foraminiferal δ¹⁸O values over the last 25 kyr in the Southern California Bight. The equations yield similar temperatures for the δ¹⁸O values of Globigerina bulloides and Neogloboquadrina pachyderma. In contrast, applying a single paleotemperature equation to G. bulloides and N. pachyderma δ¹⁸O yields different temperatures, which has been used to suggest that these species record the surface-to-thermocline temperature gradient. In Santa Barbara Basin, an isotopically distinct morphotype of G. bulloides dominates during glacial intervals and yields temperatures that appear too cold when using a paleotemperature equation calibrated for the morphotype common today. When a more appropriate paleotemperature equation is used for glacial G. bulloides, we obtain more realistic glacial temperatures. Glacial–interglacial temperature differences (G–I ΔT) calculated in the present study indicate significant cooling (8–10°C) throughout the Southern California Bight during the last glacial maximum (LGM). The magnitude of glacial cooling varies from 8°C near the middle of the Southern California Bight (Tanner Basin and San Nicolas Basin) to 9°C in the north (Santa Barbara Basin) and 9.5–10°C in the south (Velero Basin and No Name Basin). Our temperature calculations agree well with previous estimates based on the modern analog technique. In contrast, studies using N. pachyderma coiling ratios, U^k′₃₇ indices, and transfer functions estimate considerably warmer LGM temperatures and smaller G–I ΔT.     

Spectroscopic analysis of DNA base-pair opening by Escherichia coli RNA polymerase. Temperature and ionic strength effects

The interaction of Escherichia coli RNA polymerase with poly[d(A-T)] and poly[d-(I-C)] was studied by difference absorption spectroscopy at temperatures, from 5 to 45°C in the absence and presence of Mg²⁺. The effect of KCl concentration, at a fixed temperature, was studied from 12.5 to 400 mM. Difference absorption experiments permitted calculation of the extent of DNA opening induced by RNA polymerase and estimation of the equilibrium constant associated with the isomerization from a closed to an open RNA polymerase-DNA complex. ΔH⁰ and ΔS⁰ for the closed-to-open transition with poly[d(A-T)] or poly[d(I-C)] complexed with RNA polymerase are significantly lower than the values associated with the helix-to-coil transition for the free polynucleotides. For the RNA polymerase complexes with poly[d(A-T)] and poly[d(I-C)] in 50 mM KCl, ΔH⁰ ≈ 15–16 kcal/mol (63–67 kJ/mol) and ΔS⁰ ≈ 50–57 cal/K per mol (209–239 J/K per mol). The presence of Mg²⁺ does not change these parameters appreciably for the RNA polymerase-poly[d(A-T)] complex, but for the RNA polymerase-poly[d(I-C)] complex in the presence of Mg²⁺, the ΔH⁰ and ΔS⁰ values are larger and temperature-dependent, with ΔH⁰ ≈ 22 kcal/mol (92 kJ/mol) and ΔS⁰ ≈ 72 cal/K per mol (approx. 300 J/K per mol) at 25°C, and ΔC_p⁰ 2 kcal/K per mol (approx. 8.3 kJ/K per mol). The circular dichroism (CD) changes observed for helix opening induced by RNA polymerase are qualitatively consistent with the thermally induced changes observed for the free polynucleotides, supporting the difference absorption method. The salt-dependent studies indicate that two monovalent cations are released upon helix opening. For poly[d(A-T)], the temperature-dependence of enzyme activity correlates well with the helix opening, implying this step to be the rate-determining step. In the case of poly[d(I-C)], the same is not true, and so the rate-determining step must be a process subsequent to helix opening.     

Metabolic rate, maximum metabolism, and advantages of torpor in the fat mouse Steatomys pratensis natalensis Roberts, 1921 (Dendeomurinae)

1. The fat mouse Steatomys pratensis natalensis (mean body mass 37.4±0.43 (se)) has a low euthermic body temperature T_b=30.1–33.8 °C and a low basal metabolic rate (BMR)=0.50 ml O₂ g⁻¹ h⁻¹.

2. Below an ambient temperature (T_a)=15 °C, the mice were hypothermic.

3. The lowest survivable T_a=10 °C.

4. Torpor is efficient in conserving energy between T_a=15–30 °C, below T_a=15 °C, the mice arouse.

5. Euthermic and torpid mice were hyperthermic at T_a=35 °C.

6. Thermal conductance was 0.159 ml O₂ g⁻¹ h⁻¹ °C⁻¹, 98.8% of the expected value.

7. Non-shivering thermogenesis (NST) was 2.196 ml O² g⁻¹ h⁻¹ (3.69×BMR).

8. Maximal oxygen consumption, however, was 3.83 ml O₂ g⁻¹ h⁻¹ (6.44×BMR), indicating that other methods of heat production are additive.

9. Because fat mice conserve energy by torpor only between T_a=15–30 °C, we suggest that torpor may be a more important mechanism for surviving food shortages than for surviving cold weather.

