Specificity and valency of concanavalin A in neuron-substratum adhesion and redistribution of cell surface receptors

Neurons seeded in culture as spherical cells flatten partially to form lamellipodia by which they adhere to the substratum. Lamellipodium formation is stimulated specifically by concanavalin A (Con A) and other mannose-binding lectins in several types of neuronal cells, but not in similarly treated fibroblasts. Conditions that block much of the adsorption of Con A to the substratum have no effect on stimulation of lamellipodium formation by Con A. This suggests that Con A acts in solution on neurons and does not directly bind them to their substrata. Succinylated-Con A (bivalent) binds to the same receptors as native Con A (tetravalent) but does not elicit lamellipodium extension unless crosslinked with anti-Con A IgG. Treatment of neurons with Con A produces local changes in the composition of the cell surface resulting from redistribution of lectin receptor complexes. This redistribution is not as great with SCon A and, like lamellipodium formation, is sensitive to the valency of Con A. A variety of treatments (4 degrees C, trifluoperazine, nordihydroguaiaretic acid, 4-bromphenacyl bromide, and cytochalasin D), inhibit both Con A-receptor redistribution and lamellipodium extension by neurons. Other treatments (colchicine and cycloheximide) prevented neither lamellipodium formation nor redistribution.     

Relative accessibility of N-acetylglucosamine in trimers of the adenovirus types 2 and 5 fiber proteins.

Fiber is an adenovirus capsid protein responsible for virus attachment to the cell surface and contains O-linked N-acetylglucosamine (GlcNAc). Results of both amino acid analysis and Dionex chromatography indicated that 3 to 4 and 1.7 to 2.5 mol of GlcNAc are attached per mol of affinity-purified adenovirus type 2 (Ad2) and Ad5 fibers, respectively. Fiber shares an epitope with nuclear pore proteins containing O-linked GlcNAc, as shown by reactivity to monoclonal antibody RL2 directed against these pore proteins. GlcNAc on fiber was found to serve as an acceptor for the transfer of galactose from UDP-galactose by 4 beta-galactosyl-transferase in Ad2 and Ad5 but not in Ad7; quantitation by labeling with UDP-[U-14C]galactose in this reaction gave a 100-fold-lower estimate of the GlcNAc content of fiber, suggesting that these monosaccharides are buried within fiber trimers and are not accessible to the transferase. Affinity chromatography on lectin-bound Sepharose beads showed that Ad2 and Ad5 fibers bound weakly to wheat germ agglutinin and did not bind to ricin or concanavalin A; weak binding to wheat germ agglutinin suggests either that GlcNAc is not easily accessible or that there are not sufficient GlcNAcs for efficient binding. These data suggest that O-linked GlcNAc might be important for Ad2 and Ad5 fiber assembly or stabilization.     

Adhesive specificity of developing cerebellar cells on lectin substrata

Ten lectins, each with a different carbohydrate-binding specificity, have been coupled to tissue culture substrata with carbodiimide [1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide-metho-p-toluene sulfonate] and assayed for their efficacy as substrates for the carbohydrate-specific adhesion of cells dissociated from mouse cerebellum at embryonic Day 13 and postnatal Days 0 and 7. On surfaces treated with concanavalin A, succinyl-concanavalin A, Lens culinaris agglutinin, and wheat germ agglutinin, both embryonic and early postnatal cerebellar cells formed a monolayer. On surfaces coupled with Ricinus communis_I agglutinin (120,000 daltons) both embryonic and postnatal cells formed cellular aggregates with extensive fiber outgrowth. On surfaces treated with peanut agglutinin, Dolichos bifloris agglutinin, Wistaria floribunda agglutinin, soybean agglutinin, or Ulex europaeus_I agglutinin, embryonic cerebellar cells formed cellular aggregates with a cell viability of 25–35% and little or no fiber outgrowth. Postnatal cerebellar cells, in contrast, formed cellular aggregates with a cell viability of 60–70% and extensive fiber outgrowth. On surfaces treated with Ulex europaeus_I agglutinin, cells from postnatal Day 7 formed limited areas of monolayer in addition to cellular aggregates. After 12 hr in vitro the specific attachment of cerebellar cells to lectin-derivatized substrata was inhibited 60–80% by the inclusion of free hapten carbohydrate (50–100 mM) in the growth medium. The addition of soluble concanavalin A or Ricinus communis_I agglutinin (100 μg/ml) was toxic. These studies suggest the presence of glycoconjugate-binding sites for concanavalin A, Lens culinaris agglutinin, and wheat germ agglutinin which promote cerebellar cellular adhesion.     

