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.     

Cell surface charge alterations occurring during dimethylsulfoxide-induced erythrodifferentiation of Friend leukemia cells

Dimethylsulfoxide-induced erythrodifferentiation of Friend leukemia cells caused a decrease in net negative cell surface charge which began two days after exposure to the polar solvent and continued throughout the maturation process. Neuraminidase treatment caused a marked reduction in mobility of both untreated and dimethylsulfoxide-treated cells suggesting that sialic acid residues are the major anionogenic moieties of the surface membrane of Friend cells. A decrease in the content of total glycosidically bound sialic acid in dimethylsulfoxide-treated cells also occurred. The findings provide evidence to support an association between erythrodifferentiation of Friend cells and net negative surface charge dependent upon sialic acid residues.     

Cytochalasin B and the sialic acids of Ehrlich ascites cells

The effect of cytochalasin B (CB) on the electrophoretic mobility and density of ionized sialic acid groups at the surface of Ehrlich ascites cells was examined together with a biochemical assay of the total sialic acid content of treated and control cells. Sialic acid assays indicated that CB-treated cells had a greater amount of total sialic acid and sialic acid sensitive to neuraminidase than control cells/cell. Equal amounts of sialic acid were removable by neuraminidase treatment from control cells and cells pretreated with neuraminidase and subsequently cultured with CB. The electrophoresis results showed a decrease in electrophoretic mobility in the presence of CB which could be reversed by growth in CB-free medium. Neuraminidase treatment did not make a significant additional reduction in the mobility of CB-treated cells. CB also prevented the recovery of electrophoretic mobility of neuraminidase treated cells. The results suggest that while CB does not inhibit sialic acid synthesis, it does alter the expression of ionized sialic acid groups at the electrokinetic surface. CB-containing culture media could be re-utilized several times suggesting that CB is not significantly bound or metabolized by Ehrlich ascites cells.     

Demonstration of sialic acid groups in the glomerular basement membrane of the rat with phosphotungstic acid at low pH

Summary This paper reports an unrecognized aspect of phosphotungstic acid staining at low pH. It provides an on-section staining method in which sialic acid-containing molecules can be demonstrated in the laminae rarae of the rat glomerular basement membrane. The staining in the basement membrane became negative after perfusion with the following cations: protamine sulphate, hexadimethrine, Alcian Blue, Ruthenium Red and Toluidine Blue. Blocking ws not achieved with Alcian Blue at about pH 1. The staining was also abolished after mild methylation and demethylation restored the contrast. This is suggestive of the involvement of carboxyl groups. Prior digestion with pronase, trypsin and neuraminidase rendered the laminae rarae negative, whereas hyaluronidase, chondroitinase ABC and crude heparinase were without effect. This indicates that sialic acid groups are detected by this method and that heparan sulphate does not interfere. The staining of the epithelial plasma membrane, also carrying sialic acid groups, remained positive after neuraminidase treatment. It is presumed that this method can be applied successfully for detecting changes in the sialic acid content of the laminae rarae in rat glomerular basement membranes under normal and pathological conditions.     

Robonucelic acid within the cellular peripheral zone and the binding of calcium to ionogenic sites

Ribonuclease was shown to reduce the electrophoretic mobility of a line of cultured mammalian cells (RPMI no. 41), and Ehrlich ascites tumour cells. No reduction was detected in the case of human, mouse or embryonic chick erythrocytes. These data, taken with the various controls, support the hypothesis that RNA is a structural component of the peripheries of two types of cells, but not of erythrocytes from three species. Calcium-binding was studied in RPMI no. 41 cells, Ehrlich ascites tumour cells, and human and mouse eryhrocytes, by measurement of reduction in cellular electrophoretic mobility in suspending solutions containing various concentrations of calcium chloride. The effect of treating cells with neuraminidase and/or ribonuclease on calcium-binding was also studied. The results suggest that less calcium binds to the carboxyl groups of peripheral sialic acids than to the phosphates of peripheral, structural RNA. However, calcium apparently binds most avidly to as yet unidentified anionic sites.     

