Urea permeability of human red cells

The rate of unidirectional [14C]urea efflux from human red cells was determined in the self-exchange and net efflux modes with the continuous flow tube method. Self-exchange flux was saturable and followed simple Michaelis-Menten kinetics. At 38 degrees C the maximal self-exchange flux was 1.3 X 10(-7) mol cm-2 s-1, and the urea concentration for half-maximal flux, K1/2, was 396 mM. At 25 degrees C the maximal self-exchange flux decreased to 8.2 X 10(-8) mol cm-2 s-1, and K1/2 to 334 mM. The concentration-dependent urea permeability coefficient was 3 X 10(-4) cm s-1 at 1 mM and 8 X 10(-5) cm s-1 at 800 mM (25 degrees C). The latter value is consonant with previous volumetric determinations of urea permeability. Urea transport was inhibited competitively by thiourea; the half-inhibition constant, Ki, was 17 mM at 38 degrees C and 13 mM at 25 degrees C. Treatment with 1 mM p-chloromercuribenzosulfonate inhibited urea permeability by 92%. Phloretin reduced urea permeability further (greater than 97%) to a "ground" permeability of approximately 10(-6) cm s-1 (25 degrees C). This residual permeability is probably due to urea permeating the hydrophobic core of the membrane by simple diffusion. The apparent activation energy, EA, of urea transport after maximal inhibition was 59 kJ mol-1, whereas in control cells EA was 34 kJ mol-1 at 1 M and 12 kJ mol-1 at 1 mM urea. In net efflux experiments with no extracellular urea, the permeability coefficient remained constantly high, independent of a variation of intracellular urea between 1 and 500 mM, which indicates that the urea transport system is asymmetric. It is concluded that urea permeability above the ground permeability is due to facilitate diffusion and not to diffusion through nonspecific leak pathways as suggested previously.     

Permeability of human red cells to a homologous series of aliphatic alcohols. Limitations of the continuous flow-tube method

Human red cell permeability to the homologous series of methanol, ethanol, n-propanol, n-butanol, and n-hexanol was determined in tracer efflux experiments by the continuous flow tube method, whose time resolution is 2-3 ms. Control experiments showed that unstirred layers in the cell suspension were less than 2 X 10(-4) cm, and that permeabilities less than or equal to 10(-2) cm s-1 can be determined with the method. Alcohol permeability varied with the chain length (25 degrees C): Pmeth 3.7 X 10(-3) cm s-1, Peth 2.1 X 10(-3) cm s-1, Pprop 6.5 X 10(-3) cm s-1, Pbut less than or equal to 61 X 10(-3) cm s-1, Phex 8.7 X 10(-3) cm s-1. The permeability for methanol, ethanol, and n- propanol was concentration independent (1-500 mM). The permeability to n-butanol and n-hexanol, however, increased above the upper limit of determination at alcohol concentrations of 100 and 25 mM, respectively. The activation energies for the permeability to methanol, n-propanol, and n-hexanol were similar, 50-63 kJ mol-1. Methanol permeability was not reduced by p-chloromercuribenzene sulfonate (PCMBS), thiourea, or phloretin, which inhibit transport of water or hydrophilic nonelectrolytes. It is concluded (a) that all the alcohols predominantly permeate the membrane lipid bilayer structure; (b) that both the distribution coefficient and the diffusion coefficient of the alcohols within the membrane determine the permeability, and (c) that the relative importance of the two factors varies with changes in the chain length.     

Relationship between water transfers and the salinity of the environment in the isopod crustacean Sphaeroma serratum (Fabricius)]

The isopod Crustacean Sphaeroma serratum is isotonic to the medium in sea water and hypertonic in diluted media. The drinking rate is 15.9 microliter in SW and 3.2 microliter 24 h-1 100 mg-1 wet weight in 50% SW. The extracellular space is 28.4% in SW and 27.0% of the wet weight in 50% SW. The rate of urine production, calculated from the half time for the loss of sodium diatriazoate is 11.8 mg in SW and 42.9 mg 24 h-1 100 mg-1 wet weight in 50% SW. 95% of the diffusion fluxes of water take place through the pleopods: their surface is about 62.6 mm2 in a 100 mg weighing animal. The activation energy of water molecules is 15.0 kcal/mol between +5 degrees C and +15 degrees C and 6.7 kcal/mol between +15 degrees C and +25 degrees C. The diffusion permeability coefficient Pd is 1.71 X 10(-4) cm/sec in SW and 0.70 X 10(-4) cm/sec in 50% SW. The osmotic permeability coefficient Pos has a mean value of 1.91 X 10(-4) cm/sec in SW and 1.24 X 10(-4) cm/sec in 50% SW. Our results are compared with those obtained in other Crustaceans and in Fishes. Their validity is discussed (influence of unstirred layers, solute-solvent interaction). They are explained according to the different theories dealing with the water transit through the membranes.     