Stimulation of the membrane-bound, magnesium-dependent adenosine triphosphatase of mouse neuroblastoma by concanavalin A and wheat germ agglutinin.

The effect of the plant lectins concanavalin A and wheat germ agglutinin on the membrane-bound Mg⁺²-dependent ATPase of an adrenergic clone of mouse neuroblastoma was examines. When cell membranes were treated with concanavalin A or wheat germ agglutinin, a dose-related increase in ATPase-specific activity was observed. Maximal stimulation was greater with wheat germ agglutinin than with concanavalin A; half-maximal and maximal stimulation occurred at similar lectin concentrations. Concanavalin A-dependent stimulation was blocked by α-methylmannoside but not by N-acetylglucosammine. Conversely, stimulation with wheat germ agglutinin was prevented by N-acetylglucosamine but not by α-methylmannoside. The combined effects of concanavalin A and wheat germ agglutinin were greater than the individual effects of either, but were not additive. The results suggest that these lectins interact specifically with membrane glycoproteins or glycolipids, resulting in enhancement of Mg⁺²-dependent ATPase activity.     

Regulation of surfactant phospholipid secretion from isolated rat alveolar type II cells by lectins

The major surfactant-associated protein is a potent inhibitor of surfactant phospholipid secretion from isolated type II cells. Since the major surfactant-associated protein contains a carboxy terminal polypeptide domain which is homologous to the lectin-like liver mannose-binding protein, we tested whether lectins inhibit surfactant phospholipid secretion from rat alveolar type II cells. Concanavalin A, wheat germ agglutinin and Maclura pomifera agglutinin were potent inhibitors of surfactant phospholipid secretion. When adenosine 5'-triphosphate (ATP) was utilized as a secretagogue, the IC50 values for inhibition of surfactant phospholipid secretion were 5.10(-7) (wheat germ agglutinin), 1.10(-6) (concanavalin A) and 2.5.10(-5) M (M. pomifera agglutinin). Similar results were obtained when 12-O-tetradecanoylphorbol 13-acetate was utilized as a secretagogue: IC50 values of 1.10(-6) M for concanavalin A and wheat germ agglutinin and 2.5.10(-5) M for M. pomifera agglutinin. Hapten sugars were utilized to antagonize the inhibitory effect of the lectins. N-Acetyl-D-glucosamine significantly reversed inhibition of phospholipid secretion by wheat germ agglutinin in a dose-dependent fashion and methyl alpha-D-mannoside significantly reversed inhibition of phospholipid secretion by concanavalin A. N-Acetyl-D-galactosamine had no significant effect on inhibition of secretion produced by any of the lectins. The inhibitory effect of the lectins did not appear to be due to cytotoxicity since lactate dehydrogenase was not released above control levels and the inhibition of the surfactant phospholipid secretion by wheat germ agglutinin could be reversed after treatment of cells with wheat germ agglutinin by washing the lectin from the cells followed by treatment of the cells with ATP. These studies demonstrate a direct inhibitory effect of plant lectins on phospholipid secretion from type II cells in vitro.     

