Passive electrical properties of the membrane and cytoplasm of cultured rat basophil leukemia cells. I. Dielectric behavior of cell suspensions in 0.01-500 MHz and its simulation with a single-shell model

Frequency dependence of relative permittivity (dielectric constant) and conductivity, or the 'dielectric dispersion', of cultured cells (RBL-1 line) in suspension was measured using a fast impedance analyzer system capable of scanning 92 frequency points over a 10 kHz-500 MHz range within 80 s. Examination of the resulting dispersion curves of an improved reliability revealed that the dispersions consisted of at least two separate components. The low-frequency component (dispersion 1) had a permittivity increment (delta epsilon) of 10(3)-10(4) and a characteristic frequency (fc) at several hundred kHz; for the high-frequency component (dispersion 2), delta epsilon was smaller by a factor of 10(2) and fc = 10-30 MHz. Increments delta epsilon for both components increased with the volume fraction of cell suspension, while fc did not change appreciably as long as the conductivity of suspending medium was fixed. By fitting a model for shelled spheres (the 'single-shell' model) to the data of dispersion 1, the dielectric capacity of the plasma membrane phase (Cm) was estimated to be approx. 1.4 microF/cm2 for the cells in an isotonic medium. However, simulation by this particular shell model failed to reproduce the entire dispersion profile leaving a sizable discrepancy between theory and experiment especially at frequencies above 1 MHz where dispersion 2 took place. This discrepancy could not be filled up even by taking into consideration either the effect of cell size distribution actually determined or that of possible heterogeneity in the intracellular conductivity. The present data strongly indicate the need for a more penetrating model that effectively accounts for the behavior of dispersion 2.     

Impulse photoconductivity of solutions of chlorophyll and its analogs. II. Effect of medium polarity on the yield of ion-radicals during the photoxidation of chlorophyll a ]

The effect of the dielectric constant epsilon of the solvent on the yield delta n of ion-radicals during photooxidation of chlorophy-l a with n-benzoquinone was studied by the method of impulse photoconductivity. It was shown that in the studied range of dielectric constant valve (epsilon congruent to 5-25) delta n monotonously increased with an increase of epsilon the relationship delta n (epsilon) being of clearly pronounced S-like character with a bend in the region of epsilon congruent to 10. A half-quantitative explanation of the data obtained is presented.     

Effect of nitrogen and oxygen active compounds on exchange of Ca2+ and H+ through the myometrial cell membrane

While using myometrium sarcolemma vesicles the action of sodium nitroprusside, NO2-, NO3- and H+ on delta pH-dependent Ca(2+)-transport and passive permeability for H+ vesicles sarcolemma was estimated in the wide concentration range (10(-10)-10(-3) M) of the substances tested. In order of studying calcium transport 45Ca2+ was used, while for H+ translocation registrating via sarcolemma delta pH-indicator 14C-methylamine was applied. Sodium nitroprusside was displayed as weakly effective, while nitrite-anions essentially increased delta pH-dependent Ca2+ transport in the physiologically significant nanomolar concentration region, however in the micromolar region these substances effect failed to differ from the control and restored its intensity starting at 10(-4) M and more. Under the experiment sodium nitroprusside produces considerable quantities of NO2-. Effectory action of NO3- was similar as of NO2-. In the micromolar region the compounds estimated increased considerably sarcolemma passive permeability for H+. Hydrogen peroxide decreased delta pH-dependent Ca(2+)-transport by 10(-8) M and 10(-3) M while at the concentration equal to 10(-3) M increased the sarcolemma passive permeability for proton. Sodium nitroprusside and NO2(-)-effect on the vesicles passive permeability for proton failed to be prevented by dithiotriitol, while H2O2 action was completely removed. The conclusion about the complex concentration-dependent character of the active oxygen metabolities to the sarcolemma transport processes was made, and it's noticeable that the important role in vivo, probably could be played by NO (NO2-) stable nitric metabolities.     

