Negative Surface Charge Density Research Articles

Adsorption of human beta 2-microglobulin at a water/mercury interface.

Adsorption of human beta 2-microglobulin from a neutral solution of 0.15 M NaCl on a mercury surface was studied at 25 degrees C by measurement of the differential capacity of the electrical double layer. From the diffusion-controlled adsorption kinetics, the surface concentration and hence the area occupied by the adsorbed beta 2-microglobulin molecule were determined at various potentials of the mercury surface. The results indicate unfolding or flattening of beta 2-microglobulin molecules adsorbed in particular on the electrically uncharged surface. The extent of this interfacial conformational rearrangement was reduced with growing positive or negative surface charge density.

Properties of the oxidizing site of Photosystem I

The bimolecular electron transfer from plastocyanin to P-700 + after flash excitation was investigated in inside-out vesicles from spinach chloroplast lamellae with randomly distributed photosystems and in vesicles from stroma membranes. Incubation of the vesicles with increasing amounts of Triton X-100 at room temperature revealed two opposing effects on the electron transfer rate. The maximum value of the rate constant which was 1.4 · 10 8 M −1 · s −1 in the absence of detergent, increased to the extremely high value of 5.3 · 10 8 M −1 · s −1 at 0.05% ( w v ) Triton X-100. However, at 1% ( w v ) Triton X-100 the rate constant dropped to the range of 10 5 M −1 · s −1. The effect of monovalent and divalent ions on the rate constant was used to characterize the electrostatic interaction between plastocyanin and the oxidizing site of Photosystem I under the following three conditions. (i) In intact membranes, the rate constant increased with increasing salt concentration as would be expected from the negative charges on both plastocyanin and Photosystem I. (ii) In the presence of 0.05% Triton X-100, the rate constant decreased with increasing salt concentrations, indicating two oppositely charged reactants. Extrapolation of the rate constant to infinite salt concentration gave a value of 2.5 · 10 8 M −1 · s −1 in the absence as well as in the presence of 0.05% Triton X-100. It is concluded that Triton X-100 solubilizes at this concentration a negatively charged component leaving a positively charged oxidizing site of Photosystem I, but does not change the local structure which is essential for efficient reaction with plastocyanin. (iii) In the presence of 1% Triton X-100, high cation concentrations (more than 0.5 M) were found to stimulate the severely inhibited rate constant to values of 10 6 M −1 · s −1. The effect of different cations indicates an about 10-times more negative surface charge density than in inside-out vesicles. The data are consistently described by a subunit which provides a positive charge at the highly negative oxidizing site on the Photosystem I reaction center protein, thereby facilitating the fast electron transfer from plastocyanin, and is removed by high Triton X-100 concentrations. The identity of the subunit and its occurrence in maize is discussed.

The negative surface charge density is a maturation marker of human B lymphocytes

Small resting B lymphocytes were highly enriched and completely depleted of all preactivated large B lymphocytes using countercurrent centrifugal elutriation and free flow electrophoresis. They required T lymphocytes, monocytes, and a mitogen to produce antibodies after 5 days of preincubation. Large activated B lymphocytes were obtained in cell fractions which were free of resting ones. They produced antibodies even in the absence of a mitogen. Two groups were distinguished, differing in their stage of differentiation and their negative surface charge density. The cells of one group had an electrophoretic mobility (EM) like resting B lymphocytes ranging from 0.85 to 0.99 × 10 −4 (cm 2V −1 s −1). They took 2 to 3 days of preincubation before they started to secrete antibodies. Interleukin 2 and pokeweed mitogen enhanced their antibody production capability. The cells of the other group had an EM between 0.99 and 1.13 × 10 −4 (cm 2 V −1 s −1). They secreted antibodies even during the first day of incubation. The quantity of the antibodies which they produced depended only on the blood donor. It could not be influenced by a mitogen or by interleukin 2. The study shows that large B lymphocytes with high negative surface charge density are in a later maturation stage than those with lower negative surface charge density.

Adhesion, Morphology, and Locomotion of Paramoeba pemaquidensis Page (Amoebida, Paramoebidae): Effects of Substrate Charge Density and External Cations1

ABSTRACTMorphology and locomotive behavior in the marine amoeba, Paramoeba pemaquidensis Page, was examined under different environmental conditions. Paramoeba requires a minimum surface negative charge density for adhesion of amoebae to substrata. Once adhesion to the substratum has been attained, however, surface negative charge density has no effect on morphology or locomotive rate. Divalent cations are not required for adhesion, but external calcium is required for normal locomotion. In the presence of calcium, Paramoeba often assumes a locomotive form with a broad, well‐developed anterior hyaline region and truncate posterior region. Locomotive forms vary from those with only a well‐developed hyaline region (Flabellula‐like) to forms with long digitiform sub‐pseudopodia (Vexillifera‐like), with intermediate morphotypes. Locomotive rates decrease and anteroposterior polarity disappears in the presence of living or heat‐killed bacteria, indicating that phagocytosis temporarily interferes with locomotion and alters form.