A stepwise mechanism for the permeation of phloretin through a lipid bilayer

The thermodynamics of interactions between phloretin and a phosphatidylcholine (PC) vesicle membrane are characterized using equilibrium spectrophotometric titration, stopped-flow, and temperature- jump techniques. Binding of phloretin to a PC vesicle membrane is diffusion limited, with an association rate constant greater than 10(8) M-1s-1, and an interfacial activation free energy of less than 2 kcal/mol. Equilibrium binding of phloretin to a vesicle membrane is characterized by a single class of high-affinity (8 micro M), noninteracting sites. Binding is enthalpy driven (delta H = -4.9 kcal/mol) at 23 degrees C. Analysis of amplitudes of kinetic processes shows that 66 +/- 3% of total phloretin binding sites are exposed at the external vesicle surface. The rate of phloretin movement between binding sites located near the external and internal interfaces is proportional to the concentration of un-ionized phloretin, with a rate constant of 5.7 X 10(4) M-1s-1 at 23 degrees C. The rate of this process is limited by a large enthalpic (9 kcal/mol) and entropic (-31 entropy units) barrier. An analysis of the concentration dependence of the rate of transmembrane movement suggests the presence of multiple intramembrane potential barriers. Permeation of phloretin through a lipid bilayer is modeled quantitatively in terms of discrete steps: binding to a membrane surface, translocation across a series of intramembrane barriers, and dissociation from the opposite membrane surface. The permeability coefficient for phloretin is calculated as 1.9 X 10(-3) cm/s on the basis of the model presented. Structure- function relationships are examined for a number of phloretin analogues.     

Permeabilization of phospholipid bilayer membranes induced by gas-liquid flow in an airlift bubble column

The permeability of 5(6)-carboxyfluorescein (CF) through the phospholipid bilayer membranes was measured by using the system in which the CF-containing phospholipid vesicles (liposomes) were suspended in the gas-liquid flow in an external loop airlift bubble column. The airlift was operated at various superficial gas velocities UG up to 2.4 cm/s at 25 and 40 degrees C. The CF-containing liposomes composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) had the nominal diameters of 50, 100, and 200 nm. The 50- and 100-nm liposomes were stable at 40 degrees C for 5 h even at a high UG value of 2.4 cm/s based on the observed turbidity of the liposome suspension in the airlift. On the other hand, the 200-nm liposomes were stable at a low UG value of 1.4 cm/s, although a progressive decrease in size of the liposomes was implied at the high UG value of 2.4 cm/s. The permeability coefficient PCF of CF through the lipid membrane of the 100-nm liposomes was significantly increased with increasing UG at a high temperature of 40 degrees C, while at a low temperature of 25 degrees C the PCF value was little dependent on UG. As a typical result on the above liposomes, the PCF value (9.2 x 10(-11) cm/s) at 40 degrees C and UG = 2.4 cm/s in the airlift was more than 15 times larger than that at 25 degrees C in the static liquid corresponding to UG = 0. In addition, the dependence of the PCF value on UG at 40 degrees C became more significant with increasing the size of liposomes suspended. The results obtained revealed that the permeability of the liposome membranes could be regulated by suspending the liposomes in the gas-liquid flow in the airlift without modulating the membrane composition of liposomes.     