Influence of Concanavalin A, wheat germ agglutinin, and soybean agglutinin on the fusion of myoblasts in vitro

Although muscle cell fusion was shown to be an energy-requiring process, release of myoblasts from an EGTA fusion block could be accomplished with Earle's balanced salt solution (containing 1.8 mM Ca++) free of glucose or any other energy-produced metabolite. The effect of concanavalin A, abrin, and the lectins from wheat germ, soybean, and Lens culinaris on myoblast fusion was examined with synchronized myoblast cultures upon release from fusion block. At a concentration of 15 mug/ml, these lectins were found to inhibit the fusion process to the extent of 62%, 41%, 32%, 8%, and 19%, respectively. Concanavalin A inhibition could be prevented by alpha-methyl-D-mannoside. The inhibitory effect of all the lectins except abrin could be reversed by changing to the normal, serum-containing medium. The number of binding sites was 3.4 X 10(7), 6.1 X 10(7), and 1.7 X 10(6), respectively. Although myoblasts were found to have about twice as many binding sites for wheat germ agglutinin as for concanavalin A, concanavalin A was determined to be twice as effective as wheat germ agglutinin as an inhibitor of myoblast fusion. These findngs raise the possibility that specific cell surface glycoproteins may be an important factor in this process.     

Synergistic effects of lectins in the interaction of thrombin with human platelets

Several lectins have been studied for their effects on the interaction of thrombin with human platelets. Wheat germ agglutinin, concanavalin A and Ricinus communis lectin increased the number of high affinity sites for diisopropylphosphothrombin on washed platelets from 3000 to about 12 000 but the binding affinities were unchanged (Kd approx 4 nM). Two other lectins, Lens culinaris and Bandieria simplicifolia, were without effect. (2) Using formalinized platelets to avoid possible complications of the platelet release reaction, wheat germ agglutinin showed a marked increase (5-fold) in the binding of active thrombin, peanut agglutinin had no effect while Ricinus communis and :Bandieria simplicifolia showed marginal increases (2-fold). Thrombin binding was decreased to about one quarter with Lens culinaris, Phaseolus vulgaris and concanavalin A. (3) Wheat germ agglutinin caused a synergistic increase of platelet aggregation at low concentrations of thrombin (12.5 mU/ml) and ADP (1 microM), both in the absence and presence of added fibrinogen, but had no effect on ristocetin-induced aggregation.     

Trypanosoma brucei brucei, T. brucei gambiense, and T. brucei rhodesiense: common glycoproteins and glycoprotein oligosaccharide heterogeneity identified by lectin affinity blotting and endoglycosidase H treatment

Whole cell extracts of 10 clones of bloodstream forms of African trypanosomes representing two strains of Trypanosoma brucei gambiense, one strain of T. b. rhodesiense and one strain of T. b. brucei were fractionated on sodium dodecyl sulfate-polyacrylamide gels, electrophoretically transferred to nitrocellulose paper, and probed with horseradish peroxidase conjugated lectins to detect glycoproteins. Variant specific glycoproteins of all 10 clones bound peroxidase labeled concanavalin A, but peroxidase labeled wheat germ agglutinin bound to the variant specific glycoproteins of only 3 of the 10 clones examined. In addition, 22 other glycoproteins expressed in common by all clones bound peroxidase labeled concanavalin A; 19 common glycoproteins bound peroxidase labeled wheat germ agglutinin. Lectin binding to transferred glycoproteins was specifically inhibited by appropriate monosaccharides, alpha-methyl mannoside for concanavalin A and N-acetyl glucosamine for wheat germ agglutinin. Prior incubation of blots in endo-beta-N-acetylglucosaminidase H eliminated binding of peroxidase-labeled concanavalin A to most of the 22 common glycoproteins. Two glycoproteins, designated Gp 81 and Gp 110, were the major Endoglycosidase H resistant components. Endoglycosidase H treatment also reduced binding of peroxidase labeled concanavalin A to the variant specific glycoproteins of 7 clones. The variant specific glycoproteins from the 3 clones that bound peroxidase labeled concanavalin A following enzyme treatment were those that bound peroxidase labeled wheat germ agglutinin. These results show that African trypanosomes express a greater number of glycoproteins than has been reported previously and that only a limited number of these glycoproteins bear Endoglycosidase H resistant oligosaccharides.     