Plasmodium lophurae: membrane proteins of erythrocyte-free plasmodia and malaria-infected red cells

Plasma membranes of normal duckling erythrocytes were prepared by blender homogenization and nitro-en decompression. Surface membrane vesicles of red cells infected with the avian malaria Plasmodium lophurae were produced by nitrogen decompression. Membranes of erythrocyte-free malaria parasites were removed from cytoplasmic constituents by Dounce homogenization. These membranes were collected by centrifugation in a sucrose step gradient and purified on a linear sucrose gradient. Red cell membranes had a buoyant density of 1.159 g/cm3, whereas plasmodial membranes banded at 2 densities: 1.110 g/cm3 and 1.158 g/cm3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the isolated red cell membranes revealed 7 major protein bands with molecular weights (MW) ranging from 230, 000 to 22,000, and 3 glycoprotein bands with MW of 160,000, 88,000 and 37,000. Parasite membranes also had 7 major bands with MW ranging from 100,000 to 22,000. No glycoproteins were identifiable in these membranes. The proteins of the surface membranes from infected red cells had MW similar to those from normal red cells; however, there was some evidence of a reduction in the amount of the high MW polypeptides. The red cell membrane contained 79 nmoles sialic acid/mg membrane protein, whereas plasmodial membranes had 8 nmoles sialic acid/mg membrane protein. The sialic acid content of the surface membranes of infected red cells was significantly smaller than that of normal cells. Lactoperoxidase-glucose oxidase-catalyzed iodination of intact normal and malaria-infected erythrocytes labeled 7 surface components. Although no observable differences in iodinatable proteins were seen in these preparations, there was a striking reduction in the iodinatability of erythrocytic membranes obtained from P. lophurae-infected cells. Erythrocyte-free plasmodia bound very little radioactive iodine; the small amount of radioactivity was distributed among 3 major bands with MW of 42,000, 32,000 and 28,000. It is suggested that the alterations of the surface of the P. lophurae-infected erythrocyte do not occur by a wholesale insertion of plasmodial membrane proteins into the red cell plasma membrane, but rather that there are parasite-mediated modifications of existing membrane polypeptides.     

Ultrastructural observations on the cell surface of the intestinal epithelium of the nematode,Ascaris suum

Summary The intestinal epithelium of Ascaris suum consists of a single layer of tall columnar epithelial cells that rest on a thick basal membrane in contact with the pseudocoelomic cavity. Experiments were conducted on glutaraldehyde-fixed tissue to ascertain the nature of the electronegative charges associated with both the apical microvillar surface and basal membrane.A strong electronegative charge was demonstrated on the microvillar surface and basal membrane with ruthenium red and cationic ferritin staining. The ionic nature of ferritin binding was demonstrated with poly-L-lysine, a polycation that interacts with anionic groups on the membrane and thus blocks the subsequent binding of ferritin. Tissue thus treated was devoid of reaction product. Methylation with diazomethane completely abolished staining. Since the stronger acidic groups of sulfates or phosphates would not be protonated under the conditions employed in this study, and therefore susceptible to methylation, staining by ferritin is thought to be due to its interaction with carboxyl groups. Prior enzymatic treatment of tissue with neuraminidase or phospholipase C had no effect on subsequent ferritin binding. Tissue exposed to colloidal iron at various pH values showed maximal reactivity at a pH of 2.5 or above. Above pH 2.5, the dissociation of protons from free carboxyl groups of protein-bound amino-acid residues with pK's of 3.8 and 4.2 would be maximal, and the ionized carboxyl groups are then available to interact with iron micelles. These results suggest the presence of weaker acidic groups, such as the carboxyl groups of acidic amino acids or uronic acid residues. The stronger acidic groups of sialic acid and the esterified sulfate groups, if present, contribute only minimally to overall staining. These results demonstrate that a high electronegative charge density exists, despite the apparent lack of sialic acid. Staining is believed to be due to carboxyl groups of acidic amino acids and/or carboxyl groups or uronic acid residues.Part of this work was conducted at the Department of Zoology, Louisiana State University, Baton Rouge, Louisiana     

Relative susceptibilities to neuraminidase of the glycoproteins of the human erythrorcyte

Release of sialic acid from the glycoproteins of the normal human erythrocyte surface by neuraminidase was investigated. The glycoproteins of the membrane were separated by electrophoresis in sodium dodecylsulfate polyacrylamide gels. Sialic acid was determined in the sliced gel by a modification of the 2-thiobarbituric acid method, revealing three sialic acid-containing glycoproteins. Treatment of intact erythrocytes with neuraminidase to remove varying amounts of sialic acid indicates that all the glycoproteins are essentially equally accessible to the neuraminidase when 20%–60% of the sialic acid is removed. Similar but not quite identical results were obtained with isolated erythrocyte membranes.Treatment of intact cells with the lectins concanavalin A or phytohemagglutinin-P resulted in shielding of about 25% and 50%, respectively, of the sialic acid from neuraminidase. Concanavalin A blocked sialic acid release over long time periods and with high concentrations of neuraminidase. In contrast, the sialic acid shielding by phytohemagglutinin-P can be overcome by high concentrations of neuraminidase. Both lectins were found to shield the various glycoproteins selectively, with different patterns of shielding. Wheat germ agglutinin exhibited no detectable effect on the susceptibility of the erythrocyte sialic acid to neuraminidase.     