Negative surface charge density near heart calcium channels. Relevance to block by dihydropyridines.

We have measured the density of negative surface charges near the voltage sensor for inactivation gating of (L-type) Ca channels in intact calf Purkinje fibers and in isolated myocytes from guinea pig and rat ventricles. Divalent cation-induced changes in the half-maximal voltage for inactivation were determined and were well described by curves predicted by surface potential theory. We measured shifts in inactivation induced by Ca, Sr, and Ba in the single cells, and by Sr in the Purkinje fibers. All of the data were consistent with an estimated negative surface charge density of 1 electronic charge per 250 A2. In addition, the data suggest that Ca, but neither Ba nor Sr, binds to the negative charges with an association constant on the order of 1 M-1. We find that divalent ion-induced changes in surface potential can account for most of the antagonism between these ions and Ca channel block by 1,4-dihydropyridines.

Polarization Induced Instability in a Glass Passivated p‐n Junction

The polarization induced instability of a glass passivated p+‐n junction under bias‐temperature (BT) stress aging has been investigated by using a gate controlled diode (GCD) and a p+‐n diode. It is shown that positive and negative gate bias‐temperature (±GBT) stress aging causes the net surface charge density to shift symmetrically due to polarization in the glass layer. Plateau and stepped reverse current‐voltage characteristics, owing to excess positive and negative net surface charge densities, respectively, were observed. The subsequent junction bias‐temperature (JBT) stress aging on the GCD enhances the stepped reverse current‐voltage characteristics. Similar stepped current‐voltage characteristics can be found in a p+‐n diode without a gate electrode after JBT stress aging. Photoresponse data on these phenomenon are presented for the p+‐n diode after JBT stress aging, and a simple model is given to account for the polarization induced instability behavior of a glass passivated p+‐n junction.

Surface Properties of Right Side-Out Plasma Membrane Vesicles Isolated from Barley Roots and Leaves

Highly purified plasma membrane vesicles were obtained from roots and leaves of 7-day-old light-grown barley (Hordeum vulgare L. cv Kristina) seedlings by partitioning of crude microsomal fractions in a dextran-polyethylene glycol two-phase system. Sodium dodecylsulfate polyacrylamide gel electrophoresis showed the polypeptide composition of plasma membranes from the two organs to be qualitatively similar, but with different relative amounts of some of the polypeptides. Between 80 and 100% of the K(+),Mg(2+)-ATPase activity was latent indicating that the vesicles were sealed and right side-out. The isoelectric points of the outer surface of root and leaf plasma membranes as determined by cross-partitioning were similar and quite acidic-about pH 3.6. In contrast, the net negative surface charge density at pH 7.0 as measured by 9-aminoacridine fluorescence differed significantly, being -29 mC.m(-2) for the leaf plasma membrane and only -19 mC.m(-2) for the root plasma membrane. As isolated, both types of plasma membrane vesicles had Ca(2+) and Mg(2+) bound to the outer surface as shown by the combined use of chelators and 9-aminoacridine fluorescence; however, the leaf plasma membrane had a relatively higher proportion of Ca(2+) bound (0.57) than did the root plasma membrane (0.45). This difference probably reflects differences in the in vivo conditions as no chelator was present during the isolation procedure. Also Ni(2+) could bind to the root vesicles as indicated by the effect of Ni(2+) on 9-aminoacridine fluorescence, and by the binding of (63)Ni(2+) (44 nanomoles bound per milligram protein) at 100 micromolar NiCl(2).

Rheological Model of Pore Water

A rheological model of the flow of an aqueous electrolyte solution near a polar interface is presented, which is based on general equations of electrostatics and mechanics of an isotropic inhomogeneous continuum. The main purpose of the theory is to account for the consequences of an additional orientation of water molecules caused by the electrical fields of the double layer. Equations of the rheological model are merged with a modified Poisson‐Boltzmann equation in which hydration effects and dielectric saturation are included. Results of a numerical simulation of the flow of 10−5N KCl solution in capillary slits of varying widths between 104 and 10 nm, with negative surface charge densities assumed to be 6.7, 0.3, and 0.13 μC/cm2, under pressure gradients in the range of 104 to 109 Pa/m, are presented and discussed.