Lectin binding to the porcine and human ileal receptor of intrinsic factor-cobalamin

The purified porcine recpptor for the intrinsic factor-cobalamin complex bound to concanavalin A, lentil lectin and wheat germ lectin covalently coupled to Sepharose and was eluted with the corresponding soluble sugars. In contrast, human intrinsic factor bound efficiently to concanavalin A, to some extent to lentil lectin, but only slightly to wheat germ agglutinin. The binding of IF-Cbl to the receptor was inhibited when the receptor was pre-incubated with soluble wheat germ aglutinin, with an inhibition constant estimated to be 1.9 mol/l. After transfer of the purified receptor from SDS-PAGE to Immobilon, ligand blotting of the purified receptor with iodinated lectin showed that concanavalin A and lentil lectin bound to three (75, 56 and 43 kDa) components but that wheat germ agglutinin bound only to the 75 kDa component. These results showed that the subunit of the receptor could bind to wheat germ agglutinin, resulting in an inhibition of its binding with intrinsic factor. Both binding sites of intrinsic factor and of wheat germ agglutinin could be located near to each other.     

The cortical actomyosin system of cytochalasin D-treated lymphoblasts

Global cytoskeleton dynamics is likely to exist in animal cells and some experimental evidence for this has recently been obtained in cells from the human lymphoblastic cell line KE37. We have further investigated the dramatic and reversible microtubule-dependent cell elongation which occurs upon treatment of KE37 cells with cytochalasin D. This phenomenon results in a non-locomotory cell with definite polarity. It involves a sustained equatorial myosin II-dependent contraction of cortical, most of the myosin II being accumulated on segments of the main cellular extension. We report here that such a cell lengthening is energy-dependent and can be inhibited, or suppressed, by surface ligands such as wheat germ agglutinin but not by concanavalin A. Suppression of the cytochalasin D effect by wheat germ agglutinin is rapid and appears to be collapse of the cell extension and relocalization of the contracted actomyosin as a whole. It suggests that the binding of the wheat germ agglutinin to the cell surface results in the transient disassembly of microtubules, a possibility also raised by the potent antagonist effect of taxol on wheat germ agglutinin action. Taken together, the data are consistent with a specific role of microtubules in the control of the activity of the cortical actomyosin system.     

Effects of wheat germ agglutinin and concanavalin A on parameters of highly purified sodium-potassium adenosine triphosphatases from Squalus acanthias and Electrophorus electricus.

The effects of two lectins, wheat germ agglutinin and concanavalin A, were studied on a variety of parameters of two highly purified (Na+ + K+)-ATPases (ATP phosphohydrolase, EC 3.6.1.3), from the rectal salt gland of Squalus acanthias and from the electroplax of Electrophorus electricus. Both lectins agglutinated the rectal gland enzyme equally, but wheat germ agglutinin inhibited (Na+ + K+)-ATPase activity much more. The electroplax enzyme was only marginally agglutinated and inhibited by the lectins. Neuraminidase treatment of the rectal gland (Na+ + K+)-ATPase had no effect on germ agglutinin inhibition. The inhibition of the rectal gland (Na+ + K+)-ATPase by wheat germ agglutinin could be reversed by N,N'-diacetylchitobiose, which has a high affinity for wheat germ agglutinin. Neither ouabain inhibition nor ouabain binding to the rectal gland enzyme was affected by wheat germ agglutinin. The p-nitrophenylphosphatase activity of the rectal gland enzyme was not inhibited by wheat germ agglutinin. Na+-ATPase activity, which reflects ATP binding and phosphorylation at the substrate site was inhibited by wheat germ agglutinin and this inhibition was reversed by potassium. Evidence is cited (Pennington, J. and Hokin, L.E., in preparation) that the inhibition of the (Na+ + K+)-ATPase by wheat germ agglutinin is due to binding to the glycoprotein subunit.     