Purification and characterization of adenosine deaminase from camel skeletal muscle

Adenosine deaminase was purified (780-fold) from skeletal muscle of camel (Camelus Dormedarius) to homogeneity level by using DEAE Sephadex chromatography, ammonium sulfate precipitation, gel filtration and ion exchange chromatography. The enzyme appeared to be monomeric with subunit molecular weight of 43kDa and isoelectric point of 4.85. The enzyme showed specificity for adenosine and exhibited Michaelis-Menten Kinetics with kappa(cat) of 1112.41 min(-1) and K(m) of 14.7 microM at pH 7.5. The pH and temperature optima for enzyme activity were 7-7.5 and 25 degrees C, respectively. Free energy (DeltaG*), enthalpy (DeltaH*) and entropy (DeltaS*) of activation for denaturation of adenosine deaminase at 50 degrees C were 88.94, 99.65 kJmol(-1) and 33.16 Jmol(-1), respectively. The purified enzyme had half-lives of 636 and 61 min at 25 and 50 degrees C, respectively. The activation energy for catalysis of camel skeletal muscle adenosine deaminase was 9.13 kJmol(-1). Free energy (DeltaG#), enthalpy (DeltaH#) and entropy (DeltaS#) of activation for hydrolysis of adenosine deaminase at 25 degrees C were 50.35, 6.65 kJmol(-1) and -146.62 Jmol(-1), respectively. Purine riboside inhibited the enzyme competitively with K(i) of 16 microM.     

Thermodynamic and kinetic aspects on water vs. organic solvent as reaction media in the enzyme-catalysed reduction of ketones

The stereoselective reduction of ketones catalysed by alcohol dehydrogenase from Thermoanaerobium brockii was studied in different reaction media, hexane at controlled water activities, hexane with 2. 5% water (biphasic) and pure water. The reactions were studied in the temperature range from -1 to 50 degrees C. Increasing the water activity from 0.53 to 0.97 increased the reaction rate 16-fold. The rate was further enhanced in hexane when exceeding the water solubility and in pure water the rates were even higher. This was general for all ketones studied. At controlled water activity the entropy of activation (DeltaSdouble dagger) was the dominating factor. Large negative DeltaSdouble dagger values caused low reaction rates at low aw. When increasing the carbon chain length of the substrate, for reactions in hexane, the decrease of reaction rate was mainly due to a decrease in DeltaSdouble dagger. In the comparison between hexane and pure water, DeltaGdouble dagger values were higher in hexane due to higher DeltaHdouble dagger (activation enthalpy) values. The enantioselectivity (E value) increased from 2.6 at water activity 0. 53 to 4.6 at water activity 0.97. Changing media from hexane (2.5%, v/v water) to pure water was not affecting the enantioselectivity or the specificity for different ketones.     

Membrane lipid fluidity as rate limiting in the concanavalin A-mediated agglutination of pyBHK cells.

The initial rate of concanavalin A-mediated agglutination of polyoma transformed Baby Hamster Kidney (pyBHK) cells follows Arrhenius kinetics. There is a smooth decrease in the agglutination rate from 37 degrees C to 22 degrees C with an activation energy of 11.8 +/- 0.2 kcal/mol in this region. There is a sharp decrease in agglutination rate below 22 degrees C. The addition of 0.1 mM 1,3-di-tert-2-hydroxyl-5-methylbenzene, a lipid perturber, increases the agglutination rate by a factor of two and increases the membrane lipid fluidity as determined by the spin label method. The rotational correlation time of the spin label 2N14 (2,2-dimethyl-5-dodecyl-5-methyloxazolidine-N-oxide) was measured. The sum of the enthalpy of activation of rotational diffusion and the enthalpy of activation of translational diffusion is very nearly equal to the enthalpy of activation of agglutination. This is consistent with the rate limiting step of agglutination being receptor diffusion, which is probably limited in pyBHK cells by membrane lipid fluidity.     

Thermodynamic analysis of catalysis by the dihydroorotases from hamster and Bacillus caldolyticus, as compared with the uncatalyzed reaction