Sialic acid is a cell surface component of Entamoeba invadens trophozoites

The surface anionic groups of Entamoeba invadens were analysed by cell electrophoresis, by ultrastructural cytochemistry, and by identification of sialic acids using paper and gas-liquid chromatography. Binding of colloidal iron hydroxide (CIH) and of cationized ferritin (CF) particles at pH 1.8 and 7.2, respectively, was observed on the cell surface. E. invadens has a highly negative surface charge (-0.96 microns s-1 V-1 cm). Treatment of the cells with trypsin and neuraminidase significantly reduced the electrophoretic mobility by 24% and 40%, respectively. Treatment of the amoebae with neuraminidase also markedly decreased the binding of CIH to the cell surface. This finding suggests that sialic acid residues are the major anionogenic groups exposed on the surface of E. invadens. Paper and gas-liquid chromatography showed that N-acetylneuraminic acid was the only derivative characterized in E. invadens.     

Plasmodium lophurae: Membrane Proteins of Erythrocyte-free Plasmodia and Malaria-Infected Red Cells*

SYNOPSIS. Plasma membranes of normal duckling erythrocytes were prepared by blender homogenization and nitrogen decompression. Surface membrane vesicles of red cells infected with the avian malaria Plasmodium lophurae were produced by nitrogen decompression. Membranes of erythrocyte-free malaria parasites were removed from cytoplasmic constituents by Dounce homogenization. These membranes were collected by centrifugation in a sucrose step gradient and purified on a linear sucrose gradient. Red cell membranes had a buoyant density of 1.159 g/cm³, whereas plasmodial membranes banded at 2 densities: 1.110 g/cm³ and 1.158 g/cm³. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the isolated red cell membranes revealed 7 major protein bands with molecular weights (MW) ranging from 230,000 to 22,000, and 3 glycoprotein bands with MW of 160,000, 88,000 and 37,000. Parasite membranes also had 7 major bands with MW ranging from 100,000 to 22,000. No glycoproteins were identifiable in these membranes. The proteins of the surface membranes from infected red cells had MW similar to those from normal red cells; however, there was some evidence of a reduction in the amount of the high MW polypeptides. The red cell membrane contained 79 nmoles sialic acid/mg membrane protein, whereas plasmodial membranes had 8 nmoles sialic acid/mg membrane protein. The sialic acid content of the surface membranes of infected red cells was significantly smaller than that of normal cells. Lactoperoxidase-glucose oxidase-catalyzed iodination of intact normal and malaria-infected erythrocytes labeled 7 surface components. Although no observable differences in iodinatable proteins were seen in these preparations, there was a striking reduction in the iodinatability of erythrocytic membranes obtained from P. lophurae-infected cells. Erythrocyte-free plasmodia bound very little radioactive iodine; the small amount of radioactivity was distributed among 3 major bands with MW of 42,000, 32,000 and 28,000. It is suggested that the alterations of the surface of the P. lophurae-infected erythrocyte do not occur by a wholesale insertion of plasmodial membrane proteins into the red cell plasma membrane, but rather that there are parasite-mediated modifications of existing membrane polypeptides.     

Modifications of single acetylcholine-activated channels in BC3H-1 cells. Effects of trimethyloxonium and pH