Determination, using a piezo-impulse method, of iso-osmotic permeability of the apical membrane of epithelium

A frog skin, mechanically held on the mucosal side separates two Ringer solutions. It is submitted to an hydrostatic pressure difference delta P varying between 2 and 120 mb. Water permeability P (delta P) delta pi = 0 is determined with a piezo-impulse method. The rapid variation of permeability within the 2-25 mb range indicates a reversible closing of the junctions. The limiting value Pisol for high delta P is the isoosmotic permeability of the apical membrane.     

The properties of liquid water in electric and magnetic fields

A new model of liquid water is proposed, which involves the liquid-crystalline phase, namely, the presence of linearly ordered chains of water molecules and large clusters of these molecules. The presence of linearly ordered chains and clusters in liquid water makes itself evident after the exposure of water to low-frequency electromagnetic radiation. The kinetics of the linearly ordered chains is described by nonlinear excitations (solitons). A hydrodynamic equation describing the behavior of this model system was derived. On the basis of the model, the dimensions of clusters were determined (approximately 600 A). The calculated values of dielectric permeability in the range of low frequencies appeared to be close to their experimentally obtained values. A tenfold increase in the stationary value of dielectric permeability compared with the known value of the dielectric constant in the frequency range of 10(4)-10(6) Hz was predicted. The frequency dependence of dielectric permeability in this range was studied.     

Binding of the recombinant proteinase inhibitor eglin c from leech Hirudo medicinalis to human leukocyte elastase, bovine alpha-chymotrypsin and subtilisin Carlsberg: thermodynamic study

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the recombinant proteinase inhibitor eglin c from leech Hirudo medicinalis to human leukocyte elastase (EC 3.4.21.37), bovine alpha-chymotrypsin (EC 3.4.21.1) and subtilisin Carlsberg (EC 3.4.21.14) has been investigated. On lowering the pH from 9.5 to 4.5, values of Ka for eglin c binding to the serine proteinases considered decrease thus reflecting the acid-pK shift of the invariant histidyl catalytic residue (His57 in human leukocyte elastase and bovine alpha-chymotrypsin, and His64 in subtilisin Carlsberg) from congruent to 6.9, in the free enzymes, to congruent to 5.1, in the enzyme:inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for eglin c binding are: human leukocyte elastase - Ka = 1.0 x 10(10) M-1, delta G phi = -13.4 kcal/mol, delta H phi = +1.8 kcal/mol, and delta S phi = +52 entropy units; bovine alpha-chymotrypsin -Ka = 5.0 x 10(9) M-1, delta G phi = -13.0 kcal/mol, delta H phi = +2.0 kcal/mol, and delta S phi = +51 entropy units; and subtilisin Carlsberg - Ka = 6.6 x 10(9) M-1, delta G phi = -13.1 kcal/mol, delta H phi = +2.0 kcal/mol, and delta S phi = +51 entropy units (values of Ka, delta G phi and delta S phi were obtained at 21 degrees C; values of delta H phi were temperature independent over the range explored, i.e. between 10 degrees C and 40 degrees C; 1 kcal = 4184J).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of the intramolecular stacking of the fluorosulfonylbenzoyl derivatives of 1,N6-ethenoadenosine, adenosine, and guanosine