Dihydroorotase (DHOase, EC 3.5.2.3) from the extreme thermophile Bacillus caldolyticus has been subcloned, sequenced, expressed, and purified as a monomer. The catalytic properties of this thermophilic DHOase have been compared with another type I enzyme, the DHOase domain from hamster, to investigate how the thermophilic enzyme is adapted to higher temperatures. B. caldolyticus DHOase has higher Vmax and Ks values than hamster DHOase at the same temperature. The thermodynamic parameters for the binding of L-dihydroorotate were determined at 25 degrees C for hamster DHOase (deltaG = -6.9 kcal/mol, deltaH = -11.5 kcal/mol, TdeltaS = -4.6 kcal/mol) and B. caldolyticus DHOase (deltaG = -5.6 kcal/mol, deltaH = -4.2 kcal/mol, TdeltaS = +1.4 kcal/mol). The smaller enthalpy release and positive entropy for thermophilic DHOase are indicative of a weakly interacting Michaelis complex. Hamster DHOase has an enthalpy of activation of 12.3 kcal/mol, similar to the release of enthalpy upon substrate binding, rendering the kcat/Ks value almost temperature independent. B. caldolyticus DHOase shows a decrease in the enthalpy of activation from 12.2 kcal/mol at temperatures from 30 to 50 degrees C to 5.3 kcal/mol for temperatures of 50-70 degrees C. Vibrational energy at higher temperatures may facilitate the transition ES --> ES(double dagger), making kcat/Ks almost temperature independent. The pseudo-first-order rate constant for water attack on L-dihydroorotate, based on experiments at elevated temperature, is 3.2 x 10(-11) s(-1) at 25 degrees C, with deltaH(double dagger) = 24.7 kcal/mol and TdeltaS(double dagger) = -6.9 kcal/mol. Thus, hamster DHOase enhances the rate of substrate hydrolysis by a factor of 1.6 x 10(14), achieving this rate enhancement almost entirely by lowering the enthalpy of activation (delta deltaH(double dagger) = -19.5 kcal/mol). Both the rate enhancement and transition state affinity of hamster DHOase increase steeply with decreasing temperature, consistent with the development of H-bonds and electrostatic interactions in the transition state that were not present in the enzyme-substrate complex in the ground state.     

Permeability of human blood platelets to glycerol

The permeability of human platelets to glycerol was determined at 37 degrees C, 25 degrees C, and 0 degrees C from the rate of change of cell volume after abrupt addition of 0.5 mol/liter glycerol in phosphate-buffered saline. Intracellular water volume was measured employing both tritiated water and a photometric method. Intracellular glycerol was measured employing tritiated glycerol. The glycerol permeability coefficient derived from the tracer cell volume data was 4.0 +/- 0.7 X 10(-7) cm/s at 37 degrees C, and 1.1 +/- 0.4 X 10(-7) cm/s at 25 degrees C, and the photometric data gave a permeability coefficient of 5.4 +/- 0.4 X 10(-7) cm/s at 37 degrees C. The activation energy between 23 degrees C and 37 degrees C for glycerol permeation was 19.8 kcal/mol. The cells were virtually impermeable to glycerol at 0 degrees C. The minimum intracellular water volume attained after the addition of 0.5 mol/liter glycerol at 37 degrees C determined by the photometric method was 47.8% of normal water volume, whereas the minimum water volume calculated assuming that glycerol exerted its full osmotic effect (i.e., sigma = 1) was 45.6%. The reflexion coefficient was therefore assumed to be unity. Neither method of cell volume determination could be used with 1 or 2 mol/liter glycerol: adequate separation of the cells from the labeled medium could not be achieved in the tracer method; in the photometric method, it was apparent that transmittance (660 nm) was influenced by one or more variables in addition to cell volume.     

A comparative study of diffusive and osmotic water permeation across bilayers composed of phospholipids with different head groups and fatty acyl chains.

Osmotic and diffusive water permeability coefficients Pf and Pd were measured for lipid vesicles of 100-250 nm diameter composed of a variety of phospholipids with different head groups and fatty acyl chains. Two different methods were applied: the H2O/D2O exchange technique for diffusive water flow, and the osmotic technique for water flux driven by an osmotic gradient. For phosphatidylcholines in the liquid-crystalline state at 70 degrees C, permeability constants Pd between 3.0 and 5.2.10(-4) cm/s and ratios Pf/Pd 7 and 23 were observed. The observation of a permeability maximum in the phase transition region and the fact that osmotically driven water flux is higher than diffusive water exchange suggest that water is diffusing through small transient pores arising from density fluctuations in the bilayers. The Pd values depend on the nature of the head group, on the chemical structure of the chains, and on the type of chain linkage. In the case of charged lipids, the ionic strength of the solution has a strong influence. For phosphatidylethanolamines, phosphatidic acids, and ether phosphatidylcholines, permeability constants Pd were considerably lower (2-4.10(-6) cm/s at 70 degrees C). For liquid-crystalline phosphatidylcholines, a strong reduction of Pd after addition of ethanol was observed (2-4.10(-6) cm/s at 70 degrees C). The experimental values are discussed in connection with different permeation models.     