Voltage-dependent potassium channels in the molluscan neuron somatic membrane

Under voltage clamp conditions proof of the presence of two populations of potassium current channels was obtained on the molluscan neuron somatic membrane: inactivated and uninactivated. They differ from each other in their physicochemical characteristics, the property of their gating mechanisms, and the molecular structure of their current-conducting part. The inactivated potassium current is largely and selectively inhibited by cooling. Channels of the fast potassium current also are highly sensitive to temperature changes. By using parameters of gating mechanisms of the "fast" potassium channels included in the Hodgkin-Huxley model, the physicochemical properties of channels of this type were described. The density of fixd negative surface charges on the somatic membrane in the region of localization of fast potassium channels was estimated with the aid of the Gouy-Chapman theory. It is 0.3 electron charge/nm2. Data on the character of interaction of potassium channels with intracellular sodium ions revealed differences in the structure of the current-conducting part of different types of potassium channels. Experiments on intracellularly perfused molluscan neurons demonstrated the particular features of interaction between intracellular calcium ions and calcium-activated channels under conditions of strictly controlled changes in the intracellular calcium concentration.

Effect of trypsin treatment of bacteriorhodopsin on its orientation in reconstituted vesicles

The light-induced proton pump bacteriorhodopsin (bR) can be used to generate an electrochemical proton gradient (Ap) across the membrane of reconstituted vesicles, bR can be incorporated into membrane vesicles derived from the lactic acid bacterium Streptococcus eremoris by fusion of these bacterial membrane vesicles with bR-containing proteoliposomes (Driessen et al., 1985). For studies on the relationship between the Ap and solute transport (e.g. amino acids) in such model systems, a Ap, inside alkaline and negative, is required. Only one method has been described for the reconstitution of bR proteoliposomes that show light-dependent proton extrusion (Happe et al., 1977; Hellingwerf, 1982) and consequently generate a rightside-out-oriented Ap. These rightsideout bR proteoliposomes can be obtained by co-sonication of purple membranes together with phospholipid vesicles, containing acidic phospholipids using a short acidic stage (pH 2.4--3.0) during sonication. Although these preparations show light-dependent proton extrusion, a considerable part of the vesicles contains bR with an inside-out orientation. It has been suggested (Hellingwerf, 1982) that the distribution of charges over the two sides of bR is an important factor in determining the orientation of the protein in the reconstituted membrane. We therefore studied the effect of proteolytic treatment ofbR on the orientation of the protein in the reconstituted membrane. Under fully native conditions only the C-terminal part of the protein is accessible to proteolytic enzymes (e.g. trypsine). A peptide fragment is removed containing five carboxylic acid groups, negatively charged at neutral pH. Removal of this peptide results in a decrease of the negative surface charge density of the C-side (cytoplasmic side) of bR (Arrio et al., 1984) resulting in an extensive aggregation of the purple membrane sheets. Trypsin-treated bR was reconstituted into phospholipid vesicles, containing the negatively charged phospholipid cardiolipin, by the use of the rightside-out reconstitution procedure (Happe et al., 1977; Hellingwerf, 1982) at different pH values during the acid stage. An inversion of the direction of proton transloeation upon illumination can be observed when the pH of the acid stage of the reconstitution procedure is varied. This inflection point is around pH 3.0 for non-treated bR and about 3.5 for trypsin-treated bR. Trypsin-treated bR can be reconstituted in a rightside-out orientation with a higher efficiency, compared to non-treated bR. This is concluded from the following observations: (i) the extent of proton extrusion by trypsin-treated bR proteoliposomes is considerably higher than by non-treated bR, (ii) the proton extrusion extent is stimulated 1.2-2-fold by valinomycin and this is not observed in non-treated bR, (iii) tetraphenylphosphonium (TPP § is accumulated and tetraphenylboron (TPB) is extruded by proteoliposomes prepared with trypsintreated bR. Proteoliposomes prepared with native bR show some TPB- uptake, indicating the presence of proteoliposomes generating a Ap, interior positive and acid. These results support the mechanism proposed by Hellingwerf (1982) for the reconstitution of bR with an inside-out orientation in which the charge distribution over the two sides of bR plays an important role. The use of bR proteoliposomes with an inside-out orientation may be an important factor for a successful incorporation of bR into bacterial membrane vesicles, such that a Ap, inside negative and alkaline, is generated upon illumination.