Changes in the binding of concanavalin A and wheat germ agglutinin to human lymphocytes during in vitro transformation

Concanavalin A binds to human circulating lymphocytes in a complex manner suggesting the presence of multiple binding sites. Saturation of one or more of these binding sites is observed at concentrations of concanavalin A which induce blast transformation in lymphocytes. In contrast, only one saturable binding site is observed for wheat germ agglutinin. During $\text{in vitro}$ transformation, the amount of concanavalin A which can be bound by lymphocytes increases, whereas the amount of wheat germ agglutinin which can be bound remains unchanged. Since the size increases during transformation, there must be a fall in the density of surface receptors for wheat germ agglutinin whereas the density of concanavalin A receptors remains unchanged.     

Glycoprotein characteristics of the sodium channel saxitoxin-binding component from mammalian sarcolemma

The saxitoxin-binding component of the excitable membrane sodium channel exhibits glycoprotein characteristics as evidenced by its specific interaction with various agarose-immobilized lectins. The detergent-solubilized saxitoxin-binding component interacts quantitatively with immobilized wheat germ agglutinin and concanavalin A and fractionally with immobilized Lens culinaris hemagglutinin and Ricinus communis agglutinin. These lectins preferentially bind $\text{N-}\text{acetylglucosamine}$ and sialic acid (wheat germ agglutinin), mannose (concanavalin A and Lens cunilaris and galactose (Ricinus communis). Removal of terminal sialic acid residues by neuraminidase markedly decreases binding to immobilized wheat germ agglutinin but uncovers sites capable of interacting with lectins specific for galactose and $\text{N-}\text{acetylgalactosamine}$. $\text{β-N-}\text{acetylglucosaminidase}$, an exoglycosidase has no effect on the binding of the channel protein to wheat germ agglutinin. Similarly, phospholipase C has no effect on binding of the solubilized toxin binding component to this lectin. Neither wheat germ agglutinin nor concanavalin A free in solution alters the number of toxin binding sites or their affinity for toxin. The sodium channel saxitoxin-binding component appears to be a glycoprotein containing terminal sialic acid residues and internal mannose, galactose, $\text{N-}\text{acetylglucosamine}$, and $\text{N-}\text{acetylgalactosamine}$ residues. The toxin binding site is spatially separated from the binding sites for the lectins studied. The effect of these sugar moieties must be considered when evaluating the biophysical parameters of the sodium channel.     

Location of the carbohydrates present in the HK-ATPase vesicles isolated from hog gastric mucosa

The glycosylation of H+K(+)-ATPase vesicles isolated from hog gastric mucosa was investigated by various methods. Following protein separation on sodium dodecyl sulfate reducing gels and transfer to poly(vinyl difluoride) membranes, binding of concanavalin A was confined to the 94-kDa band which corresponds to the catalytic subunit. In contrast, wheat germ agglutinin binding occurred in a region below the 94-kDa subunit, corresponding to the 60-85-kDa region, and also to protein just above the catalytic subunit. Treatment with glycopeptidase F removed most of the concanavalin A staining and also the wheat germ agglutinin staining found below the 94-kDa region, but spared the higher molecular weight wheat germ agglutinin reactive material. During the deglycosylation experiments a protein of 35-kDa was produced. Sequencing analysis of V8 protease generated peptide fragments of the 35-kDa protein show at least 30% homology with the Na+K(+)-ATPase beta-subunits. Labeling of the carbohydrates by galactosyltransferase and [3H]uridine diphosphate-galactose showed that the sites of labeling were extracellular and were confined to the wheat germ agglutinin staining regions. Two molecular weight regions, below the 94-kDa region, of 60 and 85 kDa were identified. Electron microscopy using postembedding staining techniques showed that both concanavalin A and wheat germ agglutinin staining occurred on the extracellular face of the gastric vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trypanosoma brucei brucei and Trypanosoma brucei gambiense: stage specific differences in wheat germ agglutinin binding and in endoglycosidase H sensitivity of glycoprotein oligosaccharides