Organotin-mediated exchange diffusion of anions in human red cells

Organotin cations (R3Sn+) form electrically neutral ion pairs with monovalent anions. It is demonstrated that the tin derivatives induce exchange diffusion of chloride in red cells and resealed ghosts, without any detectable increase of membrane permeability to net movements of chloride ions. The obligatory anion exchange is believed to be due to the permeation of electroneural ion pairs, whereas the organic cation (R3Sn+) has an extremely low membrane permeability. Exchange fluxes of chloride increased with the lipophilicity of the substituting group (R3). At the same molar concentration of organotin, the relative potencies of the tin derivatives as anion carriers (with trimethyltin as a reference) were: methyl 1, ethyl 30, propyl = phenyl 1,00, and butyl 10,000. Tributyltin-mediated anion exchange was studied in detail. The organotin-induced anion transport increased through the sequence: F- less than Cl- less than Br- less than I- = SCN- less than OH-. Partitioning of tributyltin into red cell membranes was greater in iodide than in chloride media (partition coefficients 6.6 and 1.7 x 10(-3) cm, respectively). Bicarbonate, fluoride, nitrate, phosphate, and sulphate did not exchange with chloride in the presence of tributyltin. Chloride exchange fluxes increased linearly with tributylin concentrations up to 10(-5) M, and with chloride concentrations up to at least 0.9 M. The apparent turnover number for tributyltin-mediated chloride exchange increased from 15 to 1,350 s-1 between 0 and 38 degrees C. These figures are minimum turnover numbers, because it is not known what fraction of the organotin in the membrane exists as chloride ion pairs.     

Catalysis by entropic effects: the action of cytidine deaminase on 5,6-dihydrocytidine

In neutral solution, 5,6-dihydrocytidine undergoes spontaneous deamination (k25 approximately 3.2 x 10(-5) s(-1)) much more rapidly than does cytidine (k25 approximately 3.0 x 10(-10) s(-1)), with a more favorable enthalpy of activation (DeltaDeltaH# = -8.7 kcal/mol) compensated by a less favorable entropy of activation (TDeltaDeltaS# = -1.8 kcal/mol at 25 degrees C). E. coli cytidine deaminase enhances the rate of deamination of 5,6-dihydrocytidine (kcat/k(non) = 4.4 x 10(5)) by enhancing the entropy of activation (DeltaDeltaH# = 0 kcal/mol; TDeltaDeltaS# = +7.6 kcal/mol, at 25 degrees C). Binding of the competitive inhibitor 3,4,5,6-tetrahydrouridine (THU), a stable analogue of 5,6-dihydrocytidine in the transition state for its deamination, is accompanied by a release of enthalpy (DeltaH = -7.1 kcal/mol, TDeltaDeltaS = +2.2 kcal/mol) that approaches the estimated enthalpy of binding of the actual substrate in the transition state for deamination of 5,6-dihydrocytidine (DeltaH = -8.1 kcal/mol, TDeltaDeltaS = +6.0 kcal/mol). Thus, the shortcomings of THU in capturing all of the binding affinity expected of an ideal transition-state analogue reflect a less favorable entropy of association. That difference may arise from the analogue's inability to displace a water molecule from the "leaving group site" at which ammonia is generated in the normal reaction. The effect on binding of removing the 4-OH group from the transition-state analogue THU, to form 3,4,5,6-tetrahydrozebularine (THZ) (DeltaDeltaH = -2.1 kcal/mol, TDeltaDeltaS = -4.4 kcal/mol), is mainly entropic, consistent with the inability of THZ to displace water from the "attacking group site". These results are consistent with earlier indications [Snider, M. J., and Wolfenden, R. (2001) Biochemistry 40, 11364] that site-bound water plays a prominent role in substrate activation and inhibitor binding by cytidine deaminase.     