We have examined the effects of chemical modification with trimethyloxonium (TMO) and changes in external pH on the properties of acetylcholine (ACh)-activated channels in BC3H-1 cells, a clonal muscle cell line. TMO reacts covalently and specifically with carboxylic acid moieties in proteins to convert them to neutral methyl esters. In BC3H-1 cells TMO modification reduces the whole-cell response to ACh measured at negative membrane potentials by approximately 60%. G omega seal patch-clamp recordings of single ACh channel currents showed that the reduction in ACh sensitivity is due to alterations in both the current-carrying and the kinetic properties of the channels. Under all our experimental conditions, i.e., in external solutions of normal or low ionic strength, with or without external divalent cations, and at external pHs between 5.5 and 8.1, TMO treatment reduced ACh single-channel conductance to 70-90% of normal. The effects of TMO on channel kinetics were dependent on the ionic conditions. In normal ionic strength solutions containing both calcium and magnesium ions TMO modification reduced the channel average open time by approximately 25%. A similar reduction in open time was seen in calcium-free solution, but was not present when both calcium and magnesium ions were absent from the external solution. Lowering the ionic strength of the solution increased the mean open time in normal channels by about threefold, but did not affect the kinetics of modified channels. In low ionic strength solutions normal ACh channel open times were maximal at approximately pH 6.7 and decreased by three- to fourfold at both acid and alkaline pH. TMO modification removed the pH dependence of channel kinetics, and average open times were short at all pHs between 5.5 and 8.1. We suggest that TMO modifies normally titratable groups on the external surface of ACh channels that help to determine both the gating and permeability properties of ACh channels.     

The determination and localization of sialic acid in guinea-pig granulocytes.

When intact guinea-pig granulocytes (polymorphonuclear leucocytes) disrupted by sonication or with detergent were treated with neuraminidase from Vibrio cholerae, 3.1--3.2 nmol of sialic acid/10(7) cells was released. By using a chromatographic procedure for the specific determination of total cell sialic acid, this releasable portion was found to constitute 70% of the total sialate. All of the neuraminidase-releasable sialic acid of the cells could be removed by enzymic treatment of intact cells with neuraminidase. It thus seemed likely that the neuraminidase-releasable sialic acid is all on the cell surface. To make sure that the result was not due to entry of neuraminidase into the cells, the enzyme was bound covalently to Sepharose 6B, and intact polymorphonuclear leucocytes were treated with the bound enzyme. All of the neuraminidase-releasable sialic acid could still be removed, though more slowly. The cells remained intact and only 1.5--2% of the bound enzyme was released from the Sepharose during incubation. Freed enzyme could have been responsible, at the very most, for release of 18% of the sialic acid. Fractionation studies showed that the nucleus and cytoplasm contain low amounts of sialic acid and that the neuraminidase-releasable sialic acid distributes in a manner similar to the distribution of 5'-nucleotidase, an unambiguous marker for the plasma membrane in these cells. Thus neuraminidase-releasable sialate constitutes a clear marker for the membrane of polymorphonuclear leucocytes. Most of the neuraminidase-insensitive sialate was present in the granule fraction. Removal of sialic acid from intact polymorphonuclear leucocytes did not affect their ecto-AMPase, -ATPase and -p-nitrophenyl phosphatase activities.     

Modulation of WGA binding sites on marrow sinus endothelium in state of stimulated erythropoiesis: a possible mechanism regulating the rate of cell egress

To study the regulation of cellular and molecular traffic across the marrow-blood barrier, rat marrow endothelial surface was incubated with ferritin-conjugated concanavalin A, wheat germ agglutinin (WGA), recinus communis agglutinin I, and phytohemagglutinin. Normal animals were compared with those after erythropoietic stimulation (phenylhydrazine-induced hemolysis, phlebotomy). A selective and significant reduction in the density of WGA receptors, but not other lectins was noted congruent to the degree of reticulocytosis. Neuraminidase treatment also reduced WGA binding sites and the surface negative charge as detected by polycationic ferritin (PCF). Thus, the reduction in WGA binding sites, may reflect a decrease in the density of membrane sialic acid, rendering the endothelial surface charge less negative and providing an electrostatic attraction for the negatively charged surface of reticulocytes. The findings may also be explained by an increase in the frequency of WGA-excluding fenestrae in the endothelium. These areas, lacking sialic acid, may provide unstable areas in the membrane suitable for the passage of cells and molecules in both directions. It is concluded that, by modulating the density of sialic acid residues, the endothelium may regulate the traffic of cells and molecules across the marrow-blood barrier.     

Mitochondrial autonomy. Sialic acid residues on the surface of isolated rat cerebral cortex and liver mitochondria

