Surface charge of mammalian neurones as revealed by microelectrophoresis

Summary The surface charge of isolated rat dorsal root ganglion neurones was studied by microelectrophoresis technique. The increase of Ca concentration caused greater reduction of the electrophoretic mobility compared to that produced by an equivalent amount of divalent organic cations, dimethonium or hexamethonium. No charge reversal for Ca concentrations up to 80 mM was observed. These data fit the suggestion that two anion groups of the outer membrane surface can bind one Ca ion with apparent binding constant of about 50 M^–1. In solutions of low pH the electrophoretic mobility of cells decreased corresponding to titration of acidic groups with apparent pK=4.2. Trypsin treatment in mild conditions markedly reduced the surface charge: however, neuraminidase and hyaluronidase did not change it. N-bromosuccinimide (a specific reagent for carboxylic groups of proteins) decreased the electrophoretic mobility about 60%. However, no increase of the surface charge after the action of specific reagents for amino groups (2,4,6-trinitrobenzenesulfonic acid and maleic anhydride) was observed. It was shown that the surface charge depends also on the intracellular metabolism. If 1 mM dibutyryl cAMP or theophilline was added to the culture medium (thus, raising the concentration of cAMP inside the cell) the surface charge increased. This effect developed slowly and reached its maximum on the third day of incubation. Treatment of cells by 5 mM tolbutamide (an inhibitor of some protein kinases) did not change cell mobility. Addition of 5 mM N-ethylmaleimide (an inhibitor of adenylate cyclase) to the culture medium produced some decrease of the surface charge. On the basis of data obtained it is suggested that the charge of the outer membrane surface of neurones studied is mainly determined by carboxylic groups of membrane proteins, and changes in intracellular cAMP concentration influence the synthesis and reconstruction of these membrane components.     

Investigation of surface properties of rat spinal ganglion neuron by microelectrophoresis

The surface charge of isolated neurons in rat spinal ganglia was studied by microelectrophoresis. The surface potential of these neurons was shown to be caused by anionic groups which form complexes with calcium ions with a binding constant of between 10 and 50 liters/mole, and to titrate with hydrogen ions in accordance with pK=3.8. After treatment with trypsin under "mild" conditions many of these groups are removed from the surface. Tosyl chloride (a reagent for amino groups) leads to a small increase, and N-bromosuccinimide (a reagent for carboxyl groups) leads to a marked decrease in surface charge. It is suggested that the surface charge of neurons in rat spinal ganglia, determined by microelectrophoresis, is due to carboxyl groups of peripheral proteins. These groups are evidently located in the glycoprotein layer covering the outer side of the membrane. According to estimates the distance between the groups is about 2 nm and the thickness of the glycoprotein layer is 10 nm.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSSR, Kiev. Translated from Neirofiziologiya, Vol. 16, No. 2, pp. 176–182, March–April, 1984.     

Characterisation of the surface of bovine milk fat globule membrane using microelectrophoresis

The nature of the ionogenic groups on the surface of the milk fat globule membrane was studied by microelectrophoresis of intact fat globules after chemical and enzymic modification. The changes in pH-mobility curves effected by formaldehyde and 2,4-dinitrofluorobenzene showed that the membrane surface contained amine groups. These were identified as arising from lysine and arginine by chromatography of their dinitrophenyl derivatives. The contribution of N-acetylneuraminic acid and phosphate to the surface charge was demonstrated by their specific removal by neuraminidase and phospholipase C, respectively. After removal of N-acetylneuraminic acid and phosphate, anionogenic effects remained which were attributed to protein carboxyl groups. These groups could be partially esterified using diazomethane. The effect of sodium dodecyl sulphate and of ionic strength on electrophoretic mobility indicated that the surface contains little neutral lipid and is predominantly ionogenic. The results obtained concerning the nature of the surface of the milk fat globule membrane support the hypothesis that the milk fat globule membrane originates from the plasmalemma of the mammary alveolar cell.     

Surface charges on chloroplast membranes as studied by particle electrophoresis.