Heat capacity and entropy changes of the two major isotypes of bullfrog (Rana catesbeiana) parvalbumins induced by calcium binding

The possible structural changes of the two major isotypes (PA1 and PA2) of parvalbumins from bullfrog (Rana catesbeiana) skeletal muscle caused by Ca2+ binding have been analyzed by microcalorimetric titrations. Titrations of the parvalbumins with Ca2+ have been made in both the absence and presence of Mg2+ at pH 7.0 and at 5, 15, and 25 degrees C. The reactions of the parvalbumins with Ca2+ are exothermic in both the presence and absence of Mg2+ and at every temperature. But the contributions of enthalpy and entropy changes are variable; Mg2+-Ca2+ exchange on PA1 at 25 degrees C is driven almost entirely by a favorable enthalpy change, whereas Ca2+ binding to PA2 at 5 degrees C is driven for the most part by a favorable entropy change. The magnitudes of the hydrophobic and internal vibrational contributions to the heat capacity and entropy changes of the parvalbumins on Ca2+ binding and Mg2+-Ca2+ exchange have been estimated by the empirical method of Sturtevant [Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2236-2240]. Although PA1 (beta) and PA2 (alpha) belong to genetically different lineages, the parvalbumins indicate very similar conformational changes to each other on both Ca2+ binding and Mg2+-Ca2+ exchange. On Mg2+-Ca2+ exchange, the vibrational as well as hydrophobic entropy is slightly increased in a parallel manner. In contrast, on Ca2+ binding, the hydrophobic entropy increases but the vibrational entropy decreases. The increase in the hydrophobic entropy indicates the sequestering of nonpolar groups from the surface to the interior of molecules, while the changes in the vibrational entropy suggest that the overall structures are tightened on Ca2+ binding but loosened on Mg2+-Ca2+ exchange.     

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.     

Comparative NMR studies of diffusional water permeability of red blood cells from different species: XIV. Little Penguin, Eudyptula minor

As part of a programme of comparative measurements of diffusional water permeability (Pd) the red blood cells (RBC) from Little Penguin (Eudyptula minor) were studied. The cell dimensions were measured with light and electron microscopy, and by a newly described non-invasive technique, NMR q-space analysis. In view of its relative novelty for cell biologists, an overview of this technique is presented. The RBC revealed an ellipsoidal shape that is characteristic of avian RBC, with axis lengths ("diameters") estimated to be: a=16.0 microm; b=9.6 microm; c=5.0 microm. The values of P(d)were: 2.0 x 10(-3)cm s(-1)at 5 degrees C, 3.3 x 10(-3)cm s(-1)at 10 degrees C, 4.6 x 10(-3)cm s(-1)at 15 degrees C and approximately 5.4 x 10(-3)cm s(-1)at 20, 25, 30, 37 and 42 degrees C.There was a lack of inhibition of water permeability by p-chloromercuribenzensulfonate (PCMBS), the well-known inhibitor of RBC aquaporin. It was notable that in the temperature range 5-20 degrees C the NMR parameters, and hence the permeability, varied linearly as is found for other species, but at temperatures higher than 20 degrees C there was no temperature-dependence of Pd. Consequently, there was an obvious break at approximately 20 degrees C in the Arrhenius plot, of the mean residence life time of water inside the cells, 1/Te, versus temperature. For temperatures less than 20 degrees C the activation energy E(a,d) was 45.6 +/- 6.6 kJ/mol. For temperatures higher than 25 degrees C E(a,d) was zero. The lack of inhibition of water permeability by PCMBS and the very high value of E(a,d) for diffusive water exchange suggests that the water permeation occurs primarily via the membrane bilayer per se, i.e., there is no aquaporin in Little Penguin RBC. The discontinuity at approximately 20 degrees C in the Arrhenius plot is an interesting finding, not seen before in other species, and we suggest that it reflects a phase transition in the membrane lipids.     

Thermodynamic analysis of binding between mouse major urinary protein-I and the pheromone 2-sec-butyl-4,5-dihydrothiazole

The mouse pheromone 2-sec-butyl-4,5-dihydrothiazole (SBT) binds to an occluded, nonpolar cavity in the mouse major urinary protein-I (MUP-I). The thermodynamics of this interaction have been characterized using isothermal titration calorimetry (ITC). MUP-I-SBT binding is accompanied by a large favorable enthalpy change (DeltaH = -11.2 kcal/mol at 25 degrees C), an unfavorable entropy change (-TDeltaS = 2.8 kcal/mol at 25 degrees C), and a negative heat capacity change [DeltaC(p)() = -165 cal/(mol K)]. Thermodynamic analysis of binding between MUP-I and several 2-alkyl-4,5-dihydrothiazole ligands indicated that the alkyl chain contributes more favorably to the enthalpy and less favorably to the entropy of binding than would be expected on the basis of the hydrophobic desolvation of short-chain alcohols. However, solvent transfer experiments indicated that desolvation of SBT is accompanied by a net unfavorable change in enthalpy (DeltaH = +1.0 kcal/mol) and favorable change in entropy (-TDeltaS = -1.8 kcal/mol). These results are discussed in terms of the possible physical origins of the binding thermodynamics, including (1) hydrophobic desolvation of both the protein and the ligand, (2) formation of a buried water-mediated hydrogen bond network between the protein and ligand, (3) formation of strong van der Waals interactions, and (4) changes in the structure, dynamics, and/or hydration of the protein upon binding.     

