Biochimica et Biophysica Acta 1798, 1689–1697(2010)

ELECTROPHORETIC MOBILITY OF SARCOPLASMIC RETICULUM VESICLES IS DETERMINED BY AMINO ACIDS
OF A+P+N DOMAINS OF CA²⁺-ATPASE

Pavel Smejtek*, Laura E. Satterfield, Robert C. Word, and Jonathan J. Abramson.

Department of Physics and Molecular Biosciences Group, Portland State University, Portland, Oregon 97207-0751, USA

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 0.1 M so that the Helmholtz-Smoluchowski model is applicable. 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, μ, and the total computed charge, Q, on three cytoplasmic domains of Ca²⁺-ATPase: A, P, and N. It is given by μ = α + β*Q where β = 0.0330 ± 0.0017 m²V^-1s^-1e^-1 and α = 0.113 ± 0.023 m²V^-1s^-1. Since one value of the proportionality constant is applicable from pH 4 to pH 9, one concludes that the hydrodynamic friction of the cytoplasmic domains of SR is independent of their charge.

Chemical Research in Toxicology 21, 911-927 (2008)

TOWARD A CLASS-INDEPENDENT QUANTITATIVE STRUCTURE-ACTIVITY RELATIONSHIP MODEL FOR UNCOUPLERS
OF OXIDATIVE PHOSPHORYLATION

Simon Spycher¹, Pavel Smejtek², Tatiana I. Netzeva³ and Beate I. Escher⁴

^1,4Department of Environmental Toxicology, UTOX, Swiss Federal Institute of Aquatic Science and Technology, EAWAG, CH-8600 Dübendorf, Switzerland, ²Department of Physics, Portland State University, Portland, Oregon 97207, and ³European Chemicals Bureau, Institute for Health and Consumer Protection, Joint Research Centre, 21020 Ispra (VA), Italy

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, ΔG_{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 ΔG_{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.

Journal of Membrane Biology 203,127-142 (2005)

PARTITIONING OF TETRACHLOROPHENOL INTO LIPID BILAYERS AND SARCOPLASMIC RETICULUM: EFFECT OF LENGTH OF ACYL CHAINS, CARBONYL GROUP OF LIPIDS AND BIOMEMBRANE STRUCTURE

R. C. Word and P. Smejtek

Department of Physics, Portland State University, Portland, Oregon 97207

We report results of a partitioning study of 2,3,4,6-tetrachlorophenol (TeCP). In the study we explored (1) the effect of the length of acyl chains of lipids (C16:1 - C24:1) and alkanes (C6-C16), (2) the role of carbonyl group of lipids, and (3) the effect of molecular structure of sarcoplasmic reticulum membrane on TeCP partitioning. Mole fraction partition coefficients have been measured using equilibrium dialysis for un-ionized (HA), and ionized (A-) species, KpxHA, KpxA . Their values are concentration-dependent. Partition coefficients were analyzed in terms of a model that accounts for saturation of membrane associated with the finite area of partition site, and electrostatic interactions of (A-) species with charged membrane. Limiting values of partition coefficients, corresponding to infinite dilution of solute, Kpx0HA, Kpx0A were obtained. Kpx0HA and Kpx0A measure the strength of solute-membrane interactions. Studies were done with single-layered vesicles of lipids with variable chain length: 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (C16:1), 1,2-dioleoyl-sn-glycero-3-phosphocholine (C18:1), 1,2-dierucoyl-sn-glycero-3-phosphocholine (C22:1), and 1,2-dinervonoyl-sn-glycero-3-phosphocholine (C24:1), and egg-PC. Kpx0 for transfer of TeCP from water into lipid membranes were found to be independent of the length of acyl chains, whereas Kpx0 for transfer from water into alkanes increased with the length of alkane. The effect of carbonyl CO group of lipids on partitioning was measured using 1,2-di-o-octadecenyl-sn-glycero-3-phosphocholine (CO absent) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (CO present) liposomes. Carbonyl groups, known to change dipolar potential, had no effect on partitioning. Partition coefficients of un-ionized and ionized forms of TeCP were invariant to the presence of proteins and other membrane components of sarcoplasmic reticulum (SR) membrane.

Journal of Chemical Physics 120, 1383-1394 (2004)

ENVIRONMENTAL SWAP ENERGY AND ROLE OF CONFIGURATIONAL ENTROPY IN TRANSFER OF SMALL MOLECULES FROM WATER INTO ALKANES

Pavel Smejtek and Robert C. Word

Department of Physics, Portland State University, Portland, Oregon 97207

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°C, 11°C, 20°C, 33°C, and 47°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.