1. Particle microelectrophoresis mobility studies have been conducted with chloroplast thylakoid membranes and with isolated intact chloroplasts. 2. The pH dependence of the electrophoretic mobility indicated that at pH values above 4.3 both membrane systems carry a net negative charge. 3. Chemical treatment of thylakoids has shown that neither the sugar residues of the galactolipids in the membrane nor the basic groups of the membrane proteins having pK values between 6 and 10 are exposed at the surface. 4. However, treatment with 1-ethyl-3(3-dimethylaminopropyl)carbodiimide, together with glycine methyl ester, neutralized the negative charges on the thylakoid membrane surface indicating the involvement of carboxyl groups which, because of their pH sensitivity, are likely to be the carboxyl groups of aspartic and glutamic acid residues. 5. The nature of the protein giving rise to the negative surface charges on the thylakoids is not known but is shown not to involve the coupling factor or the light harvesting chlorophyll a/chlorophyll b pigment . protein complex. 6. No significant effect of light was observed on the electrophoretic mobility of either thylakoids or intact chloroplasts. 7. The striking difference in the ability of divalent and monovalent cations to screen the surface charges was demonstrated and explained in terms of the Gouy-Chapman theory. 8. Calculations of the zeta-potentials for thylakoid membranes gave values for the charge density at the plane of shear to be in the region of one electronic charge per 1500--2000 A2. 9. The significance of the results is discussed in terms of cation distribution in chloroplasts and the effect of cations on photosynthetic phenomena.     

Sialic acid, electrophoretic mobility and transmembrane potentials of the Amphiuma red cell

Red cells from the giant salamander Amphiuma means are shown to contain sialic acid. The amount removed by the action of neuraminidase is equal to that released by acid hydrolysis, indicating that all of the sialic acid is present on the outer surface of the plasma membrane. These cells have a negative electrophoretic mobility and 100% enzymatic removal of sialic acid results in a 40% reduction in the mobility, suggesting that either a fraction of the sialic acid carboxyl groups are unavailable to the action of external electric fields, or other negatively charged groups contribute to the surface charge. A further reduction in mobility of normal and sialic acid-free cells is caused by an increased extracellular calcium concentration. The negative groups affected by calcium are most likely to be phosphate groups, since the isoelectric point of the cells is found to lie between the pK values for H₂PO₄⁻ groups and the carboxyl groups of sialic acid. Membrane potentials of single cells, from which 80% or more of the total sialic acid had been removed, were identical to those measured in normal cells, confirming that sialic acid plays little, if any, direct role in the maintenance of membrane potentials and ionic permeabilities.     

Characterization and Implications of the Cell Surface Reactivity of Calothrix sp. Strain KC97

The cell surface reactivity of the cyanobacterium Calothrix sp. strain KC97, an isolate from the Krisuvik hot spring, Iceland, was investigated in terms of its proton binding behavior and charge characteristics by using acid-base titrations, electrophoretic mobility analysis, and transmission electron microscopy. Analysis of titration data with the linear programming optimization method showed that intact filaments were dominated by surface proton binding sites inferred to be carboxyl groups (acid dissociation constants [pK_a] between 5.0 and 6.2) and amine groups (mean pK_a of 8.9). Sheath material isolated by using lysozyme and sodium dodecyl sulfate generated pK_a spectra similarly dominated by carboxyls (pK_a of 4.6 to 6.1) and amines (pK_a of 8.1 to 9.2). In both intact filaments and isolated sheath material, the lower ligand concentrations at mid-pK_a values were ascribed to phosphoryl groups. Whole filaments and isolated sheath material displayed total reactive-site densities of 80.3 × 10⁻⁵ and 12.3 × 10⁻⁵ mol/g (dry mass) of cyanobacteria, respectively, implying that much of the surface reactivity of this microorganism is located on the cell wall and not the sheath. This is corroborated by electrophoretic mobility measurements that showed that the sheath has a net neutral charge at mid-pHs. In contrast, unsheathed cells exhibited a stronger negative-charge characteristic. Additionally, transmission electron microscopy analysis of ultrathin sections stained with heavy metals further demonstrated that most of the reactive binding sites are located upon the cell wall. Thus, the cell surface reactivity of Calothrix sp. strain KC97 can be described as a dual layer composed of a highly reactive cell wall enclosed within a poorly reactive sheath.     