Diffusion of Salts across a Butanol-Water Interface

Diffusion of the chloride salts of Li, Na, K, Rb, and Cs from water into 1-butanol and from 1-butanol into water was examined at temperatures from 13-40 degrees C. Distribution coefficients, interfacial transfer coefficients, Arrhenius activation energy, free energy of activation, enthalpy, and entropy of activation were determined for the diffusion of these salts across the alcohol-water interface. The results indicate that the entropy decrease made the major contribution to the change in the free energy of activation.     

Effects of trifluoperazine on calcium binding by calmodulin. A microcalorimetric study

Microcalorimetric titrations of calmodulin with Ca2+ and trifluoperazine (TFP) at various molar ratios have been carried out at 25 degrees C and at pH 7.0. Ca2+ binding to calmodulin produces heat (-delta H) in the presence of TFP, while heat is absorbed in the absence of TFP. The total heat produced by Ca2+ binding to all four sites is increased at increasing TFP-to-calmodulin ratios, attaining a plateau at about 7. These results indicate that at the higher ratios, the enthalpy changes (delta H) associated with Ca2+ binding are affected by TFP molecules bound at both high- and low-affinity sites. In addition, the Ca2+ binding reaction of the calmodulin-TFP complex is driven solely by a favorable enthalpy change of -27 kJ/mol of site; the entropy change (delta S) is -35 J/mol/K. These thermodynamic changes are opposite to those for TFP-free calmodulin and distinctly different from other Ca2+ binding proteins such as skeletal and cardiac troponin C and parvalbumin, where the reaction is driven by favorable changes of entropy as well as enthalpy.     

Ontogeny of rabbit proximal tubule urea permeability

Urea transport in the proximal tubule is passive and is dependent on the epithelial permeability. The present study examined the maturation of urea permeability (P(urea)) in in vitro perfused proximal convoluted tubules (PCT) and basolateral membrane vesicles (BLMV) from rabbit renal cortex. Urea transport was lower in neonatal than adult PCT at both 37 and 25 degrees C. The PCT P(urea) was also lower in the neonates than the adults (37 degrees C: 45.4 +/- 10.8 vs. 88.5 +/- 15.2 x 10(-6) cm/s, P < 0.05; 25 degrees C: 28.5 +/- 6.9 vs. 55.3 +/- 10.4 x 10(-6) cm/s; P < 0.05). The activation energy for PCT P(urea) was not different between the neonatal and adult groups. BLMV P(urea) was determined by measuring vesicle shrinkage, due to efflux of urea, using a stop-flow instrument. Neonatal BLMV P(urea) was not different from adult BLMV P(urea) at 37 degrees C [1.14 +/- 0.05 x 10(-6) vs. 1.25 +/- 0.05 x 10(-6) cm/s; P = not significant (NS)] or 25 degrees C (0.94 +/- 0.06 vs. 1.05 +/- 0.10 x 10(-6) cm/s; P = NS). There was no effect of 250 microM phloretin, an inhibitor of the urea transporter, on P(urea) in either adult or neonatal BLMV. The activation energy for urea diffusion was also identical in the neonatal and adult BLMV. These findings in the BLMV are in contrast to the brush-border membrane vesicles (BBMV) where we have previously demonstrated that urea transport is lower in the neonate than the adult. Urea transport is lower in the neonatal proximal tubule than the adult. This is due to a lower rate of apical membrane urea transport, whereas basolateral urea transport is the same in neonates and adults. The lower P(urea) in neonatal proximal tubules may play a role in overall urea excretion and in developing and maintaining a high medullary urea concentration and thus in the ability to concentrate the urine during renal maturation.