The differences in conformation in solution of fluorosulfonylbenzoyl nucleosides were analyzed by fluorescence and proton nuclear magnetic resonance spectroscopy. The quantum yield of 5'-p-fluorosulfonylbenzoyl-1,N6-ethenoadenosine (5'-FSB epsilon A) in aqueous solution is low (? = 0.01) as compared to that of its parent nucleoside, ethenoadenosine (? = 0.54), and increases approximately 5-fold when measured in a series of solvents of decreasing dielectric constant. The quantum yield of 5'-p-sulfonylbenzoyl-1,N6-ethenoadenosine covalently bound to glutamate dehydrogenase and pyruvate kinase is also 0.01, suggesting that the analogue may exist in the same conformation when enzyme-bound as when free in solution. In D2O, the resonances of the purine ring protons on 5'-FSB epsilon A, 5'-p-fluorosulfonylbenzoyl adenosine (5'-FSBA), and 5'-p-fluorosulfonylbenzoyl guanosine (5'-FSBG) are shifted upfield by about 0.1-0.3 ppm relative to the corresponding protons of their parent nucleosides. The calculated difference in chemical shift (delta delta) decreases as the dielectric constant of the solvent decreases. The delta delta decreases with increasing temperature. These data indicate that 5'-FSB epsilon A, 5'-FSBA, and 5'-FSBG exist in aqueous solution in a conformation in which the purine ring is intramolecularly stacked with the benzoyl moiety. From the magnitude of change in delta delta for 5'-FSB epsilon A, 5'-FSBA, and 5'-FSBG as a function of solvent, it appears that the three analogues differ in their sensitivity to disruption of stacking. The solution conformation of these three fluorosulfonylbenzoyl nucleoside analogues may be an important determinant of their reaction with various enzymes and may explain differences among the analogues in their reaction with a single enzyme.     

Thermolysis of coenzymes B12 at physiological temperatures: activation parameters for cobalt-carbon bond homolysis and a quantitative analysis of the perturbation of the homolysis equilibrium by the ribonucleoside triphosphate reductase from Lactobacillus leichmannii

The kinetics of the thermolysis of 5'-deoxyadenosylcobalamin (AdoCbl, coenzyme B12) in aqueous solution, pH 7.5, have been studied in the temperature range 30-85 degrees C using AdoCbl tritiated at the adenine C2 position and the method of initial rates. Combined with a careful analysis of the distribution of adenine-containing products, the results permit the dissection of the competing rate constants for carbon-cobalt bond homolysis and heterolysis. After correction for the temperature-dependent occurrence of the much less reactive base-off species of AdoCbl, the activation parameters for homolysis of the base-on species were found to be delta H++homo,on = 33.8 +/- 0.2 kcal mol-1 and delta S++homo,on = 13.5 +/- 0.7 cal mol-1 K-1, values not significantly different from those determined by Hay and Finke (J. Am. Chem. Soc. 108 (1986) 4820), in the temperature range 85-115 degrees C. In contrast, the heterolysis of base-on AdoCbl was characterized by a much smaller enthalpy of activation (delta H++het,on = 18.5 +/- 0.2 kcal mol-1) and a negative entropy of activation (delta S++het,on = -34.0 +/- 0.7 cal mol-1 K-1) so that heterolysis, which is minor pathway at elevated temperatures, is the dominant pathway for AdoCbl decomposition at physiological temperatures. Using literature values for the rate constant for the reverse reaction, the equilibrium constant for AdoCbl homolysis at 37 degrees C was calculated to be 7.9 x 10(-18). Comparison with the equilibrium constant for this homolysis at the active site of the ribonucleoside triphosphate reductase from Lactobacillus leichmannii shows that the enzymes shifts the equilibrium constant towards homolysis products by a factor of 2.9 x 10(12) (17.7 kcal mol-1) by binding the thermolysis products with an equilibrium constant of 7.1 x 10(16) M-2, compared to the bonding constant for AdoCbl of 2.4 x 10(4) M-1.     

Kinetics of carboxymethylation of histidine hydantoin.