Some properties of the chloroplast envelope as revealed by electrophoretic mobility studies of intact chloroplasts

The electrophoretic mobility of mature spinach (Spinacia oleracea L. var. Americana) chloroplasts sampled over a 7-month period was between −2.03 and −2.45 micrometers per second per volt per centimeter when suspended in a solution containing 1 millimolar CaCl₂. The surface charge density of EDTA-treated chloroplasts was calculated to be −7,400 electrostatic units per square centimeter representing, on the average, one electronic charge per 645 square Angstroms. Electrophoretic mobility increases during plastid maturation. Calcium, but not magnesium, generally stabilized the envelope of isolated plastids against small increases in surface charge that occur with time in the absence of calcium. Pronase caused a sharp, but temporary, decrease in the electrophoretic mobility of chloroplasts. This was interpreted as representing a transient binding of pronase to the envelope surface during proteolysis. No −SH groups were detected on the surface of the plastid envelope. Inasmuch as the isoelectric point of intact chloroplasts was found to be at pH 4.5, it is likely that the major part of the total surface charge results from the presence of exposed carboxyl groups of intrinsic envelope proteins that are not readily hydrolyzed by mild pronase treatment.     

Surface charges on chloroplast membranes as studied by particle electrophoresis

1. Particle microelectrophoresis mobility studies have been conducted with chloroplast thylakoid membranes and with isolated intact chloroplasts.2. The pH dependence of the electrophoretic mobility indicated that at pH values above 4.3 both membrane systems carry a net negative charge.3. Chemical treatment of thylakoids has shown that neither the sugar residues of the galactolipids in the membrane nor the basic groups of the membrane proteins having pK values between 6 and 10 are exposed at the surface.4. However, treatment with 1-ethyl-3(3-dimethylaminopropyl)carbodiimide, together with glycine methyl ester, neutralized the negative charges on the thylakoid membrane surface indicating the involvement of carboxyl groups which, because of their pH sensitivity, are likely to be the carboxyl groups of aspartic and glutamic acid residues.5. The nature of the protein giving rise to the negative surface charges on the thylakoids is not known but is shown not to involve the coupling factor or the light harvesting $\text{chlorophyl}\text{a}\text{chlorophyll}\text{b}\text{pigment · protein complex}$.6. No significant effect of light was observed on the electrophoretic mobility of either thylakoids or intact chloroplasts.7. The striking difference in the ability of divalent and monovalent cations to screen the surface charges was demonstrated and explained in terms of the Gouy-Chapman theory.8. Calculations of the ζ-potentials for thylakoid membranes gave values for the charge density at the plane of shear to be in the region of one electronic charge per 1500–2000 Ų.9. The significance of the results is discussed in terms of cation distribution in chloroplasts and the effect of cations on photosynthetic phenomena.     

Seasonal activity of frog erythrocytes by data of electrophoretic mobility

The peculiarities of frog erythrocyte electrophoretic mobility, coupled to the seasonal course of temperatures, have been studied. At the periods of anabiosis and of burst of hemopoiesis, in the vascular bed there increases the portion of functionally young erythrocytes (up to 22%) with increased values of the cell membrane surface charge. Preparation to winter is accompanied by a rise of the number of circulating functionally worn-down blood cells (up to 60%) with low values of the superficial charge and low mobility in electrical field. Use of the cell microelectrophoresis method of evaluation of seasonal activity of frog erythrocytes allows obtaining objective data about the cellular surface charge and its depending functional cell activity without submitting the erythrocytes to modifying actions.     