Effects of Additives on ZnO ‐ B 2 O 3 ‐ SiO2 Passivation Glasses

The addition of , , or to glass A with composition (weight percent [w/o]) resulted in more negative surface charge density and increase in the thermal expansion coefficient (α). The glass B, showed finer crystals by the addition of (1 w/o) or glass A without affecting the surface charge density or α. The effects of additives on the DTA characteristic temperatures of the two glasses are also presented.

The Natural Higher Fatty Acid Soaps in Natural Rubber Latex and Their Effect on the Mechanical Stability of the Latex

Abstract The natural HFA soaps present in ammoniated NR latex concentrates are derived from the hydrolysis of the saponifiable lipids on the surfaces of the latex particles. A method based on cold extraction of the soaps has been developed for determining the natural HFA soaps in freshly concentrated latices. This method is better than the hot extraction method, which has been shown to cause some hydrolysis of the lipids. The natural HFA soaps in NR latex increase with time and reach a constant value in 3 to 6 weeks. The rate of increase of the soaps is divided into two linear regions and is more rapid in the first 1 to 2 weeks. More than 92% of the natural HFA soaps are adsorbed on the surfaces of the latex particles. This results in an increase in the negative electrical surface charge density of the particles and causes an increase in the mechanical stability of the latex. In fact, the increase in log MST is linearly proportional to the increase in natural HFA soaps in the latex. After the natural HFA soaps become constant, further increase in MST value is likely to be caused partly by the oxygen from the air.

The negative surface charge density of plasmalemma vesicles from wheat and oat roots

Plasmalemma vesicles were isolated in a sucrose-containing medium from wheat and oat roots and the net negative surface charge density was determined with 9-aminoacridine fluorescence [Chow, W.S. and Barber, J. (1980) J. Biochem. Biophys. Methods 3, 173-185]. The outer surface of the vesicles (measured in the presence of sucrose) had densities of − 16 to − 20 mC·m −2 and −29 mC·m −2 for wheat and oat roots, respectively. The inner surface - presumed to be the cytoplasmic side and calculated from the values measured in the presence and absence of sucrose - was more negative, and its size depended on the salt status of the roots.

Biorheological aspect in relation to endoendothblial fibrin lining

In connection with the findings of Copley et al. on the reduction of the apparent viscosity of blood, plasma and serum in contact with fibrin, a theoretical analysis of a Newtonian flow through a fibrin-coated tube was developed, taking into account the electric double layer between the fibrin layer and blood. The Stern model for the diffuse double layer was introduced. It is shown that the effect is governed by an “ electric double layer parameter” α, which is proportional to the term | σ + σ′ | ζ/R. ζ is the zeta potential, R is the radius of the tube, and σ is the negative surface charge density fixed on the fibrin layer, σ′ is the positive surface charge density within the immobile Stem layer. Our theory predicts that the apparent viscosity is increased or decreased according as the zeta potential is positive or negative.

Role of membrane lipids in the immunological killing of tumor cells: I. Target cell lipids.

The metabolic and physical properties of tumor cells that are associated with their ability to resist or escape from immune attack have been investigated. The susceptibility of P815 murine mastocytoma cells to immune killing can be modulated. Culturing the cells with adriamycin or with hydrocortisone increases or decreases, respectively, the sensitivity of the cells to killing by antibody (Ab) plus complement (C); in addition, culturing the cells with mitomycin C or hydrocortisone increases or decreases, respectively, the sensitivity of the cells to killing by cytotoxic T lymphocytes (CTL). The susceptibility of the cells to Ab-C killing correlates with the ability of the cells to synthesize complex cellular lipids, but not DNA, RNA, protein, or carbohydrate. Further, tumor cells rendered sensitive to Ab-C killing by adriamycin are decreased in total lipid content and in their cholesterol/phospholipid mole ratio; hydrocortisone-treated resistant cells showed the opposite effects. The ability of tumor cells to resist CTL killing did not correlate with their total cellular lipid synthesis, but did correlate with the synthesis and composition of specific cellular phospholipids. In addition, tumor cells increased in sensitivity to Ab-C killing exhibited an increase in cell surface membrane fluidity, whereas cells increased in susceptibility to CTL attack showed an increase in their net negative cell surface charge density. These data show certain unique chemical and physical properties of tumor cells to be of fundamental importance for their ability to resist either humoral or cell-mediated immunologic attack; modulation of one or another of these cellular properties results in a change in the cells' susceptibility to immune killing by antibody plus C or by cytotoxic T lymphocytes.

Bacteriophage P22 in vitro DNA packaging monitored by agarose gel electrophoresis: rate of DNA entry into capsids.