N-acetylneuraminic acid at the surfaces of rat cerebral cortex and liver mitochondria and derived mitoplasts (inner membrane plus matrix particles) was studied biochemically and electrokinetically. Rat cerebral cortex mitochondria in 0.0145 M NaCl, 4.5% sorbitol, pH 7.2 ± 0.1, 0.6 mM NaHCO₃, had an electrophoretic mobility of - 2.88 ± 0.01 µ/sec per v per cm. In the same solution the electrophoretic mobility of rat liver mitochondria was - 2.01 ± 0.02, of rat liver mitoplasts was - 1.22 ± 0.07, and of rat cerebral cortex mitoplasts - 0.91 ± 0.04 µ/sec per v per cm. Treatment of these particles with 50 µg neuraminidase/mg particle protein resulted in the following electrophoretic mobilities in µ/sec per v per cm: rat cerebral cortex mitochondria, - 2.27; rat liver mitochondria, - 1.40; rat cerebral cortex mitoplasts, - 0.78; and rat liver mitoplasts, - 1.10. Rat liver mitochondria, mitoplasts, and outer mitochondrial membranes contained 2.0, 1.1, and 4.1 nmoles of sialic acid/mg protein, respectively. 10% of the liver mitochondrial protein and 27.5% of the sialic acid was solubilized in the mitoplast and outer membrane isolation procedure. Rat cerebral cortex mitochondria, mitoplasts, and outer mitochondrial membranes contained 3.1, 0.8, and 6.2 nmoles sialic acid/mg protein, respectively; 10% of the brain mitochondrial protein and 49 % of the sialic acid was solubilized in the mitoplast and outer membrane isolation solution procedure. Treatment of both the rat liver and cerebral cortex mitochondria with 50 µg neuraminidase (dry weight) /mg protein resulted in the release of about 50% of the available outer membrane sialic acid residues. Treatment of all of the particles with trypsin caused release of sialic acid but did not greatly affect the particle electrophoretic mobility. In each instance, curves of pH vs. electrophoretic mobility indicated that the particle surface contained an acid dissociable group, most likely a carboxyl group of sialic acid with pK_a ∼ 2.7. Treatment of either the rat liver or the cerebral cortex mitochondria with trypsinized concanavalin A did not affect the particle electrophoretic mobility but did cause a decrease in the electrophoretic mobility of L5178Y mouse leukemic cells.     

Calcium binding by human erythrocyte membranes. Significance of carboxyl, amino and thiol groups

1. The role of the ionized carboxyl groups of proteins of the erythrocyte membrane as Ca(2+) receptor sites was investigated. A water-soluble carbodi-imide [1-cyclohexyl-3-(2-morpholinoethyl)carbodi-imide methotoluene-p-sulphonate], referred to as carbodi-imide reagent, and glycine methyl ester were used to modify the free carboxyl groups of the membrane. The degree of modification was estimated from amino acid analyses, which showed the amount of glycine incorporated. As the concentration of carbodi-imide reagent was raised (0.1-0.4m) incorporation of glycine increased and Ca(2+) binding decreased by about 77%. At 0.4m-carbodi-imide reagent all of the binding of Ca(2+) to protein was abolished and it was estimated that about 37% of the side-chain carboxyl groups of aspartic acid plus glutamic acid had been blocked by glycine. 2. Acetylation of all of the free amino groups was achieved by incubating the erythrocyte ;ghosts' at pH10.3 with acetic anhydride (10-15mg/10mg of ;ghost' protein). Acetylation increased by 1.5-fold the capacity of the ;ghost' to bind Ca(2+), indicating that the remaining carboxyl groups of aspartic acid and glutamic acid were made available for Ca(2+) binding by this procedure. These findings support the concept that in normal ;ghosts', at pH7.4, Ca(2+) binding to free carboxyl groups is partially hindered by the presence of charged amino groups. 3. Treatment of ;ghosts' with N-acetylneuraminidase, which removed 94% of sialic acid residues, and treatment with 1mm-p-chloromercuribenzoate did not alter Ca(2+) binding. The major effect of 5.8mm-p-chloromercuribenzoate upon ;ghosts' was to cause a solubilization of a calcium-membrane complex, which included about one-third of the ;ghost' protein. The molar ratio of Ca(2+): protein in the solubilized material was the same as that in the intact (untreated) ;ghosts'.     

The binding of calcium ions by erythrocytes and `ghost''-cell membranes

1. Washed human erythrocytes, suspended in iso-osmotic sucrose containing 2.5mm-calcium chloride, bind about 400mug-atoms of calcium/litre of packed cells. Sucrose may be replaced by other sugars. 2. Partial replacement of sucrose by iso-osmotic potassium chloride diminishes the uptake of calcium, 50% inhibition occurring at about 50mm-potassium chloride. 3. Other univalent cations behave like potassium, whereas bivalent cations are much more inhibitory. The tervalent cations, yttrium and lanthanum, however, are the most effective inhibitors of calcium uptake. 4. An approximate correlation exists between the calcium uptake and the sialic acid content of erythrocytes of various species and of human erythrocytes that have been partially depleted of sialic acid by treatment with neuraminidase. However, even after complete removal of sialic acid, human erythrocytes still bind about 140mug-atoms of calcium/litre of packed cells. 5. A Scatchard (1949) plot of calcium uptake at various Ca(2+) concentrations in the suspending media shows the presence of three different binding sites on the external surface of the human erythrocyte membrane. 6. Erythrocyte ;ghost' cells, the membranes of which appear to be permeable to Ca(2+) ions, can bind about 1000mug-atoms of calcium per ;ghost'-cell equivalent of 1 litre of packed erythrocytes. This indicates that there are also binding sites for calcium on the internal surface of the erythrocyte membrane.     