Gel electrophoresis, lectin affinity blotting, and endoglycosidase H digestion have been used to analyze the glycoprotein profiles of bloodstream and procyclic forms of Trypanosoma brucei brucei and T. b. gambiense. Proteins resolved by polyacrylamide gel electrophoresis were stained with silver nitrate or electrophoretically transferred to nitrocellulose and probed with a horseradish peroxidase conjugate of either concanavalin A or wheat germ agglutinin. Silver staining showed, as expected, that the expression of the variant specific glycoprotein was restricted to the bloodstream forms. Twenty-three concanavalin A binding proteins were resolved in blots of bloodstream forms. Concanavalin A binding molecules corresponding in electrophoretic mobility to 21 of these 23 bloodstream form glycoproteins were detected in blots of procyclic forms. The two concanavalin A binding glycoproteins present only in bloodstream form extracts were variant specific glycoprotein and an 81-kDa protein designated glycoprotein 81b. One concanavalin A binding molecule of 84 kDa, glycoprotein 84p, was detected only in procyclic forms. The 19 major wheat germ agglutinin binding glycoproteins expressed by bloodstream forms were not detected in procyclic forms; only small proteins or protein fragments in procyclic form extracts bound wheat germ agglutinin. Incubating transferred proteins in endoglycosidase H eliminated subsequent binding of concanavalin A to most of the 22 common glycoproteins of bloodstream forms. Three major concanavalin A binding glycoproteins of bloodstream forms, variant specific glycoprotein, glycoprotein 81b, and a 110-kDa molecule (glycoprotein 110b), and other minor glycoproteins carried sugar chains that resisted endoglycosidase H digestion. In contrast, concanavalin A did not bind to any procyclic form glycoproteins, including a 110-kDa concanavalin A binding molecule (glycoprotein 110p) after endoglycosidase H treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Some physicochemical aspects of oligosaccharide binding to concanavalin A and wheat germ agglutinin

The binding of fluorescently labelled carbohydrates to concanavalin A and wheat germ agglutinin was studied at equilibrium and by the stopped-flow and temperature jump relaxation methods. Ligand were mainly die 4-methylumbelliferyl glycosides of α (1 → 2)-linked manno-oligosaccharides and of β (1 → 4)-linked chito oligosaccharides as limited homologous series. They offer distinct advantages, parti cularly for kinetic studies.Enthalpie and kinetic considerations suggest that concanavalin A specifically binds a single mannopyranosyl group in α (1 →2)-linked manno-oligosaccharides. This occurs preferentially at the non-reducing end. Glycosylation of a carbohydrate withe.g. an aryl group does not afect die binding kinetics and for all carbohydrates the association rate is comparable but relatively slow, which indicates that a common process is involved in the binding of all carbohydrates to concanavalin A. The affinity of a carbohydrate for concanavalin A is determined by the dissociation-rate parameter, resulting in a longer residence time for a better ligand.Interaction of chito-oligosaccharides with wheat germ agglutinin is complex. With the larger members of the 4-methylumbelliferyl chito-oligosaccharides, binding studies were only possible at low fractional saturation to avoid formation of unsoluble complexes. The binding kinetics of wheat germ agglutinin are faster than with concanavalin A and are consistent with a wheat germ agglutinin binding region composed of two adjacent subsites. For binding of the monoside as well as the bioside, two consistent kinetic models apply. They have common that for each ligand there exist two complexes with comparable population.     