The reaction of the imidazole group of histidine hydantoin with bromoacetate was studied as a model for carboxymethylation of histidine residues in proteins. pK values of 6.4 and 9.1 (25 degrees C) and apparent heats of ionization of 7.8 and 8.7 kcal/mol were determined for the imidazole and hydantoin rings, respectively. At pH values corresponding to the isoelectric points for histidine hydantoin, the rates of carboxymethylation at 12, 25, 37, and 50 degrees C were determined; the modified hydantoins were hydrolyzed to the corresponding histidine derivatives for quantitative amino acid analysis. At pH 7.72 and 25 degrees C, the imidazole tele-N was alkylated (k = 3.9 X 10(-5) M-1 s-1) twice as fast as the pros-N. The monocarboxymethyl derivatives were carboxymethylated at the same rate at the pros-N (k = 2.1 X 10(-5) M-1 s-1) but 3 times faster at the tele-N (k = 11 X 10(-5) M-1 s-1). The enthalpies of activation determined for carboxymethylation of the imidazole ring and its monocarboxymethyl derivatives were similar (15.9 +/- 0.7 kcal/mol). delta S for the four carboxymethylations was -25 +/- 2 eu. The electrostatic component of delta S (delta S es) was calculated from the influence of the dielectric constant on the reaction rate at 25 degrees C. delta S es was slightly negative (-4 +/- 1 eu) for mono- or dicarboxymethylations, indicating some charge separation in the transition state. The nonelectrostatic entropy of activation was -21 +/- 2 eu for all four carboxymethylations.     

Binding of the recombinant proteinase inhibitor eglin c, of the soybean Bowman-Birk proteinase inhibitor and of its chymotrypsin and trypsin inhibiting fragments to Leu-proteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves: thermodynamic study.

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the recombinant proteinase inhibitor eglin c (eglin c), of the soybean Bowman-Birk proteinase inhibitor (BBI) and of its chymotrypsin and trypsin inhibiting fragments (F-C and F-T, respectively) to Leu-proteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves, has been investigated. On lowering the pH from 9.5 to 4.5, values of Ka (at 21 degrees C) for complex formation decrease thus reflecting the acidic pK-shift of the hystidyl catalytic residue from approximately 6.9, in the free Leu-proteinase, to approximately 5.1, in the enzyme: inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for the proteinase:inhibitor complex formation are: Leu-proteinase:eglin c-Ka = 2.2 x 10(11) M-1, delta G degree = -64 kJ/mol, delta H degree = +5.9 kJ/mol, and delta S degree = +240 kJ/molK; Leu-proteinase:BBI-Ka = 3.2 x 10(10) M-1, delta G degree = -59 kJ/mol, delta H degree = +8.8 kJ/mol, and delta S degree = +230 J/molK; and Leu-proteinase:F-C-Ka = 1.1 x 10(6) M-1, delta G degree = -34 kJ/mol, delta H degree = +18 J/mol, and delta S degree = +180 J/molK (values of Ka, delta G degree and delta S degree were obtained at 21.0 degrees C; values of delta H degree were temperature-independent over the range explored, i.e. between 10.0 degrees C and 40.0 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)     

Stoichiometry and voltage dependence of the sodium pump in voltage- clamped, internally dialyzed squid giant axon