Journal of Membrane Biology 167, 151-163 (1999)

ELECTROKINETIC PROPERTIES OF THE SARCOPLASMIC RETICULUM MEMBRANE OBTAINED FROM RECONSTITUTION STUDIES

P. Smejtek¹, M. Mense², R. Word¹, S. Wang¹, ¹Department of Physics and Molecular Biosciences Group, Portland State University, Portland, OR 97207, USA; ²Department of Cellular & Molecular Physiology, Yale School of Medicine, New Haven, CT 06510, USA

Electrophoretic mobility data of SR vesicles reconstituted with uncharged and two mixtures of charged and uncharged lipids (Brethes, D., Dulon, D., Johannin, G., Arrio, B., Gulik-Krzywicki, T., Chevallier, J. 1986. Study of the electrokinetic properties of reconstituted sarcoplasmic reticulum vesicles. Arch. Biochem. Biophys. 246:355-356) were analyzed in terms of four models of the membrane-water interface: (I) a smooth, negatively charged surface; (II) a negatively charged surface of lipid bilayer covered with an electrically neutral surface frictional layer; (III) an electrically neutral lipid bilayer covered with a neutral frictional layer containing a sheet of negative charge at some distance above the surface of the bilayer; (IV) an electrically neutral lipid bilayer covered with a homogeneously charged frictional layer. The electrophoretic mobility was predicted from the numerical integration of Poisson-Boltzmann and Navier-Stokes equations. Experimental results were consistent only with predictions based on Model-Ill with charged sheet about 4 nm above the bilayer and frictional layer about 10 nm thick. Assuming that the charge of the SR membrane is solely due to that on Ca⁺⁺-ATPase pumps, the dominant SR protein, the mobility data of SR and reconstituted SR vesicles are consistent with 12 electron charges of ATPase. This value compares well to the net charge of the cytoplasmic portion of ATPase estimated from the amino acid sequence (-11e). The position of the charged sheet suggests that the charge on the ATPase is concentrated in the middle of the cytoplasmic portion. The frictional layer of SR can be also assigned to the cytoplasmic portion of Ca⁺⁺-ATPase. The layer has been characterized with hydrodynamic shielding length of 1.1 nm. Its thickness is comparable to the height of the cytoplasmic portion of Ca⁺⁺-ATPase.

Chemosphere 33, 177-201 (1996)

HYDROPHOBICITY AND SORPTION OF CHLOROPHENOLATES TO LIPID MEMBRANE

Pavel Smejtek, Andreas Blochel and Shanru Wang, Department of Physics and Environmental Sciences and Resources Doctoral Program, Portland State University, Portland, Oregon 97207, U.S.A.

We have studied sorption of ionized species of chlorophenols and pentahalophenols to lipid membranes using egg-phosphatidylcholine (egg-PC) vesicles and measuring their zeta-potential as a function of aqueous concentration of the phenolates. The zeta-potential isotherms can be understood in terms of a sorption model that is a combination of the Gouy-Chapman model of the electrical double layer at the membrane-water interface and the Langmuir model for sorption. Two intrinsic sorption parameters were determined: the linear partition coefficient b_m, which relates the membrane surface density of the phenolates to their aqueous concentration and the area of the adsorption site, P_s. The linear partition coefficient is the measure of the affinity of phenolates to the lipid membrane. It depends strongly on the molecular structure:

2,6-dichlorophenolate b_m = (0.45±0.08)x10^-7m;

3,5-dichlorophenolate b_m = (0.22±0.02)x10^-6 m;

2,4,6-trichlorophenolate b_m = (0.63±0.06)x10^-6 m;

2,4,5-trichlorophenolate b_m = (0.11±0.01)x10^-5 m;

2,3,5,6-tetrachlorophenolate b_m = (0.56±0.07)x10^-5 m;

2,3,4,5-tetrachlorophenolate b_m = (0.55±0.06)x10^-5 m;

pentachlorophenolate b_m = (0.34±0.05)x10^-4 m;

pentafluorophenolate b_m = (1.00±0.13)x10^-7 m; and

pentabromophenolate b_m = (0.19±0.04)x10^-3 m.

P_s was found to be independent of phenolate structure, P_s = 3.3±0.1 nm². The membrane affinity of chlorophenolates was compared with the octanol-water partition coefficients of un-ionized chlorophenols. It was shown that the free energy of transfer of chlorophenolates from water into the lipid membrane can be divided into non-electrostatic and electrostatic contributions. The non-electrostatic contribution corresponds to the hydrophobicity parameter a = -3.94±0.08 kcal per nm² of molecular surface area. The electrostatic contribution contains a term inversely proportional to the molecular radius of the phenolate ion which has the physical meaning of the work of transfer of the phenolate ion from water into the membrane. The polarity of the sorption region of egg-PC membranes is given in terms of the dielectric constant and was estimated to be 12.4 (range 10.5-13.4).