Bacteriophage P22, like other double-stranded DNA bacteriophages, packages DNA in a preassembled, DNA-free procapsid. The P22 procapsid and P22 bacteriophage have been electrophoretically characterized; the procapsid has a negative average electrical surface charge density (sigma) higher in magnitude than the negative sigma of the mature bacteriophage. Dextrans, sucrose, and maltose were shown to have a dramatic stimulatory effect on the in vitro packaging of DNA by the P22 procapsid. However, sedoheptulose, smaller sugars, and smaller polyols did not stimulate in vitro P22 DNA packaging. These and other data suggest that an osmotic pressure difference across some particle, probably a capsid, stimulates P22 DNA packaging. After in vitro packaging was optimized by including dextran 40 in extracts, the entry kinetics of DNA into P22 capsids were measured. Packaged DNA was detected by: (i) DNA-specific staining of intact capsids after fractionation by agarose gel electrophoresis and (ii) agarose gel electrophoresis of DNase-resistant DNA after release of DNase-resistant DNA from capsids. It was found that the first DNA was packaged by 1.5 min after the start of incubation. The data further suggest that either P22 capsids with DNA partially packaged in vitro are too unstable to be detected by the above procedures or entry of DNA into the capsid occurs in less than 0.25 min.

The application of a potential-sensitive cyanine dye to rat small intestinal brush border membrane vesicles

The sensitivity of the fluorescent dye, 3,3′-diethylthiadicarbocyanine (DiS-C 2(5)), was too low for the detection of membrane potential changes in rat small intestinal membrane vesicles. Only after adding LaCl 3 or after fractionation of the intestinal membranes by free-flow electrophoresis could the dye be used to monitor electrogenic Na +-dependent transport systems. It is concluded that the response of this potential-sensitive dye is influenced by the negative surface charge density of the vesicles.

Differing electrical surface charge and transplantation properties of genetically variant sublines of the TA3 murine adenocarcinoma tumor

Comparative measurements have been made of the transplantation and electrical surface charge properties of the near-diploid TA 3-Ha ascites tumor and a new hypotetraploid ascites subline designated TA 3-L. The negative surface charge density and the density of electrophoretically detectable sialic acid residues were determined to be twice as great for TA 3-L as TA 3-Ha cells. The two TA 3 sublines were found to have identical growth properties in isogeneic mice and to exhibit progressive growth in three allogeneic strains of mice. The lethal tumor cell inoculum in allogeneic mice was lower in all cases for the TA 3-L cells. From a comparison of several TA 3 ascites tumor sublines, it can be concluded that their transplantability into allogeneic strains of mice is correlated with the density of negative surface charge but is independent of the degree of tumor aneuploidy.

The Cell Surface Sulphydryl Content of Metastatic Variants of B16 Murine Melanoma

The cell surface sulphydryl content of three metastatic variants of the B16 murine melanoma has been determined using isoelectric equilibrium techniques. The F1 variant, which has no ability for natural metastasis, and the F10 variant with moderate metastatic ability appeared to have no detectable surface thiol groups. The variant BL6, which shows a high degree of natural metastasis, possessed surface thiol groups. The variants were found to be heterogeneous in isoelectric distribution. Three subpopulations were identified based on isoelectric criteria. The size of the pI 5.0 subpopulation appeared to increase with metastatic ability. A proportion of this subpopulation, approximately 4% in the F10 and 10% in the BL6, was found to possess surface thiol groups. In the BL6 line, 10-20% of the pI 4.6 subpopulation possessed surface thiol groups. The surface negative charge density of the cell types showed no correlation with their natural metastatic ability.

Membrane effects of vitamin E deficiency: bioenergetic and surface charge density studies of skeletal muscle and liver mitochondria.

Vitamin E deficiency in rats increased the sensitivity of liver and muscle mitochondria to damage during incubation at various temperatures, irradiation with visible light, or steady-state respiration with substrates. In all cases, vitamin E-deficient mitochondria exhibited increased lipid peroxidation, reduced transmembrane potential, decreased respiratory coupling, and lower rates of electron transport compared to control mitochondria. Muscle mitochondria always showed greater negative inner membrane surface charge density, and were also more sensitive to damage than were liver mitochondria. Vitamin E-deficient mitochondria also showed slightly more negative inner membrane surface charge density compared to controls. The relationship observed between greater negative surface potential and increased sensitivity to damage provides for a new and sensitive method to probe further the role of surface charge in membrane structure and function. Implications of these new findings for the well-known human muscle myopathies and those experimentally induced by vitamin E deficiency in animals are discussed.