Surface charge of eosinophils. Binding of cationic particles and measurement of cellular electrophoretic mobility

Summary The surface charge of eosinophils, isolated from the peritoneal exudate of rats by the use of a Metrizamide gradient, was analysed by ultrastructural cytochemistry and cellular electrophoretic mobility. Binding of colloidal iron hydroxide and of cationized ferritin particles at pH 1.8 and 7.2 respectively, was observed on the surface of the eosinophils. An electrophoretic mobility of –1.08 and –1.39 m·s^–1·V^–1·cm was determined for living and glutaraldehyde-fixed eosinophils, respectively. Treatment of the cells with neuraminidase reduced the electrophoretic mobility to –0.64 m·s^–1·V^–1·cm (glutaraldehyde-fixed), reduced significantly and abolished completely the binding of both colloidal iron hydroxide and cationized ferritin particles to the surface of the cells. These results indicate that sialic acid exists on the surface of eosinophils, where it accounts for part of the negative surface charge.     

Salt-induced pH changes in spinach chloroplast suspension. Changes in surface potential and surface pH of thylakoid membranes

A pH decrease in chloroplast suspension in media of low salt concentration was observed when a salt was added at pH values higher than 4.4, while at lower pH values a pH increase was observed. The salt-induced pH changes depended on the valence and concentration of cations of added salts at neutral pH values (higher than 4.4) and on those of anions at acidic pH values (lower than 4.4). The order of effectiveness was trivalent > divalent > monovalent. The pH value change by salt addition was affected by the presence of ionic detergents depending on the sign of their charges. These characteristics agreed with those expected from the Gouy-Chapman theory on diffuse electrical double layers. The results were interpreted in terms of the changes in surface potential, surface pH and the ionization of surface groups which result in the release (or binding) of H⁺ to (or from) the outer medium.The analysis of the data of KCl-induced pH change suggests that the change in the surface charge density of thylakoid membranes depends mainly on the ionization of carboxyl groups, which is determined by the surface pH. When the carboxyl groups are fully dissociated, the surface charge density reaches ?1.0 ± 0.1 · 10^?3 elementary charge/square Å.Dependence of the estimated surface potential on the bulk pH was similar to that of electrophoretic mobility of thylakoid membrane vesicles.     

The Surface Charge of Isolated Toad Bladder Epithelial Cells : Mobility, effect of pH and divalent ions

The surface charge of epithelial cells isolated from the toad bladder has been determined by the microscope method of cell electrophoresis. The cells possess a net negative charge, and a net surface charge density of 3.6 x 10⁴ electronic charges per square micron at pH 7.3. Estimates of net surface charge over the alkaline pH range indicate (a) that an average distance of the order of 40 A separates the negatively charged groups, and (b) that amino as well as acid groups are present at the electrophoretic surface of shear. A significant increase in mobility following cyanate treatment of the cells suggests that a large proportion of the amino groups are the ε-amino groups of lysine. In view of the known effects of calcium and other divalent ions on cell permeability and cell adhesion, the extent of binding of calcium and magnesium to the cell surface was determined by the electrophoretic technique. Mobility was significantly decreased in the presence of calcium or magnesium, indicating that these ions are bound by surface groups. When the pH was lowered from 7.3 to 5.2, calcium binding was markedly decreased, an observation consistent with competition between calcium and hydrogen ions for a common receptor site.     

Recurrent decreases of erythrocyte electrophoretic mobility in rats exposed to 1.0 R whole-body X-radiation

Circulating erythrocytes from rats were examined up to 30 weeks post whole-body exposures of 1.0 R for alterations in the expression of net negative surface charge as measured by whole-cell microelectrophoresis in saline sorbitol. Erythrocyte electrophoretic mobility was determined in an apparatus composed of a horizontal transilluminated cylindrical chamber, equipped with a reversible, blacked platinum electrode, immersed in a water bath maintained at 25.0±0.1°C (Rank Brothers). In two separate experiments, recurrent decreases in the expression of net negative surface charge occurred at 10, 17, and 30 weeks post-irradiation. At these times distribution analyses of recorded erythrocyte electrophoretic mobility (EEPM) values revealed a skewing of the normally distributed EEPM population values to lower EEPM. Total sialic acid content released from hydrolyzed erythrocyte membrane preparations revealed no significant differences between erythrocytes from sham and irradiated animals. In vivo post-irradiation labeling of erythrocytes with diisopropyl-[³²P] phosphorofluoridate at 4 and 33 weeks (separate experiments) indicated only a minor abbreviated erythrocyte life span at 33 weeks. Therefore, effects from low dose (1.0 R) whole-body irradiation would appear to include a recurrent defect in the expression of the net negative surface charge.     