Biophysical Journal 70, 818-830 (1996)

ADSORPTION OF RUTHENIUM RED TO PHOSPHOLIPID MEMBRANES

Dirk Voelker and Pavel Smejtek, Department of Physics, Environmental Sciences and Resources Doctoral Program, Portland State University, Portland, Oregon 97207, USA

We have measured the distribution of the hexavalent ruthenium red cation (RuR) between water and phospholipid membranes, have shown the critical importance of membrane negative surface charge for RuR binding, and determined the association constant of RuR for different phospholipid bilayers. The studies were performed with liposomes made of mixtures of zwitterionic L-a-phosphatidylcholine (PC), and one of the negatively charged phospholipids L-a-phosphatidylserine (PS), L-a-phosphatidylinositol (PI), or L-a-phophatidylglycerol (PC). Lipid composition of PC:PX membranes was 1:0, 19:1, 9:1, and 4:1. Liposomes were processed using freeze-and-thaw treatment, and their size distribution was characterized by light scattering and electron microscopy. Experimental distribution isotherms of RuR obtained by ultracentrifugation and spectrophotometry can be reproduced with the Langmuir-Stern-Grahame model, assuming that RuR behaves in the diffuse double layer as an ion with effective valency <6. In terms of this model, PC-PS, PC-PI, and PC-PG membranes were found to be electrostatically equivalent and the intrinsic association constants of RuR were obtained. RuR has highest affinity to PS-containing membranes; its association constant for PC-Pl and PC-PG membranes is about 5 times smaller than that for PC-PS membranes. From the comparison of RuR binding to mixed negatively charged phospholipid membranes and RuR binding to sarcoplasmic reticulum (SR), we conclude that the low-affinity RuB binding sites may indeed be associated with the lipid bilayer of SR.

Biochimica et Biophysica Acta 1140, 262-270 (1993)

AHA^- HETERODIMER OF A CLASS-2 UNCOUPLER: PENTACHLOROPHENOL

Arthur W. Barstad^a , David H. Peyton^b, and Pavel Smejtek^a; Environmental Sciences and Resources Program, ^aPhysics Department, and^bChemistry Department, Portland State University, Portland, OR (USA)

AHA^- heterodimers formed by association of neutral molecules of weak acid (HA) with its conjugate anion (A^-) have been proposed to be the charged membrane-permeable species of class-2 uncouplers. Past attempts to extract and identify AHA^- heterodimers failed. We have measured optical spectra of HA + A^- (1:1) solutions of pentachlorophenol (PCP) in various solvents and in the presence of PC liposomes. Optical studies were supplemented by nuclear magnetic resonance measurements of HA + A^- (1:1) solutions of PCP in dichloroethane to gain insight into the formation of AHA^- species in lipid membranes. From these experiments, we found evidence for AHA^- formation in non-hydrogen-bonding solvents, then reported the AHA^- formation constant K_f and the molar absorptivity e_AHA-(l).

K_f decreases with increasing dielectric constant, k, from 1210 ± 130 M^-1 for dichloroethane (k=10.7), to 340 ± 34 M^-1 for acetonitrile (k=37.5); K_f also decreases with increasing concentration of water. In hydrogen-bonding solvents, octanol (k=10.3) and methanol (k= 33.5) and in liposomes, AHA^- heterodimers are not observed optically.

We estimate K_f for PCP in lipid bilayers from a combination of data on membrane electrical conductivity and surface density of adsorbed PCP. Our estimate for lipid bilayer, 0.005 <K_f < 0.5 M^-1, is consistent with our inability to detect the AHA^- species optically in liposome suspensions. 

We propose that penetration of water into the membrane inhibits formation of AHA^- in lipid bilayers.

Archives of Environmental Contamination and Toxicology 25, 394-404 (1993)

DISTRIBUTION OF HYDROPHOBIC IONIZABLE XENOBIOTICS BETWEEN WATER AND LIPID MEMBRANES: PENTACHLOROPHENOL AND PENTACHLOROPHENATE.

A COMPARISON WITH OCTANOL-WATER PARTITION

Pavel Smejtek and Shanru Wang; Department of Physics and Environmental Sciences and Resources Doctoral Program, Portland State University, Portland, Oregon 97207-715, USA

We have studied distribution of pentachlorophenol (PCP) - a major environmental pollutant - between egg-phosphatidylcholine (egg-PC) membranes and water. The objectives were (1) to compare the membrane-water partition of the un-ionized (HA) and ionized (A) PCP, and (2) to establish similarities and differences between the partition of PCP into lipid membranes and into octanol. The studies were made with egg-PC liposomes. It is shown that the distribution isotherms can be understood in terms of the Langmuir-Stern-Grahame adsorption model. The model is applicable to both the HA and A species; it takes into account the electrostatic interactions at the membrane-water interface charged by the adsorbed pentachlorophenate. Relationships between the membrane surface adsorption and bulk partition characteristics were presented and used to relate the partition of PCP into egg-PC membranes to those for octanol-water systems. Results (egg-PC membranes): bulk distribution coeff. g_HA=2.9x10⁵, g_A=1.6x10⁴,association constant K_mHA=2.9x10⁵ M^-1, K_mA= 0.7x10⁵ M^-1, adsorption site area P_sHA=0.6nm², P_sA=3.5 nm², and linear partition coeff. b_mHA=550 mm,b_mA=30 mm. Comparable to g_HA and g_A for octanol-water are P_ow(HA)@1.3x10⁵ and P_ow(A)@30. The major difference is in the distribution of ionized PCP, which is several hundred times greater for egg-PC membranes compared to octanol. The difference is associated with the properties of the membrane-water interface.

