Cell surface charge. Correlation of partition with electrophoresis

Critical mixtures of aqueous solutions os polymers separate into two or more immiscible phases. Particulate materials distribute in such phase systems generally between one bulk phase and the interface between bulk phases. The distribution is described by a simple partition law, and is qunatitatively determined by, inter alia, the nature of the particle surface, particularly net electrical charge. The partition behaviour of various cells, native or modified by treatment with trypsin, neurominidase or maleic anhydride, correlate strongly with electrophoretic mobility. Partition behaviour and electrophoretic mobility are both dependent upon cell surface charge. Thus, in appropriate conditions, changes in surface charge may be registered as changes in partition.     

Free-flow electrophoresis for purification of plant mitochondria by surface charge

Sample purity is the key for a successful in-depth analysis of any given subcellular proteome. The suitability of free-flow electrophoresis to assist conventional, centrifugation-based techniques in the preparation of plant mitochondria from green and non-green tissue was assessed by various means, including functional assays, immunoblots, electron microscopy and differential gel electrophoresis. Results indicated a significant increase in purity of the mitochondrial samples, highlighted specific contaminants previously reported as mitochondrial proteins, and also pointed to new means for separating plastids and peroxisomes from mitochondria in plant organellar extracts by exploiting differences in surface charge. This approach has the potential to allow a deeper and more comprehensive investigation of the Arabidopsis organellar proteomes, by providing a second dimension of separation based on surface charge in addition to conventional centrifugation purification protocols relying on size and density.     

Quantitative measurement of lipoprotein surface charge by agarose gel electrophoresis.

The electrophoretic mobilities of low density lipoprotein (LDL) and six pure proteins in a 0.5% agarose gel have been compared to literature electrophoretic mobility values determined by the Tiselius moving boundary method. There is a strong correlation (r = 0.99) between the electrophoretic mobilities determined by the two techniques. The electrophoretic behavior of charged particles smaller than very low density lipoproteins (VLDL) is not markedly perturbed by a 0.5% agarose matrix, and variations in mobility primarily reflect differences in particle valence and density of surface charge. Application of electrokinetic theory to derive protein and lipoprotein net charges from the electrophoretic mobilities in agarose yields a quantitative delineation of lipoprotein electrophoretic migration patterns wherein the beta mobility region comprises a surface potential range of -4.5 to -7.0 mV; the pre-beta region a range of -7.0 to -10.5 mV; the alpha mobility region a range of -10.5 to -12.5 mV and the serum albumin region a range of -12.5 to -14.0 mV. Because protein conformation and charge are critical in metabolic regulation, the agarose gel electrophoresis technique provides a valuable analytical tool that should help to elucidate further details of the structure-function relationships of serum lipoprotein particles.     

Changes of the cell surface charge of amphibian ectoderm after induction

Summary Isolated ectoderm of early gastrula stages ofTriturus alpestris was treated with vegetalizing factor for 24 h employing the sandwich method (induced ectoderm). Controls were incubated for the same period with -globulin which has no inducing activity. Explants of both series were labelled with cationized ferritin, which binds to negatively charged groups at physiological pH. In non-induced ectoderm, ferritin particles can be found as a thin layer all over the plasma membranes. In induced ectoderm the total amount of ferritin bound to the plasma membrane is much lower than in non-induced ectoderm. Ferritin is located in restricted areas only. In contrast to the controls, other membrane areas are free of ferritin particles. The correlation between these results and the change of cell affinity after induction with vegetalizing factor is discussed.     

Size-dependent scattering efficiency in dye-sensitized solar cell

Effect of scattering particle size on light scattering efficiency in dye-sensitized solar cell has been investigated with a FTO/semitransparent nano-TiO₂ layer (main-layer)/scattering layer (overlayer) structure, where two different rutile TiO₂ particles of 0.3 μm (G1) and 0.5 μm (G2) were used for a scattering overlayer and 20-nm anatase particle for nano-TiO₂ main-layer. The conversion efficiency of 7.55% for the 7 μm-thick main-layer film is improved to 8.94% and 8.78% when G1 and G2 particulate overlayers are introduced, respectively, corresponding to 18.4% and 16.3% increments. While the conversion efficiency of the 14 μm-thick main-layer is slightly improved from 8.60% to 9.09% and 9.15% upon depositing G1 and G2 particulate overlayers, respectively. Significant improvement and strong size-dependence upon deposition of scattering overlayer on the relatively thinner TiO₂ main-layer film are associated with the quantity and wavelength of transmitted light and the difference in reflectivity of G1 and G2 scattering particles.     

