Electrophoretic mobility of sarcoplasmic reticulum vesicles - analytical model includes amino acid residues of A+P+N domain of Ca(2+)-ATPase and charged lipids.

This work is an experimental and theoretical study of electrostatic and hydrodynamic properties of the surface of sarcoplasmic reticulum (SR) membrane using particle electrophoresis. The essential structural components of SR membrane include a lipid matrix and a dense layer of Ca(2+)-ATPases embedded in the matrix. The Ca(2+)-ATPase layer both drives and impedes vesicle mobility. To analyze the experimental mobility data, obtained at pH4.0, 4.7, 5.0, 6.0, 7.5, and 9.0 in 0.1M monovalent (1:1) electrolyte, an analytical solution for the vesicle mobility and electroosmotic flow velocity distribution was obtained by solving the Poisson-Boltzmann and the Navier-Stokes-Brinkman equations. The electrophoretic mobility model includes two sets of charges that represent: (a) charged lipids of the lipid matrix of the vesicle core, and (b) charged amino acid residues of APN domains of Ca(2+)-ATPases. APN domains are assumed to form a charged plane displaced from the surface of lipid matrix. The charged plane is embedded in a frictional layer that represents the surface layer of calcium pumps. Electrophoretic mobility is driven by the charged APN domain and by lipid matrix while the surface layer provides hydrodynamic friction. The charge of APN domain is determined by ionized amino acid residues obtained from the amino acid composition of SERCA1a Ca(2+)-ATPase. Agreement between the measured and the predicted mobility is evaluated by the weighted sum of mobility deviation squared. This model reproduces the experimental dependence of mobility on pH and predicts that APN domains are located in the upper half of the SR vesicle surface layer.

Electrophoretic mobility of sarcoplasmic reticulum vesicles is determined by amino acids of A+P+N domains of Ca2+-ATPase.

Establishing the origin of electrophoretic mobility of sarcoplasmic reticulum (SR) vesicles is the primary goal of this work. It was found that the electrophoretic mobility originates from ionizable amino acids of cytoplasmic domains of the Ca2+-ATPase, the calcium pump of SR. The mobility was measured at pH 4.0, 4.7, 5.0, 6.0, 7.5, and 9.0 in the region of ionic strength from 0.05 to 0.2 M. Mobility measurements were supplemented by studies of SR vesicles by photoelectron microscopy. The median diameter of SR vesicles was 260 nm. Ca2+-ATPases were not resolved. The mobility data were standardized by interpolation to a reference ionic strength of 0.1M. The mobility of the SR vesicles is determined by the charge of the Ca2+-ATPase. It is due to the ionizable amino acids selected from the amino acid sequence of SERCA1a Ca2+-ATPase. The pH dependence of charge residing in various domains of Ca2+-ATPase was computed using pKa values in free water. The charge correlated with measured mobility. It was shown that a linear relationship exists between the mobility of the SR vesicles, mu, and the total computed charge, Q, on three cytoplasmic domains of Ca2+-ATPase: A, P, and N. It is given by mu=alpha+betaQ where the fitted values beta=(0.043+/-0.002) x 10(-8) m(2) V(-1) s(-1) e(-1) and alpha=(0.16+/-0.02) x 10(-8) m(2) V(-1) s(-1). Since beta and alpha values do not change from pH 4 to pH 9, one concludes that the hydrodynamic friction of the cytoplasmic domains of SR is independent of their charge.

Toward a class-independent quantitative structure--activity relationship model for uncouplers of oxidative phosphorylation.

A mechanistically based quantitative structure-activity relationship (QSAR) for the uncoupling activity of weak organic acids has been derived. The analysis of earlier experimental studies suggested that the limiting step in the uncoupling process is the rate with which anions can cross the membrane and that this rate is determined by the height of the energy barrier encountered in the hydrophobic membrane core. We use this mechanistic understanding to develop a predictive model for uncoupling. The translocation rate constants of anions correlate well with the free energy difference between the energy well and the energy barrier, Delta G well-barrier,A (-) , in the membrane calculated by a novel approach to describe internal partitioning in the membrane. An existing data set of 21 phenols measured in an in vitro test system specific for uncouplers was extended by 14 highly diverse compounds. A simple regression model based on the experimental membrane-water partition coefficient and Delta G well-barrier,A (-) showed good predictive power and had meaningful regression coefficients. To establish uncoupler QSARs independent of chemical class, it is necessary to calculate the descriptors for the charged species, as the analogous descriptors of the neutral species showed almost no correlation with the translocation rate constants of anions. The substitution of experimental with calculated partition coefficients resulted in a decrease of the model fit. A particular strength of the current model is the accurate calculation of excess toxicity, which makes it a suitable tool for database screening. The applicability domain, limitations of the model, and ideas for future research are critically discussed.

Environmental swap energy and role of configurational entropy in transfer of small molecules from water into alkanes.

We studied the effect of segmented solvent molecules on the free energy of transfer of small molecules from water into alkanes (hexane, heptane, octane, decane, dodecane, tetradecane, and hexadecane). For these alkanes we measured partition coefficients of benzene, 3-methylindole (3MI), 2,3,4,6-tetrachlorophenol (TeCP), and 2,4,6-tribromophenol (TriBP) at 3, 11, 20, 33 [corrected], and 47 degrees C. For 3MI, TeCP, and TriBP the dependence of free energy of transfer on length of alkane chains was found to be very different from that for benzene. In contrast to benzene, the energy of transfer for 3MI, TeCP, and TriBP was independent of the number of carbons in alkanes. To interpret data, we used the classic Flory-Huggins (FH) theory of concentrated polymer solutions for the alkane phase. For benzene, the measured dependence of energy of transfer on the number of carbons in alkanes agreed well with predictions based on FH model in which the size of alkane segments was obtained from the ratio of molar volumes of alkanes and the solute. We show that for benzene, the energy of transfer can be divided into two components, one called environmental swap energy (ESE), and one representing the contribution of configurational entropy of alkane chains. For 3MI, TeCP, and TriBP the contribution of configurational entropy was not measurable even though the magnitude of the effect predicted from the FH model for short chain alkanes was as much as 20 times greater than experimental uncertainties. From the temperature dependence of ESE we obtained enthalpy and entropy of transfer for benzene, 3MI, TeCP, and TriBP. Experimental results are discussed in terms of a thermodynamic cycle considering creation of cavity, insertion of solute, and activation of solute-medium attractive interactions. Our results suggest that correcting experimental free energy of transfer by Flory-Huggins configurational entropy term is not generally appropriate and cannot be applied indiscriminately.