Biophysical Journal 59, 1064-1073 (1991)

DOMAINS AND ANOMALOUS ADSORPTION ISOTHERMS OF DIPALMITOYLPHOSPHATIDYLCHOLINE MEMBRANES AND LIPOPHILIC IONS: PENTACHLOROPHENOLATE, TETRAPHENYLBORATE, AND DIPICRYLAMINE

Pavel Smejtek and Shanru Wang; Department of Physics and Environmental Sciences and Resources Doctoral Program, Portland State University, Portland, Oregon 97207-0715 USA

Dipalmitoylphosphatidylcholine (DPPC) vesicles acquire negative surface charge on adsorption of negatively charged pentachlorophenolate (PCP^-), and lipophilic ions tetraphenylborate (TPhB^-), and dipicrylamine (DPA^-). We have obtained (a) z-potential isotherms from the measurements of electrophoretic mobility of DPPC vesicles as a function of concentration of the adsorbing ions at different temperatures (25-42⁰C), and (b) studied the effect of PCP^- on gel-to-fluid phase transition by measuring the temperature dependence of z-potential at different PCP^- concentrations. The z-potential isotherms of PCP^- at 25, 32, and 34^°C correspond to adsorption to membrane in its gel phase. At 42^°C the z-potential isotherm corresponds to membrane in its fluid phase. These isotherms are well described by a Langmuir-Stern-Grahame adsorption model proposed by McLaughlin and Harary (1977. Biochemistry. 15:1941-1948). The z-potentiaI isotherm at 37°C does not follow the single-phase adsorption model. We have also observed anomalous adsorption isotherms for lipophilic ions TPhB^- and DPA^- at temperatures as low as 25^°C. These isotherms demonstrate a gel-to-fluid phase transition driven by ion adsorption to DPPC membrane during which the membrane changes from weakly to a strongly adsorbing state. The anomalous isotherm of PCP^- and the temperature dependence of z-potential can be described by a two-phase model based on the combination of (a) Langmuir-Stern-Grahame model for each phase, (b) the coexistence of gel and fluid domains, and (c) depression of gel-to-fluid phase transition temperature by PCP^-. Within the anomalous region the magnitude of z-potential rapidly increases with increasing concentration of adsorbing species, which was characterized in terms of an Esin-Markov coefficient. This effect can be exploited in membrane-based devices. Comments are also made on the possible effect of PCP, as an uncoupler, in energy transducing membranes.

Biophysical Journal, 58, 1285-1294 (1990)

ADSORPTION TO DIPALMITOYLPHOSPHATIDYLCHOLINE MEMBRANES IN GEL AND FLUID STATE: PENTACHLOROPHENOLATE, DIPICRYLAMINE, AND TETRAPHENYLBORATE

Pavel Smejtek and Shanru Wang; Department of Physics and Environmental Sciences and Resources Doctoral Program, Portland State University, Portland, Oregon 97207-0715 USA

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^°C to determine adsorption properties of DPPC membrane in the gel and fluid state, respectively. From the analysis of z-potential isotherms in terms of Langmuir-Stern-Grahame model we obtained the association constant, K, the area of the adsorption site, P_s,and the linear partition coefficient, b.

Results: K, (x10⁴M¹):

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);

P_s (nm²):

P_s(gel): PCP (5.4 ± 2.3), DPA (5.9 ± 2), TPhB (5.0 ± 1.7);

P_s(fluid): PCP (4.5 ± 0.4), DPA (5.2 ± 0.4), TPhB (4.1 ± 0.2);

b, (x10⁵m):

b (gel): PCP (0.15 ± 0.09), DPA (7.1 ± 0.3), TPhB (10 ± 7);

b (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 DDG⁰ = DG⁰(gel)- DG⁰(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.

Biochimica et Biophysica Acta 1029, 259-266 (1990)

ADSORPTION OF AMINOPYRIDINES TO PHOSPHATIDYLSERINE MEMBRANES

P. Smejtek¹,W.K. Riker², C. Wright¹ and M.J. Bennett³; ¹Department of Physics and Environmental Sciences and Resources Program. Portland State University. ²Department of Pharmacology, and ³Department of Anesthesiology, Oregon Health Sciences University, Portland, OR (USA.)