Temperature dependence of anion transport in the human red blood cell

Arrhenius plots of chloride and bromide transport yield two regions with different activation energies (E_a). Below 15 or 25°C (for Cl⁻ and Br⁻, respectively), E_a is about 32.5 kcal/mol; above these temperatures, about 22.5 kcal/mol (Brahm, J. (1977) J. Gen. Physiol. 70, 283–306). For the temperature dependence of SO₄²⁻ transport up to 37°C, no such break could be observed. We were able to show that the temperature coefficient for the rate of SO₄²⁻ transport is higher than that for the rate of denaturation of the band 3 protein (as measured by NMR) or the destruction of the permeability barrier in the red cell membrane. It was possible, therefore, to extend the range of flux measurements up to 60°C and to show that, even for the slowly permeating SO₄²⁻ in the Arrhenius plot, there appears a break, which is located somewhere between 30 and 37°C and where E_a changes from 32.5 to 24.1 kcal/mol. At the break, the turnover number is approx. 6.9 ions/band 3 per s. Using ³⁵Cl⁻-NMR (Falke, Pace and Chan (1984) J. Biol. Chem. 259, 6472–6480), we also determined the temperature dependence of Cl⁻-binding. We found no significant change over the entire range from 0 to 57°C, regardless of whether the measurements were performed in the absence or presence of competing SO₄²⁻. We conclude that the enthalpy changes associated with Cl⁻-or SO₄²⁻-binding are negligible as compared to the E_a values observed. It was possible, therefore, to calculate the thermodynamic parameters defined by transition-state theory for the transition of the anion-loaded transport protein to the activated state for Cl⁻, Br⁻ and SO₄²⁻ below and above the temperatures at which the breaks in the Arrhenius plots are seen. We found in both regions a high positive activation entropy, resulting in a low free enthalpy of activation. Thus the internal energy required for carrying the complex between anion and transport protein over the rate-limiting energy barrier is largely compensated for by an increase of randomness in the protein and/or its aqueous environment.     

Transition-state structure for a conformation change of ribonuclease

The rate constants k₁₂ⁿ for isomerization of the E₁H isomer (pK_a 8 in H₂O) of ribonuclease-A to the E₂H isomer (pK_a = 6.1 in H₂O), determined from proton-uptake measurements by the temperature-jump technique, in mixtures of protium and deuterium oxides (atom fraction of deuterium n), are described by the equation k₁₂ⁿ = (733 ± 16)(1 − n + [0.46 ± 0.04]n)(1 − n + 0.69n)²sec⁻¹ at 25°C. On the basis of the absolute magnitude of the rate constant, the magnitude of the solvent isotope effect and the proton inventory, it appears that the rate-determining step is proton transfer to a water molecule from the imidazolium form of a histidine residue, with a product-like activated complex resembling a hydronium ion. The subsequent motion of the protein structure to generate the new isomer (conformation change) must then occur in a time approaching a vibrational period. Alternative but less likely mechanisms include rate-limiting protein reorganization concerted with proton transfer to water, rate-limiting diffusion of hydronium ion away from the enzyme, or “solvation catalysis” of protein reorganization.     

Thyroid hormone: Aldosterone antagonism in cultured epithelial cells

Thyroid hormone (T₃) has been demonstrated to inhibit the action of aldosterone on sodium transport in toad urinary bladder and rat kidney. We have exammined the effect of T₃ on aldosterone action and specific nuclear binding in cultured epithelial cells derived from toad urinary bladder. In cell line TB6-C, addition of 5·10⁻⁸ M T₃ to culture media for up to 3 days results in no change in short-circuit current or transepithelial resistance. This concentration of T₃ completely inhibits the maximal increase in short-circuit current in response to 1·10⁻⁷ M aldosterone. The inhibition can be demonstrated with 18 h preincubation or with simultaneous addition of T₃ and aldosterone. The half-maximal concentration for the inhibition of the aldosterone effect is approx. 5·10⁻⁹ M T₃. T₃ has no effect on cyclic AMP-stimulated short-circuit current in these cells. The effect of T₃ on nuclear binding of [³H]aldosterone was examined using a filtration assay with data analysis by at least-squares curve-fitting program. Best fit was obtained with a model for two binding sites. The dissociation constants for the binding were K′_d1 = (0.82 ± 0.36)·10⁻¹⁰ M and K′_d2 = (3.2±0.60)·10⁻⁸ M.The half-maximal concentration for aldosterone-stimulated sodium transport in these cells is approx. 1·10⁻⁸ M. Analysis of nuclear aldosterone binding in cells preincubated for 18 h with 5·10⁻⁸ M T₃ showed a K′_d1 = (0.15 ± 0.10)·10⁻¹⁰ M and K′_d2 = (3.5 ± 0.10)·10⁻⁸ M. We conclude that T₃ i action of aldosterone on sodium transport at a site after receptor binding in the nucleus.     

Conformational stabilities of guinea pig OCP1 and OCP2

OCP1 and OCP2, the most abundant proteins in the cochlea, are evidently subunits of an SCF E3 ubiquitin ligase. Although transcribed from a distinct gene, OCP2 is identical to Skp1. OCP1 is equivalent to the F-box protein known as Fbs1, Fbx2, or NFB42 — previously shown to bind N-glycosylated proteins and believed to function in the retrieval and recycling of misfolded proteins. The high concentrations of OCP1 and OCP2 in the cochlea suggest that the OCP1–OCP2 heterodimer may serve an additional function independent of its role in a canonical SCF complex. At 25 °C, urea-induced denaturation of OCP1 is slow, but reversible. The data suggest that the protein possesses one or more disordered regions, a conclusion supported by analysis of the far-UV circular dichroism spectrum and the appearance of the ¹H, ¹⁵N-HSQC spectrum. Thermal denaturation of OCP1 is irreversible, evidently due to formation of high molecular weight aggregates. Analysis with a kinetic model yields an estimate for the activation energy for unfolding of 49 kcal/mol. Urea denaturation data for OCP2 returns ΔG_o and m values of 6.2 kcal/mol and 1.5 kcal mol^− 1 M^− 1, respectively. In contrast to OCP1, thermal denaturation of OCP2 is reversible. In phosphate-buffered saline, at pH 7.40, the protein exhibits a ΔH_vH/ΔH_cal ratio of 1.69, suggesting that denaturation proceeds largely from the native dimer directly to the unfolded state. OCP1 and OCP2 associate tightly at room temperature. However, DSC data for the complex suggest that they denature independently, consistent with the highly exothermic enthalpy of complex formation reported previously.