Enhancement of colloid uptake by tumor cell surface electrical charge modification

Colloidal [51Cr]chromic phosphate uptake is considerably increased by preincubation of P388 ascites leukemia cells with poly(DL-lysine). The uptake increase is in direct relationship with the concentration and the degree of polymerization of poly(DL-lysine). The probable implication of cell surface electrical charge modification in these phenomena is discussed.     

Calcium-dependent process in reduction of cell surface charge after x-irradiation.

The electrophoretic mobility (EPM) of rat erythrocytes and cultured melanoma cells decreased with time after X-irradiation in the presence of calcium at concentrations higher than 10 (-5) M. At 37 degrees C, the presence of calcium for the first 20 min of exposure was suffcient to induce the EPM reduction, and Ca 2+ administration subsequent to Ca 2+ -free incubation for 30 min following irradiation had no effect on EPM. At lower temperatures, from 10 down to 20 degrees C however, the effect of calcium on the reduction of EPM decreased drastically. If the cells were kept Ca 2+ -inonophore A23187 also induced to decrease in EPM only in the presence of Ca 2+. These results revealed the transitory existence of membrane condition reactive to extracellular Ca 2+ immediately after X-irradiation, which can be postponed at low temperatures. The reduction of EPM by Ca 2+ -ionophore might suggest that the influx of Ca 2+ is a step in the reduction of EPM after X-irradiation.     

Cell electrophoresis of amphibian blastula and gastrula cells; the relationship of surface charge and morphogenetic movement

By use of a novel modification in cell electrophoresis technique, an “electrophoretic map” has been made of selected embryonic regions at four different stages of embryogenesis in Rana pipiens. Hitherto, it had been impossible to make surface charge determinations for cells from small, selected regions of early amphibian embryos.Embryonic regions were dissected, and the tissue was disaggregated in EDTA. For cell electrophoresis, cells were suspended in low ionic strength medium with 15% Ficoll, and timed over 14.6 micrometers in a voltage gradient of 20 V/cm.The data reveal a correlation between three parameters: that of surface charge, morphogenetic movement, and adhesiveness. Embryonic regions that undergo a rapid increase in surface charge with the onset of gastrulation, are the same regions that initiate morphogenetic activity. Regions in which surface charge remains constant tend to be morphogenetically less active. Adhesive estimates were based on observations of degree of rounding of cells in situ. There is an apparent correlation, in ectoderm, mesoderm, and endoderm, of increased surface charge with decreased adhesiveness (and vice versa).     

A simple model for surface charge on ion channel proteins.

We present a simple two-parameter model for surface charge directly associated with ion channels. A spherically symmetric "charged shell" models a distribution of surface charge arrayed about the channel entrance, with a corresponding set of image charges behind the plane of the membrane. The transition between a regime of buffered conductance and a regime of rapidly falling conductance at very low ionic strength is found to depend on the magnitude of the surface charge as well as the separation between the charge and the channel entrance. This resolves an apparent discrepancy between the experimental findings of Naranjo and Latorre (1993. Biophys. J. 64:1038-1050) and previous theoretical computations. The charged-shell model is used in a comparative study of the toad skeletal muscle conductance data of Naranjo and Latorre, the rat skeletal muscle conductances of Ravindran et al. (1992. Biophys. J. 61:494-508), and a second set of rat muscle conductances presented in this paper.     

Direct determination of macromolecular charge by equilibrium electrophoresis

Charge is a fundamental property of macromolecules in solution. However, estimation of the apparent charge on polyions has confounded science for decades. Presented here is a general method to determine directly the apparent charge on a polyion, regardless of its size or shape. This new method uses equilibrium electrophoresis, a procedure in which opposing solute flows from electrophoresis and from diffusion balance everywhere as the system reaches a steady-state distribution. The method uses only small quantities of materials, is nondestructive, and requires only simple, inexpensive instrumentation. Here we describe a prototype apparatus, demonstrate the phenomenon, and present experimental examples of the procedure.     