Aminopyridines belong to the class of compounds which facilitate synaptic transmission at low calcium concentration, an effect associated with the block of K+ channels, enhanced entry of calcium into presynaptic terminals and greater release of transmitter. We have measured the zeta-potential of phosphatidylserine vesicles in the presence of aminopyridines and some related compounds in order to relate the strength of association of the aminopyridines with their biological effectiveness. The dependence of zeta-potential on the concentration of aminopyridines was analyzed in terms of the Langmuir-Stern-Grahame adsorption model. The rank order of the association constants (in M^-1) obtained in the study was as follows:

3,4-diaminopyridine (6.5),

4,5-diaminopyrimidine (3.8),

4-aminopyridine (2.6),

3-aminopyridine (1.8),

2-aminopyridine (1.6),

4-dimethylaminopyridine (0.5),

4-aminopyridine methiodide (0.2), and, as control,

calcium (12.1).

The comparison of association constants with published results of the electric potential maps obtained by the CNDO/2 method suggests that binding to phosphatidylserine membrane increases with the density of excess charge on the protonated aminopyridine ring. We find that the sequence of potencies of aminopyridines in blocking K + channels, in releasing transmitter, and in the shifts of calcium concentration dependence of synaptic transmission are about the same as the sequence of association constants with the phosphatidylserine membrane. Assuming that the binding domain for aminopyridines in the presynaptic terminal has similar adsorption properties as the phosphatidylserine membrane, we estimate the electric potential difference between the domain and the external solution to be between -300 and -340 mV.

Chemical-Biological Interactions, 71, 37-61 (1989)

PENTACHLOROPHENOL-INDUCED CHANGE OF
z-POTENTIAL AND GEL-TO-FLUID TRANSITION TEMPERATURE IN MODEL LECITHIN MEMBRANES

Pavel Smejtek, Arthur William Barstad and Shanru Wang; Environmental Sciences and Resources Doctoral Program and Department of Physics, Portland State University, Portland, OR 97207-0751 (U.S.A.)

We have determined z-potentials for dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) membranes by measuring the electrophoretic mobility of multilayered vesicles and the temperatures of the gel-to-ripple-to-fluid phase transitions of sonicated vesicles by a photometric method. Some conclusions are: (1) The z-potentials of DMPC and DPPC vesicles become negative due to adsorption of ionized pentachlorophenol (PCP), (2) their magnitude changes, step-like, on gel-to-fluid transition and (3) the temperature of the step-like change in z-potential decreases with an increase in PCP concentration. (4) PCP exhibits a large effect on membrane structure: It induces an isothermal phase change from the ordered to disordered state, which is enhanced by monovalent salt in the aqueous phase. (5) Both ionized and unionized PCP decrease the melting phase transition temperature and abolish the pre-transition, (6) the unionized species increases the melting transition width and (7) the ionized species is more potent in abolishing the pre-transition. (8) The shorter chain lipid (DMPC) is more sensitive to the presence of PCP; the maximum decrease in DT_t is 13 K (DMPC) and 7 K (DPPC) in the presence of ionized PCP. We have shown experimentally, by comparing the DT_t from photometric studies with the density of adsorbed PCP derived from z-potential isotherms, that (9) the shift of the melting phase transition temperature increases linearly with the density of adsorbed PCP. (10) In contrast to membranes made of negatively charged lipids, the transition temperature of DMPC and DPPC membranes in the presence of PCP further decreases in the presence of monovalent salt. The salt effect is due to screening of the membrane surface leading to enhanced adsorption of ionized PCP and a depression in transition temperature. (11) It is shown that both the adsorption and the changes of gel-to-fluid phase transition temperature can be described in terms of the Langmuir-Stern-Grahame model and (12) proposed that future studies of membrane toxicity of PCP should be focused on its pH dependence.

Journal of Membrane Science, 33, 249-268 (1987)

THE PHYSICOCHEMICAL BASIS OF THE MEMBRANE TOXICITY OF PENTACHLOROPHENOL: AN OVERVIEW

Pavel Smejtek; Portland State University, Department of Physics and Environmental Sciences and Resources Doctoral Program, P.O. Box 751, Portland, OR 97207 (U.S.A.)

This report is concerned with the phenomenon of the membrane toxicity of pentachlorophenol (PCP ) - a popular herbicide and wood preservative, and now one of the most widespread pollutants. We compare experimental data on membrane-PCP interactions from multiple studies. These data include membrane electrical conductivity, PCP toxicity, microelectrophoresis, and spectrophotometry. The membrane toxicity of PCP is associated with the PCP-induced hydrogen ion permeability of the lipid matrix of biomembranes. The onset of the toxic effect corresponds to the loss of membrane electrical resistance and the onset of measurable PCP adsorption. We show that electrophoresis of lipid vesicles can be effectively used to study PCP adsorption and that the PCP-membrane interaction can be described in terms of the Langmuir-Stern-Grahame model. We report how the solvatochromic shifts of the long wavelength UV absorption band of ionized PCP can be used to characterize the polarity of the PCP adsorption site on membranes. The dielectric constant of the site in phosphatidylcholine (PC) and in negatively charged phosphatidylglycerol (PG) membranes was found to be 8 and 20. The pK_a of the dissociation of membrane-bound PCP is different from that in water (4.74). The largest pK_a (6.7) was observed for PCP bound to negatively charged membranes. It was shown that when the membrane surface potential was taken into account, the intrinsic pK_as for the PC and PG membranes are approximately the same (5.1-5.6). This work illustrates the complementarity of studies done on lipid bilayer membranes and biological membranes.