The stoichiometry and voltage dependence of the Na/K pump were studied in internally dialyzed, voltage-clamped squid giant axons by simultaneously measuring, at various membrane potentials, the changes in Na efflux (delta phi Na) and holding current (delta I) induced by dihydrodigitoxigenin (H2DTG). H2DTG stops the Na/K pump without directly affecting other current pathways: (a) it causes no delta I when the pump lacks Na, K, Mg, or ATP, and (b) ouabain causes no delta I or delta phi Na in the presence of saturating H2DTG. External K (Ko) activates Na efflux with Michaelis-Menten kinetics (Km = 0.45 +/- 0.06 mM [SEM]) in Na-free seawater (SW), but with sigmoid kinetics in approximately 400 mM Na SW (Hill coefficient = 1.53 +/- 0.08, K1/2 = 3.92 +/- 0.29 mM). H2DTG inhibits less strongly (Ki = 6.1 +/- 0.3 microM) in 1 or 10 mM K Na-free SW than in 10 mM K, 390 mM Na SW (1.8 +/- 0.2 microM). Dialysis with 5 mM each ATP, phosphoenolpyruvate, and phosphoarginine reduced Na/Na exchange to at most 2% of the H2DTG-sensitive Na efflux. H2DTG sensitive but nonpump current caused by periaxonal K accumulation upon stopping the pump, was minimized by the K channel blockers 3,4-diaminopyridine (1 mM), tetraethylammonium (approximately 200 mM), and phenylpropyltriethylammonium (20-25 mM) whose adequacy was tested by varying [K]o (0-10 mM) with H2DTG present. Two ancillary clamp circuits suppressed stray current from the axon ends. Current and flux measured from the center pool derive from the same membrane area since, over the voltage range -60 to +20 mV, tetrodotoxin-sensitive current and Na efflux into Na-free SW, under K-free conditions, were equal. The stoichiometry and voltage dependence of pump Na/K exchange were examined at near-saturating [ATP], [K]o and [Na]i in both Na-free and 390 mM Na SW. The H2DTG-sensitive F delta phi Na/delta I ratio (F is Faraday's constant) of paired measurements corrected for membrane area match, was 2.86 +/- 0.09 (n = 8) at 0 mV and 3.05 +/- 0.13 (n = 6) at -60 to -90 mV in Na-free SW, and 2.72 +/- 0.09 (n = 7) at 0 mV and 2.91 +/- 0.21 (n = 4) at -60 mV in 390 mM Na SW. Its overall mean value was 2.87 +/- 0.07 (n = 25), which was not significantly different from the 3.0 expected of a 3 Na/2 K pump.(ABSTRACT TRUNCATED AT 400 WORDS)     

Correlation of the turnover number of the ATP synthase in liposomes with the proton flux and the proton potential across the membrane

The fluorescent indicator pyranine was used for recording the internal pH of liposomes. The proton permeability was deduced from the velocity of the internal pH increase which was caused by shifting the external pH from 7 to 9. From valinomycin titration of the proton permeability in the presence of internal and external KCl (0.1 M), the permeability coefficient of H+ (PH) was obtained as 10(-4) cm/s at 22 degrees C. The coefficient was twice this value with the ATP synthase isolated from Wolinella succinogenes present in the liposomal membrane (10 mg protein/g phospholipid). ADP and phosphate had no effect on the latter PH. The protonophore TTFB (5 mumol/g phospholipid) increased the PH by 3 orders of magnitude. The permeability coefficients of H+ and K+ were used for calculating the delta uH and the proton flux associated with the phosphorylation which was driven by gradients of H+ and K+. For the conditions of limiting permeability of K+, the following conclusions were drawn. (1) In the steady state of rapid ion flux, the electrical potential across the liposomal membrane as calculated according to the Goldman equation, is directed opposite to the corresponding Nernst potential which is calculated from the K+ gradient. (2) The maximum turnover numbers of phosphorylation require a delta uH of 200-220 mV across the liposomal membrane. These values of delta uH and the corresponding turnover numbers are close to those brought about by the bacterial electron transport and the coupled phosphorylation. (3) The velocity of phosphorylation is linearly related to the proton flux. The slope of the line can be explained on the basis of an H+/ATP ratio of approx. 3.     

DSC studies of the conformational stability of barstar wild-type.

The temperature induced unfolding of barstar wild-type of bacillus amyloliquefaciens (90 residues) has been characterized by differential scanning microcalorimetry. The process has been found to be reversible in the pH range from 6.4 to 8.3 in the absence of oxygen. It has been clearly shown by a ratio of delta HvH/delta Hcal near 1 that denaturation follows a two-state mechanism. For comparison, the C82A mutant was also studied. This mutant exhibits similar reversibility, but has a slightly lower transition temperature. The transition enthalpy of barstar wt (303 kJ mol-1) exceeds that of the C82A mutant (276 kJ mol-1) by approximately 10%. The heat capacity changes show a similar difference, delta Cp being 5.3 +/- 1 kJ mol-1 K-1 for the wild-type and 3.6 +/- 1 kJ mol-1 K-1 for the C82A mutant. The extrapolated stability parameters at 25 degrees C are delta G0 = 23.5 +/- 2 kJ mol-1 for barstar wt and delta G0 = 25.5 +/- 2 kJ mol-1 for the C82A mutant.     