Determination of protein charge by capillary zone electrophoresis

The feasibility of employing classical electrophoresis theory to determine the net charge (valence) of proteins by capillary zone electrophoresis is illustrated in this paper. An outline of a procedure to facilitate the interpretation of mobility measurements is demonstrated by its application to a published mobility measurement for Staphylococcal nuclease at pH 8.9 that had been obtained by capillary zone electrophoresis. The significantly higher valence of +7.5 (cf. 5.6 from the same series of measurements) that has been reported on the basis of a "charge ladder" approach for charge determination signifies the likelihood that the latter generic approach may be prone to error arising from nonconformity of the experimental system with an inherent assumption that chemical modification or mutation of amino acid residues has no effect on the overall three-dimensional size and shape of the protein.     

No elevated cell surface charge at mitosis in X-ray-irradiated mammalian cells

Rounded mitotic cells showed 30% enhanced electrophoretic mobility (EPM) when compared to spindle-formed interphase cells. This increase in EPM that was not present in interphase cells that had been rounded chemically by EDTA is considered to reflect a structural change in the cell membrane during mitosis. X-ray irradiation induced a dose-dependent EPM decrease in both interphase and mitotic cells during a 4-hour period. During the next 20 h of incubation, EPM recovery took place in cells irradiated with 250R, but not in cells exposed to 1000R. EPM was enhanced during mitosis in cells irradiated with low doses, but was absent in cells irradiated with 1000R. The ratio of colony-forming cells and of electrophoretically recovered mitotic cells after 24 h of exposure showed a good statistical correlation. These results indicate that unrepaired membrane damage contributes to mitotic cell death after irradiation.     

Human red blood cell aging: correlative changes in surface charge and cell properties

Red blood cells (RBCs) during microcirculation, aging and storage, lose N-acetylneuraminic acid (NANA) and other biomaterials thereby altering cell structures, some properties and functions. Such cell damage very likely underlies the serious adverse effects of blood transfusion. However, a controversy has remained since 1961-1977 as to whether with aging, the RBCs, suffering loss of NANA, do have a decreased charge density. Any correlation between the changes in the cell properties with cell aging is also not clear. Therefore, to remove the ambiguity and uncertainty, we carried out multiparameteric studies on Percoll fractions of blood of 38 volunteers (lightest-young-Y-RBCs, densest-old-O-RBCs, two middle fractions).We found that there were striking differences between the properties of Y-RBCs and O-RBCs. The ζ-potential of Y-RBCs decreased gradually with aging. Studies in parallel on RBC fractions incubated with both positively charged quantum dots and Sambucus Nigra-fluorescein isothiocyanate (FITC) along with their ζ-potentials provide for the first time direct visual evidence about the lesser amount of charge density and NANA on O-RBCs, and a collinear decrease in their respective ζ-potentials. Close correlation was found between the surface charge on an aging RBC and its structure and functions, from the cell morphology, the membrane deformability to the intracellular Hb structure and oxidation ability. This quantitative approach not only clarifies the picture but also has implications in biology and medicine.     

Separation of mammalian cell surface proteins by a two-dimensional gel electrophoresis system

Separation of externally exposed plasma membrane proteins of mammalian cells has been achieved by a new two-dimensional gel electrophoresis system. The proteins were separated in the first dimension on cylindrical polyacrylamide gels containing 0.1% sodium dodecyl sulfate (SDS) and in the second dimension on polyacrylamide slab gels containing 9 M urea, 0.1% SDS, and 0.1% Triton CF10. Using this method we have obtained reproducible high-resolution patterns of cell surface proteins of differentiated rat neuro-tumor cells in culture and of normal rat retinal cells. Different cell types show characteristic cell surface proteins in addition to ubiquitous ones. The number of common surface proteins between two cell types account for approximately half of the total surface proteins. By immunoprecipitation we have also found that rabbit anti-serum against a rat neuronal cell line can recognize most of these external proteins. Since the separation in the first dimension is done in the presence of SDS and the second dimension in the presence of SDS, a non-ionic detergent, and urea, the technique is particularly suitable for proteins that are of poor solubility. In addition to size, net charge and hydrophobicity appear to be important factors in the separation. Virtually all of the proteins that run in the first dimension can be recovered and further separated in the second.     

A model of cell surface receptor aggregation

Journal of Mathematical Biology - In this paper we construct and analyze a model of cell receptor aggregation. Experiments have shown that receptors in an aggregated state have greatly reduced...