Biochimica et Biophysica Acta 905, 213-221 (1987)

ADSORPTION OF IONIZED AND NEUTRAL PENTACHLOROPHENOL TO PHOSPHATIDYLCHOLINE MEMBRANES

Pavel Smejtek, Shanru Wang and Arthur W. Barstad; Environmental Sciences and Resources Doctoral Program and Department of Physics, Portland State University, Portland OR (US.A.)

We have studied adsorption of pentachlorophenol (PCP) to phosphatidylcholine (PC) membranes by measuring the electrophoretic mobility of multilayered lipid vesicles in PCP solutions. PC vesicles become negatively charged due to the adsorption of ionized PCP, and we have found that their zeta potential depends upon the ionic strength and pH of the aqueous suspension. We have shown that the experimental results can be adequately accounted for in terms of a two-component Langmuir-Stern-Grahame adsorption model assuming that the ‘PCP adsorption sites’ are occupied either by the neutral (HA) or the ionized (A-) species. The characteristics of adsorption isotherms of the PCP - PC membrane are as follows: the association constants are K_A- = 55000 dm³/mol, K_HA = 279000 dm³/mol; 4.3 PC molecules make up each PCP adsorption site at saturation; the linear partition coefficients are b_HA = (15.5 ± 0.7) x 10^-5 m and b_A = (3.0 ± 0.3) x 10^-5 m. The properties of PCP adsorption isotherms for PC membranes predict an increased pK_a value of membrane-bound PCP, which has been observed in related studies.

Biochimica et Biophysica Acta 902, 109-127 (1987)

DIELECTRIC PROPERTIES OF ADSORPTION/IONIZATION SITE OF PENTACHLOROPHENOL IN LIPID MEMBRANES

Pavel Smejtek, Arthur W. Barstad and Kwan Hsu; Environmental Sciences and Resources Doctoral Program and Department of Physics, Portland State University. Portland, OR 97207 (U.S.A.)

The results of three complementary studies focused on characterization of the local environment of the common pesticide pentachlorophenol (PCP) adsorbed to phosphatidylcholine (PC) and phosphatidylglycerol (PG) membranes are reported. The effect of cholesterol (Chol) was examined. These studies included:

(1) Measurements of solvatochromic shifts of the ultraviolet absorption spectra of PCP in membranes and in polar non-hydrogen-bonding (a red shift) and hydrogen-bonding (a blue shift) solvents. p-p* transition energies were analyzed in terms of the dielectric cavity models of (a) Onsager, (b) Block-Walker, which includes dielectric saturation, and (c) a soft dipole model of Suppan, which accounts for PCP’s polarizability. The estimates of dielectric constant of the PCP adsorption site yielded 8.1-8.7 for the PC and 16.8-20.1 for PG membranes. Solvatochromic effects indicate hydrogen bonding between the membrane-bound ionized PCP molecule and water, which is enhanced by the presence of cholesterol.

(2) Determinations of the pK_a of PCP adsorbed to PC, PG, PC / Chol, PG / Chol membranes and dissolved in dioxane-water solutions of a known dielectric constant. The pK_a value of PCP adsorbed to membranes was always greater than the standard pK_a value and it increased with the membrane’s negative charge. The pK_a value sequence in 0.1 M KCI was 6.68 (PG), 6.32 (PG / Chol = 70:30 mole fractions), 5.97 (PC), and 5.75 (PC / Chol = 70:30). The intrinsic pK_a values of PCP in membranes were 5.2-5.4 (PG) and 5.5-6.0 (PC). Estimates of the dielectric constant of PCP’s ionization site in membranes yielded 10-22 (PC) and 27-37 (PG). Cholesterol facilitated the release of the hydrogen ion from membrane-bound PCP.

(3) Measurements of pH dependence of PCP-induced membrane electrical conductivity. pH values of conductivity maxima were always greater than the standard pK_a of PCP, and their sequence corresponded to that of the pK_a values of membrane-bound PCP. The anomalous properties of PCP as a Class 2 uncoupler are due to PCP’s lipophilic character. In response to a low dielectric constant of the adsorption / ionization site, the physicochemical characteristics of PCP adsorbed to membranes are different from the standard values - a fact that needs to be taken into account in the development of models of PCP’s toxicity.