Reactivity with lectins of the saccharide components of rhodopsin in reconstituted membranes. Orientation of the carbohydrates

Rhodopsin-containing liposomes may provide a model for investigating the interaction of intrinsic membrane glycoproteins in biological systems. As part of the characterization of this preparation, the surface orientation of the carbohydrates of rhodopsin, assembled from purified bovine rhodopsin and egg phosphatidylcholine was examined, and is the topic of this report. The major tool used in these studies was the interaction with the carbohydrate-specific reagents, plant lectins. Two techniques were used: lectin-mediated aggregation of the liposomes, as measured by light scattering; the binding of 125I-labeled succinylated concanavalin A, and Scatchard analysis as a measure of affinity. The preparation most extensively examined had a mole ratio of rhodopsin:phospholipid of 1:100. Among a variety of lectins which were examined, only concanavalin A, succinylated concanavalin A, and wheat germ agglutinin were able to mediate the aggregation of rhodopsin-containing liposomes, as expected. The aggregation with concanavalin A was prevented by the presence of sugars having the alpha-D-glucopyranosyl configuration, and that brought about with wheat germ agglutinin, by N-acetylglucosamine (GlcNAc). In addition, the aggregation with concanavalin A was reversed with methyl alpha-D-mannoside, and with wheat germ agglutinin, by GlcNAc, suggesting that membrane fusion did not take place. On a molar basis, wheat germ agglutinin brought about a greatly reduced extent of aggregation as compared to concanavalin A, suggesting the relative inaccessibility of GlcNAc residues in the liposomes as compared to mannose. The initial rate of the aggregation, however, were similar with either lectin. The first-order rate constants were unaffected by wide variation in the concentrations of concanavalin A and wheat germ agglutinin, and by variation in the mole ratios of rhodopsin in the liposomes from 0.2 to 19 moles per 100 moles of egg lecithin. Rhodopsin-liposomes were also prepared from a total lipid extract of rod outer segments instead of egg lecithin. Similar kinetic properties were exhibited by this preparation as were obtained with the liposome prepared with the purified phospholipid. Scatchard analysis of the binding of 125I-labeled succinylated concanavalin A by rhodopsin liposomes indicated the presence of a single class of binding site as the preferred fit, with an apparent Kd of 2.8 X 10(-7) M. The binding was destroyed or extensively interfered with by trypsinization and by periodate treatment.     

Chloride influx provokes lamellipodium formation in microglial cells.

Lamellipodium extension and retraction is the driving force for cell migration. Although several studies document that activation of chloride channels are essential in cell migration, little is known about their contribution in lamellipodium formation. To address this question, we characterized chloride channels and transporters by whole cell recording and RT-PCR, respectively, as well as quantified lamellipodium formation in murine primary microglial cells as well as the microglial cell-line, BV-2, using time-lapse microscopy. The repertoire of chloride conducting pathways in BV-2 cells included, swelling-activated chloride channels as well as the KCl cotransporters, KCC1, KCC2, KCC3, and KCC4. Swelling-activated chloride channels were either activated by a hypoosmotic solution or by a high KCl saline, which promotes K(+) and Cl(-) influx instead of efflux by KCCs. Conductance through swelling-activated chloride channels was completely blocked by flufenamic acid (200 microM), SITS (1 mM) and DIOA (10 microM). By exposing primary microglial cells or BV-2 cells to a high KCl saline, we observed a local swelling, which developed into a prominent lamellipodium. Blockade of chloride influx by flufenamic acid (200 microM) or DIOA (10 microM) as well as incubation of cells in a chloride-free high K(+) saline suppressed formation of a lamellipodium. We assume that local swellings, established by an increase in chloride influx, are a general principle in formation of lamellipodia in eukaryotic cells.     

Concanavalin A and wheat germ agglutinin receptor activity of sialoglycopeptides isolated from the surface of Novikoff hepatoma cells

Novikoff ascites hepatoma cells were highly agglutinable by the plant lectins concanavalin A and wheat germ agglutinin. Treatment of the intact cells with papain released from the cell surface a glycopeptide fraction which possessed concanavalin A and wheat germ agglutinin receptor activity, as judged by its ability to inhibit lectin-induced hemagglutination. A component of the cell-surface glycopeptide fraction, excluded from Sephadex G-50, possessed lectin receptor activities reflecting the cytoagglutination properties of the intact cells from which it was derived. Further resolution of this component by pronase digestion, gel filtration, and ion-exchange chromatography resulted in the isolation of sialoglycopeptides which exhibited potent and specific concanavalin A receptor activity.