Cytochemistry of colloidal iron binding to the surface of Hela cells and human erythrocytes.

It seems from the literature that colloidal iron (C.I.) binding sites on cell surfaces cannot be completely removed by treatment with Vibrio Colerae alpha-neuraminidase. We wondered if C.I. particles bind to negative groups other than the carboxyl groups of sialic acids. Using HeLa cells from suspension cultures and fresh human erythrocytes, we examined, with the transmission electronmicroscope, the influence of the following enzymatic and histochemical treatments on C.I. staining: alpha-neuraminidase; hyaluronidase; ribonuclease; alpha-amylase; mild methylation (MM); MM + saponification (Sap.); MM + Sap +MM; MM + Sap + alpha-neuraminidase; active methylation (AM); AM + Sap; AM + Sap + AM; AM + Sap + alpha-neuraminiadase; CH3OH (80%); Sap. It seemed from these experiments that the carboxyl groups of alpha-neuraminidase sensitive sialic acids constitute the majority of binding sites for C.I. to these particular cells. The most interesting candidates for the residual binding of C.I. are carboxyl groups of alpha-neuraminidase resistant molecules, sulfon, sulfin, and sulfate groups.     

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.     

A study of the cell surface of tumour, foetal and lymph-node cells by cell electrophoresis after antibody and enzymic treatment

1. Rabbit anti-(rat foetal liver) serum, absorbed with adult rat liver cells, decreased the electrophoretic mobility of foetal liver cells by 51% and rat hepatoma cells by 45%, indicating the presence of a foetal-type antigen on the hepatoma cell membrane. 2. The chemical nature of the surface antigen was investigated. Incubation with neuraminidase had no effect on adult liver cells but decreased the electrophoretic mobility of foetal liver cells by 51% and of hepatoma cells by 34%; the effect of antiserum was decreased to one-fifth. 3. Sialic acid, or the supernatant from neuraminidase-treated cells, partially blocked the decrease in electrophoretic mobility induced by antiserum. 4. The pH-electrophoretic mobility curves of hepatoma cells treated with antisera were consistent with a sialic acidcontaining antigen on the surface of the tumour cells. 5. Treatment with ribonuclease did not decrease the electrophoretic mobility of adult-liver cells, but decreased that of the foetal liver cells by 17% and hepatoma cells by 29%. 6. In parallel studies made with mouse BP8 ascites-tumour cells ribonuclease decreased the electrophoretic mobility by 39%, that of normal mouse lymph-node cells by 4.8% and allergized mouse lymph-node cells by 13.3%. 7. Trypsin treatment also decreased the electrophoretic mobility of hepatoma cells by 22%.     

Electrokinetic properties of isolated cerebral-cortex synaptic vesicles

Synaptic vesicles isolated from guinea-pig cerebral cortex had an electrophoretic mobility of -3.55mum.s(-1).V(-1).cm in saline-sorbitol, pH7.2, at 25 degrees C (ionic strength 0.015g-ions/1). The mobility was pH-dependent, varied with ionic strength and indicated that the vesicular surface contained weak acidic functions with a pK(a) in the range 3.0-3.8. Although the vesicular surface was determined to be highly negatively charged, treatment with neuraminidase had no effect on mobility and indicated that the relatively strong carboxyl groups of sialic acid do not contribute significantly to vesicular electrokinetic properties. Treatment of synaptic vesicles with trypsin or trypsinized concanavalin A resulted in increases in mobility, but treatment with ribonuclease, deoxyribonuclease, chrondroitinase ABC or hyaluronidase had no significant effect on mobility. Mn(2+) or Ca(2+) was more effective in decreasing vesicle mobility than was Mg(2+), Sr(2+) or Ba(2+). The electrokinetic properties of the synaptic vesicle surface are discussed and contrasted with the properties of the synaptosomal membrane.