Journal of Membrane Biology 71, 119-430 (1983)

EFFECT OF 3-PHENYLINDOLE ON LIPOPHILIC ION AND CARRIER-MEDIATED ION TRANSPORT ACROSS BILAYER LIPID MEMBRANES

Barbara A. Sinha and Pavel Smejtek; Department of Physics, Environmentat Sciences and Resources Program, Portland State University, Portland, Oregon 97207

The physical effects of 3-phenylindole, an antimicrobial compound which interacts with phospholipids, on ion transport across phosphatidylcholine-cholesterol bilayers have been investigated using three lipophilic ions and one ion-carrier complex. It was found that 3-phenylindole increased membrane electrical conductance of positively charged membrane probes and decreased electrical conductance of negatively charged probes. The enhancement of conductance detected by nonactin-K⁺ complex and tetraphenylarsonium⁺ was several orders of magnitude, whereas the suppression of conductance due to tetraphenylborate^- and dipicrylamine^- was less than a factor of ten. Presence of 3-phenylindole in aqueous phase slightly decreased adsorption of tetraphenylborate^- and dipicrylamine^- at the membrane surface. From the voltage dependence of the steady-state conductance it was shown that 3-phenylindole induced kinetic limitation of membrane transport of potassium mediated by nonactin. No such limitation was found in the case of tetraphenylarsonium⁺ transport. These results are shown to be consistent with the present concept of ion diffusion in membranes and the assumption that 3-phenylindole decreases the electric potential in the membrane interior. The asymmetry of the effect of 3-phenylindole on the magnitude of conductance changes for positively and negatively charged membrane permeable ions is also discussed as a reflection of the discreteness of both the adsorbed 3-phenylindole and lipid dipoles.

Pesticide Biochemistry and Physiology 18, 197-204 (1982)

INDUCED HYDROGEN ION TRANSPORT IN LIPID MEMBRANES AS ORIGIN OF TOXIC EFFECT OF PENTACHLOROPHENOL IN AN ALGA

Ranjith Jayaweera, Richard Petersen, and Pavel Smejtek; Department of Physics and Biology. Environmental Science and Resources Program, Portland State University, Portland, Oregon 97207

Pentachlorophenol (PCP) decreases the rate of carbon assimilation in the alga Selenastrum capricornutum. In parallel with the reduction of carbon assimilation in this alga there is a decrease of electrical resistance of lipid membranes and development of negative membrane surface charge. The experimental results suggest that PCP toxicity to algae is due to adsorption of negatively charged PCP ions at the membrane surface that act as carriers of hydrogen ion across the membrane. This protonophoretic action of PCP causes the decrease of membrane electrical resistance and the dissipation of hydrogen ion electrochemical potential gradients across cellular and subcellular membranes, which reduces the ability of algae to assimilate carbon.

Biophysical Journal 26, 441-466 (1979)

MODIFICATION OF ION TRANSPORT IN LIPID BILAYER MEMBRANES IN THE PRESENCE OF 2,4-DICHLOROPHENOXYACETIC ACID

I. ENHANCEMENT OF CATI0NIC CONDUCTANCE AND CHANGES OF THE KINETICS OF NONACTIN-MEDIATED TRANSPORT OF POTASSIUM

Pavel Smejtek and Mali Paulis-Illangasekare, Environmental Science Program, Department of Physics, Portland State University, Portland, Oregon 97207 U.S.A.

We have found that herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) has the ability to increase the rate of transport of positive ions of several kinds, and to inhibit transport of negatively charged tetraphenylborate ions in lipid bilayer membranes. It has been found that only the neutral form of 2,4-D is transport active, whereas the ionized form of 2,4-D does not modify transport of ions, and does not by itself permeate through lipid membranes. The results suggest that the enhancement of transport of positively charged ions such as tetraphenylarsonium⁺ and nonactin-K⁺ is dominated by the increase of the ion translocation rate constant. It has been shown that the enhancement of nonactin-mediated transport of K⁺ by 2,4-D can be accounted for by a simple carrier model. We have observed that at 2,4-D concentration above 3 X 10^-4 M the potassium ion transport in phosphatidylcholine-cholesterol as well as in cholesterol-free glycerolmonoo!eate membranes is enhanced to such a degree that, depending upon the concentration of potassium ions, it becomes limited by the rate of recombination of K⁺ with nonactin, and/or by backdiffusion of unloaded nonactin molecules. Furthermore, the effect of 2,4-D is enhanced by ionic strength of aqueous solution. From the changes of kinetic parameters of nonactin-K⁺ transport, as well as from the changes of membrane conductance due to tetraphenylarsonium⁺ ions, we have estimated the changes of the electrical potential of the membrane interior. We have found that the potential of the interior of the membrane becomes more negative in the presence of 2,4-D, and that its change is proportional to the aqueous concentration of 2,4-D. The effect of 2,4-D on ion transport has been attributed to a layer of 2,4-D molecules absorbed within the interfacial region, and having a dipole moment directed toward the aqueous medium. The results of kinetic studies of nonactin-K⁺ transport suggest that this layer is located on the hydrocarbon side of the interface.