Thermodynamics of the conversion of penicillin G to phenylacetic acid and 6-aminopenicillanic acid

The thermodynamics of the enzymatic conversion (penicillin acylase) of aqueous penicillin G to phenylacetic acid and 6-aminopenicillanic acid have been studied using both high-pressure liquid-chromatography and microcalorimetry. The reaction was carried out in aqueous phosphate buffer over the pH range 6.0-7.6, at ionic strengths from 0.10 to 0.40 mol kg-1, and at temperatures from 292 to 322 K. The data have been analyzed using a chemical equilibrium model with an extended Debye-Hückel expression for the activity coefficients. For the reference reaction, penicillin G- (aq) + H2O(l) = phenylacetic acid-(aq) + 6-aminopenicillanic acid-(aq) + H+ (aq), the following parameters have been obtained: K = (7.35 +/- 1.5) X 10(-8) mol kg-1, delta G0 = 40.7 +/- 0.5 kJ mol-1, delta H0 = 29.7 +/- 0.6 kJ mol-1, and delta C0p = -240 +/- 50 J mol-1 K-1 at 298.15 K and at the thermochemical standard state. The extent of reaction for the overall conversion is highly dependent upon the pH.     

Non-ohmic proton conductance of mitochondria and liposomes

Direct measurements of the proton/hydroxyl ion flux across rat liver mitochondria and liposome membranes are reported. H+/OH- fluxes driven by membrane potential (delta psi) showed nonlinear dependence on delta psi both in mitochondria and in liposomes whereas delta pH-driven H+/OH- flux shows linear dependence on delta pH in liposomes. In the presence of low concentrations of a protonophore the H+/OH- flux was linearly dependent on delta psi and showed complex dependence on delta pH. The nonlinearity of H+/OH- permeability without protonophore is described by an integrated Nernst- Plank equation with trapezoidal energy barrier. Permeability coefficients depended on the driving force but were in the range 10(-3) cm/s for mitochondria and 10(-4)-10(-6) cm/s for liposomes. The nonlinear dependence of H+/OH- flux on delta psi explains the nonlinear dependence of electrochemical proton gradient on the rate of electron transport in energy coupling systems.     

Molecular motions of polysaccharides in the solid state: dextran, pullulan and amylose.

Dextran, pullulan and amylose have been investigated by differential scanning calorimetry, thermogravimetric analysis, dynamic mechanical and dielectric spectroscopy over a wide range of temperatures and frequencies. No melting or glass transition is seen below the range of thermal degradation (about 300 degrees C) for either amylose or pullulan; only dextran shows a Tg at 223 degrees C (delta cp = 0.40 J/g deg). The viscoelastic spectrum of the 'dry' polysaccharides is characterized by a low temperature relaxation that occurs at -94, -73 and -59 degrees C, at 1 kHz, (activation energy 32, 39 and 52 kJ/mol) in dextran, pullulan and amylose respectively and is assigned to small entity local motions of the polysaccharide backbone. Absorbed water strongly modifies the relaxation spectrum, inducing a new relaxation below room temperature and dissipation regions associated with water loss above room temperature. The former appears at temperatures higher than the relaxation characteristic of the dry polymer and moves to lower temperature with increasing water content. In normal 'room humidity' conditions (about 10% absorbed water) the water-induced relaxation, attributed to the motion of complex polymer-water relaxing units, is the only observable feature in the dynamic mechanical and dielectric spectrum below room temperature.     