Adsorption equilibria between liposome membrane formed of phosphatidylcholine and aqueous sodium chloride solution as a function of pH

The effect has been studied of the adsorption of ions (H(+), Na(+), OH(-), Cl(-)) which are present in solution upon the electric charge of the liposome membrane formed of phosphatidylcholine (PC). The surface charge density of the membrane was determined as a function of pH and electrolyte concentration from electrophoretic mobility measurements. The measurements were carried out by the laser-Doppler microelectrophoresis method. A four-equilibria model has been proposed to describe the phenomena occurring on the membrane surface. The equilibria in which the adsorption of other ions on the liposome membrane surface was involved were assumed to exist beside the equilibria in which the H(+) and OH(-) ions were engaged. The idea was confirmed by mathematical calculations. Association constants of the liposome membrane surface with ions of solution (K(AH), K(ANa), K(BOH), K(BCl)) were determined. The proposed model has been proved to be correct by comparing the resulting theoretic charge variation curves of the lecithin membrane with the experimental data.     

Surface potential and surface charge density of the cerebral-cortex synaptic vesicle and stability of vesicle suspension

Using a microelectrophoresis instrument employing the Lazer-Zee system, the electrophoretic mobility of synaptic vesicles isolated from Guinea-pig brain cortex was measured under several conditions. The mobility was found to depend on both pH and ionic concentration of the solution. The surface of the synaptic vesicle was shown to be negatively charged under physiological conditions. The isoelectric point was observed at pH 4.0 in 0.01 M NaCl solution. Effects of divalent cations were examined and reversal of surface charge was observed in 0.1 M CaCl₂ solution. Interaction of vesicles was also considered on the basis of the DLVO theory of colloid stability by using calculated values of surface charge density and surface potential of the synaptic vesicle.     

Recurrent decreases of erythrocyte electrophoretic mobility in rats exposed to 1.0 R whole-body x-radiation

Circulating erythrocytes from rats were examined up to 30 weeks post whole-body exposures of 1.0 R for alterations in the expression of net negative surface charge as measured by whole-cell microelectrophoresis in saline sorbitol. Erythrocyte electrophoretic mobility was determined in an apparatus composed of a horizontal transilluminated cylindrical chamber, equipped with a reversible, blacked platinum electrode, immersed in a water bath maintained at 25.0 +/- 1.0 degree C (Rank Brothers). In two separate experiments, recurrent decreases in the expression of net negative surface charge occurred at 10, 17, and 30 weeks post-irradiation. At these times distributional analyses of recorded erythrocyte electrophoretic mobility (EEPM) values revealed a skewing of the normally distributed EEPM population values to lower EEPM. Total sialic acid content released from hydrolyzed erythrocyte membrane preparations revealed no significant differences between erythrocytes from sham and irradiated animals. In vivo post-irradiation labeling of erythrocytes with diisopropyl-[32P] phosphorofluoridate at 4 and 33 weeks (separate experiments) indicated only a minor abbreviated erythrocyte life span at 33 weeks. Therefore, effects from low dose (1.0 R) whole-body irradiation would appear to include a recurrent defect in the expression of the net negative surface charge.     