Biophysical Journal 26, 467-488 (1979)

MODIFICATION OF ION TRANSPORT IN LIPId BILAYER MEMBRANES IN THE PRESENCE OF 2,4-DICHLOROPHENOXYACETIC ACID

II. SUPPRESSION OF TETRAPHENYLBORATE CONDUCTANCE AND CHANGES OF INTERFACIAL POTENTIALS

Pavel Smejtek and Mali Paulis-Illangasekare, Environmental Science Program, Department of Physics, Portland State University, Portland, Oregon 97207 U.S.A.

It has been shown that the blocking of negatively charged tetraphenylborate ion transport in phosphatidylcholine (PC)-cholesterol membranes by the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is dominated by the suppression of TPhB^- diffusion across the membrane interior, rather than by the decrease of adsorption of TPhB^- ions at the membrane surface. The blocking effect can be associated with the decrease of electric potential inside the membrane with respect to that of the aqueous medium, this decrease being proportional to the concentration of 2,4-D in the aqueous solution. It has been estimated that 25-30% of the total 2,4-D-induced change of the potential difference is between the plane of adsorption of TPhB^- and the aqueous solution, and the remaining fraction is between the membrane interior and the adsorption plane. The results of this study support the dipolar hypothesis of 2,4-D action in lipid membranes. These conclusions are further supported by measurements of changes of electric potential difference across air/water and air/lipid monolayer/water interfaces. It has been found that the electric potential of the nonpolar side of the interface decreases in the presence of neutral molecules of 2,4-D and that this effect becomes more prominent in the presence of electrolyte. We have confirmed that PC-cholesterol monolayer cannot be considered as a model for half of the bilayer membrane because of the disagreement between the changes of the interfacial potential difference of PC-cholesterol monolayers and those determined from studies of transport of positive and negative ions across bilayer membranes. In contrast, we have found close agreement between the 2,4-D-induced changes of electric potential of the lipid hydrocarbon region in glycerolmonooleate (GMO) membranes and GMO monolayers. We suggest that the action of 2,4-D in lipid membranes is not associated with the changes of orientation of dipoles of lipids constituting the membrane, but rather with a layer of 2,4-D molecules adsorbed at the nonpolar/polar membrane boundary.

Biophysical Journal 16, 319-336 (1976)

ELECTRICAL CONDUCTIVITY IN LIPID BILAYER MEMBRANES INDUCED BY PENTACHLOROPHENOL

Pavel Smejtek, Kwan Hsu, and William H. Perman, Environmental Science Program, Physics Department, Portland State University, Portland, Oregon 97207

Electrical conductivity induced in thin lipid bilayer membranes by penta-chlorophenol has been studied. The membranes were formed from phosphatidylcholine, phosphatidylethanolamine, or phosphatidylglycerol and various amounts of cholesterol. The position and the magnitude of the maximum of the conductivity vs. pH curve depend on the type of lipids and cholesterol content. At low pentachlorophenol concentrations and low pH the concentration dependence of conductivity is quadratic and becomes linear at higher pH. Above 10^-5 M of pentachlorophenol the concentration dependence of the membrane conductivity tends to saturate. Presence of pentachlorophenol enhances membrane transport of nonactin-K⁺ complex. Increase of cholesterol content increases pentachlorophenol induced conductivity in all membranes and shifts the conductivity toward lower pH. For phosphatidylcholine the largest rate of change of membrane conductivity with cholesterol occurs at 1:1 phospholipid to cholesterol molar ratio. Pentachlorophenol is found to be a class II uncoupler and the experimental results are consistent with the hypothesis that the membrane permeable species are dimers formed by combination of neutral and dissociated pentachlorophenol molecules. Several schemes of membrane conduction, including dimer formation in the aqueous phase as well as at the membrane-water interface have been considered. Arguments are given in favor of the formation of dimers within the membrane surface.

Chemistry and physics of lipids 13, 141-154 (1974)

SIMPLE MODEL OF ION TRANSPORT THROUGH ALAMETHICIN CHANNELS IN LIPID MEMBRANES

P. Smejtek, Department of Physics, Portland State University, Portland, Oregon 97207, USA

The electrical characteristics of wide membrane channels such as those induced in lipid membranes by alamethicin have been analyzed using an electrodiffusion model. The channel is considered to be a water filled cylinder in which the potential energy barrier is a result of the difference in polarization energy of the ion environment when the ion is located inside as compared to outside of the channel. In addition, an electric field related to the channel structure is assumed. It is shown that without postulating any specific chemical ion-channel interaction one can reproduce experimental membrane potentials for NaCl, KCl, and CaCl₂ concentration gradients with a single set of channel parameters. The calculations also yield experimental J-V characteristics of discrete conduction states. In addition, a simple mechanism of interchannel coupling based on the above model is discussed. The model suggests a unifying approach to the problem of the origin of interionic selectivity of membrane channels induced by polyene antibiotics.