Adsorption to dipalmitoylphosphatidylcholine membranes in gel and fluid state: pentachlorophenolate, dipicrylamine, and tetraphenylborate.

We measured the dependence of electrophoretic mobility of dipalmitoylphosphatidylcholine (DPPC) vesicles on the aqueous concentration of negatively charged ions of pentachlorophenol (PCP), dipicrylamine (DPA), and tetraphenylborate (TPhB). The objective was to determine how the physical state of hydrocarbon chains of lipids affects adsorption of lipophilic ions. The studies were done at 25 and 42 degrees C to determine adsorption properties of DPPC membrane in the gel and fluid state, respectively. From the analysis of zeta-potential isotherms in terms of Langmuir-Stern-Grahame model we obtained the association constant, K, the area of the adsorption site, Ps, and the linear partition coefficient, beta. Results: K, (x 10(4)M-1): K(gel): PCP (0.49 +/- 0.28), DPA (25 +/- 10), TPhB (31 +/- 10); K(fluid): PCP (4.5 +/- 0.9), DPA (74 +/- 21), TPhB (59 +/- 14); Ps, (nm2): Ps(gel): PCP (5.4 +/- 2.3), DPA (5.9 +/- 2), TPhB (5.0 +/- 1.7); Ps(fluid): PCP (4.5 +/- 0.4), DPA (5.2 +/- 0.4), TPhB (4.1 +/- 0.2); beta, (x 10(-5) m): beta(gel): PCP (0.15 +/- 0.09), DPA (7.1 +/- 0.3), TPhB (10 +/- 7); beta(fluid): PCP (1.7 +/- 0.3), DPA (24 +/- 7), TPhB (24 +/- 6). It was interesting to find that the adsorption site area for PCP, DPA, and TPhB were very similar for both the gel and fluid membranes; also, the areas were independent of the size and molecular structure of the adsorbing species. Using a simple discrete charge model the adsorption site areas for all species were consistent with a dielectric constant of 8-10 and with an ion adsorption depth of 0.4-0.6 nm below the water/dielectric interface. The delta delta G0 = delta G0(gel) - delta G0(fluid) was found to be about twice as large for PCP than for DPA and TPhB. This indicates that PCP will be significantly more adsorbed in the fluid and disordered regions of biomembranes, whereas the distribution of DPA and TPhB is expected to be relatively more even.     

Effect of temperature on the creatine kinase equilibrium.

The effect of temperature on the apparent equilibrium constant of creatine kinase (ATP:creatine N-phosphotransferase (EC 2.7.3.2)) was determined. At equilibrium the apparent K' for the biochemical reaction was defined as [formula: see text] The symbol sigma denotes the sum of all the ionic and metal complex species of the reactant components in M. The K' at pH 7.0, 1.0 mM free Mg2+, and ionic strength of 0.25 M at experimental conditions was 177 +/- 7.0, 217 +/- 11, 255 +/- 10, and 307 +/- 13 (n = 8) at 38, 25, 15, and 5 degrees C, respectively. The standard apparent enthalpy or heat of the reaction at the specified conditions (delta H' degree) was calculated from a van't Hoff plot of log10K' versus 1/T, and found to be -11.93 kJ mol-1 (-2852 cal mol-1) in the direction of ATP formation. The corresponding standard apparent entropy of the reaction (delta S' degree) was +4.70 J K-1 mol-1. The linear function (r2 = 0.99) between log10 K' and 1/K demonstrates that both delta H' degree and delta S' degree are independent of temperature for the creatine kinase reaction, and that delta Cp' degree, the standard apparent heat capacity of products minus reactants in their standard states, is negligible between 5 and 38 degrees C. We further show from our data that the sign and magnitude of the standard apparent Gibbs energy (delta G' degree) of the creatine kinase reaction was comprised mostly of the enthalpy of the reaction, with 11% coming from the entropy T delta S' degree term. The thermodynamic quantities for the following two reference reactions of creatine kinase were also determined. [formula: see text] The delta H degree for Reaction 2 was -16.73 kJ mol-1 (-3998 cal mol-1) and for Reaction 3 was -23.23 kJ mol-1 (-5552 cal mol-1) over the temperature range 5-38 degrees C. The corresponding delta S degree values for the reactions were +110.43 and +83.49 J K-1 mol-1, respectively. Using the delta H' degree of -11.93 kJ mol-1, and one K' value at one temperature, a second K' at a second temperature can be calculated, thus permitting bioenergetic investigations of organs and tissues using the creatine kinase equilibria over the entire physiological temperature range.     