Potential-dependent interaction of chemical reagents with the gating mechanism of the nerve fiber sodium channel

The action of two water-soluble carbodiimides and of Woodward's reagent K on the properties of the gating mechanism of sodium channels of the Ranvier node membrane was investigated. Treatment with carbodiimide solution (pH 4.8–5.2) at a potential of –80 to –100 mV was shown to delay activation and inactivation of the channels considerably and to reduce the sensitivity of both gating functions to changes in membrane potential. The effective activation charge, determined relative to the limiting logarithmic slope of the activation curve (z_ef) was reduced by 1.7 times. Treatment of the membrane under the same conditions, but at zero holding potential, induced much smaller changes in properties of the gating mechanism; under these conditions z_ef remained unchanged. Woodward's reagent at high negative potential induced the same changes in the gating system as carbodiimide at 0 mV. The action of Woodward's reagent also depended on potential, but by a lesser degree than when carbodiimides were used. The results suggest that two types of carboxyl groups exist on the outer surface of the membrane: "mobile," which perform the role of gating particles and which are moved from the surface when the channel changes into the open or inactivated state, and "immobile," which face the external solution whatever the state of the channel.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 16, No. 5, pp. 577–590, September–October, 1984.     

Sialic acid, electrophoretic mobility and transmembrane potentials of the Amphiuma red cell.

Red cells from the giant salamander Amphiuma means are shown to contain sialic acid. The amount removed by the action of neuraminidase is equal to that released by acid hydrolysis, indicating that all of the sialic acid is present on the outer surface of the plasma membrane. These cells have a negative electrophoretic mobility and 100% enzymatic removal of sialic acid results in a 40% reduction in the mobility, suggesting that either a fraction of the sialic acid carboxyl groups are unavailable to the action of external electric fields, or other negatively charged groups contribute to the surface charge. A further reduction in mobility of normal and sialic acid-free cells is caused by an increased extracellular calcium concentration. The negative groups affected by calcium are most likely to be phosphate groups, since the isoelectric point of the cells is found to lie between the pK values for H2PO-4 groups and the carboxyl groups of sialic acid. Membrane potentials of single cells, from which 80% or more of the total sialic acid had been removed, were identical to those measured in normal cells, confirming that sialic acid plays little, if any, direct role in the maintenance of membrane potentials and ionic permeabilities.     

Electrophoretic mobility of mouse cells and homologous isolated nuclei

The electrophoretic mobilities of Ehrlich ascites, sarcoma 37 ascites, mouse liver cells and their isolated nuclei were measured under similar environmental conditions. No differences in mobility were detected between cells and homologous nuclei from the same cell population and it was concluded that their surface charge densities were probably the same. The effect of neuraminidase on Ehrlich ascites and liver cells and nuclei was also determined; neuraminidase reduced the mobility of Ehrlich ascites cell nuclei as well as cells. The reduction in mobility of cells and nuclei prepared by a sucrose method was the same; however, the reduction in mobility of citric acid prepared nuclei was less than that of citric acid treated cells. The reduction in mobility of both liver cells and nuclei was small or insignificant. It is suggested that although cells and nuclei have similar electrophoretic mobilities, possibly different groups contribute to their surface charge.     

Translocation of hyaluronic acid in cell surface of cultured mammalian cells after X-irradiation and its recovery by added adenosine triphosphate

To investigate the mechanism of radiation-induced decrease in cell electrophoretic mobility and its recovery by added adenosine triphosphate, specific enzymes and buffer solutions of different ionic strength were utilized. Decrease in the mobility of irradiated cells was detected only with the buffer solution of ionic strengths higher than 0.100. In this range of ionic strengths, removal of hyaluronic acid from cell surface by hyaluronidase had no effect on the electrophoretic mobility of irradiated cells, while the enzyme treatment resulted in 27% mobility reduction in non-irradiated cells. The removal of sialic acid and chondroitin sulfate by their specific enzymes resulted in the similar decrease in mobility either in irradiated and non-irradiated cells. These results suggest that the X-ray induced translocation of hyaluronic acid from the peripheral zone of 0–7.5 Å into the deeper zone of about 10–17 Å, if we use the Debye-Hückel's thickness of ion atmosphere for an approximate estimate of effective depth of electrokinetic plane of shear. Hyaluronic acid reappeared to the peripheral zone by the subsequent incubation after small dose irradiation, or by the addition of 1 mM adenosine triphosphate with Ca²⁺.