Tris+/Na+ permeability ratios of nicotinic acetylcholine receptors are reduced by mutations near the intracellular end of the M2 region

Tris+/Na+ permeability ratios were measured from shifts in the biionic reversal potentials of the macroscopic ACh-induced currents for 3 wild-type (WT), 1 hybrid, 2 subunit-deficient, and 25 mutant nicotinic receptors expressed in Xenopus oocytes. At two positions near the putative intracellular end of M2, 2' (alpha Thr244, beta Gly255, gamma Thr253, delta Ser258) and -1', point mutations reduced the relative Tris+ permeability of the mouse receptor as much as threefold. Comparable mutations at several other positions had no effects on relative Tris+ permeability. Mutations in delta had a greater effect on relative Tris+ permeability than did comparable mutations in gamma; omission of the mouse delta subunit (delta 0 receptor) or replacement of mouse delta with Xenopus delta dramatically reduced relative Tris+ permeability. The WT mouse muscle receptor (alpha beta gamma delta) had a higher relative permeability to Tris+ than the wild-type Torpedo receptor. Analysis of the data show that (a) changes in the Tris+/Na+ permeability ratio produced by mutations correlate better with the hydrophobicity of the amino acid residues in M2 than with their volume; and (b) the mole-fraction dependence of the reversal potential in mixed Na+/Tris+ solutions is approximately consistent with the Goldman-Hodgkin-Katz voltage equation. The results suggest that the main ion selectivity filter for large monovalent cations in the ACh receptor channel is the region delimited by positions -1' and 2' near the intracellular end of the M2 helix.     

Thermodynamics of the hydrolysis of sucrose

A thermodynamic investigation of the hydrolysis of sucrose to fructose and glucose has been performed using microcalorimetry and high-pressure liquid chromatography. The calorimetric measurements were carried out over the temperature range 298-316 K and in sodium acetate buffer (0.1 M, pH 5.65). Enthalpy and heat capacity changes were obtained for the hydrolysis of aqueous sucrose (process A): sucrose(aq) + H2O(liq) = glucose(aq) + fructose (aq). The determination of the equilibrium constant required the use of a thermochemical cycle calculation involving the following processes: (B) glucose 1-phosphate2-(aq) = glucose 6-phosphate2-(aq); (C) sucrose(aq) + HPO4(2-)(aq) = glucose 1-phosphate2-(aq) + fructose(aq); and (D) glucose 6-phosphate2-(aq) + H2O(liq) = glucose(aq) + HPO4(2-)(aq). The equilibrium constants determined at 298.15 K for processes B and C are 17.1 +/- 1.0 and 32.4 +/- 3.0, respectively. Equilibrium data for process D was obtained from the literature, and in conjunction with the data for processes B and C, used to calculate a value of the equilibrium constant for the hydrolysis of aqueous sucrose. Thus, for process A, delta G0 = -26.53 +/- 0.30 kJ mol-1, K0 = (4.44 +/- 0.54) x 10(4), delta H0 = -14.93 +/- 0.16 kJ mol-1, delta So = 38.9 +/- 1.2 J mol-1 K-1, and delta CoP = 57 +/- 14 J mol-1 K-1 at 298.15 K. Additional thermochemical cycles that bear upon the accuracy of these results are examined.