The effect of temperature on concanavalin A-mediated agglutination of cells with rigid receptors.

The agglutination of a yeast, Candida albicans, by concanavalin A has been described. The agglutination was cell-number dependent. Prolonged incubation (60 min) was needed to reach maximum agglutination at 37 degrees C. The rate but not the extent of agglutination was temperature dependent. The dimeric forms of concanavalin A, obtained either at low pH or after succinylation, agglutinated the yeast cells as well as the tetramer. Temperature changes affected the agglutination of yeast cells by dimers and by tetramers to the same extent.     

Membrane lipid fluidity as rate limiting in the concanavalin A-mediated agglutination of pyBHK cells

The initial rate of concanavalin A-mediated agglutination of polyoma transformed Baby Hamster Kidney (pyBHK) cells follows Arrhenius kinetics. There is a smooth decrease in the agglutination rate from 37°C to 22°C with an activation energy of $\text{11.8 ± 0.2}\text{kcal/mol}$ in this region. There is a sharp decrease in agglutination rate below 22°C. The addition of 0.1 mM 1,3-di-tert-2-hydroxyl-5-methylbenzene, a lipid perturber, increases the agglutination rate by a factor of two and increases the membrane lipid fluidity as determined by the spin label method. The rotational correlation time of the spin label 2N14 $\text{(2,2-}\text{dimethyl-5-dodecyl-5-methyloxazolidine}\text{-N-}\text{oxide}$) was measured. The sum of the enthalpy of activation of rotational diffusion and the enthalpy of activation of translational diffusion is very nearly equal to the enthalpy of activation of agglutination. This is consistent with the rate limiting step of agglutination being receptor diffusion, which is probably limited in pyBHK cells by membrane lipid fluidity.     

The role of the membrane skeleton in concanavalin A-mediated agglutination of human erythrocytes

In the erythrocyte membrane, the mobility of band 3 protein, the receptor for concanavalin A (Con A), is drastically reduced by the membrane skeleton. Yet, the vesicles free of membrane skeletal proteins, isolated from the highly agglutinable proteinase-treated cells, are found to be devoid of Con A agglutinability. The vesicles bind Con A in normal amounts, and remain agglutinable with the wheat germ and Ricinus agglutinins. Intracellular entrapment of monospecific antibodies to spectrin and 4.1 protein (two of the major skeletal components of the membrane) is also found to inhibit agglutination by 30-50%. Thus the membrane skeleton appears to play a positive role in the agglutination of the cells with Con A. The anti-ankyrin antibodies are found to be without any effect. The anti-band 3 (cytoplasmic domain) antibodies are also inhibitory to agglutination. Since Con A binding to cells alters the shape responses and deformability of the cells, and the cells resist fragmentation at 49 degrees C, the properties of the whole skeleton, especially spectrin, appear to be changed. The Con A-bound membranes also do not release the complex of spectrin-band 4.1-actin when extracted with a hypotonic medium. It appears that Con A binding leads to interaction of the cytoplasmic domain of the receptor with a skeletal component, possibly spectrin. Subsequent to this, the receptor molecules and the skeletal proteins undergo aggregation in the membrane, which is detected by their crosslinking by an 8.6-A span bifunctional reagent. The contractility believed to be associated with the membrane skeleton may be responsible for the aggregation.     

Threshold effects on the concanavalin A-mediated agglutination of modified erythrocytes.

Bovine erythrocytes, which are not concanavalin A (ConA)-agglutinable, can be rendered so by attaching alpha-D-mannose residues to their outer membrane. The sugars are incorporated by mildly oxidizing the cells with periodate followed by coupling the liberated aldehyde groups with an alpha-thiomannosyl containing hydrazide (I). The rate and extent of ConA-mediated aggregation of the modified cells are not linearly dependent on the amount of sugar incorporated. For example, treatment of the erythrocytes with 0.075 mM periodate for 5 min followed by I led to the introduction of 1.05 x 10(6) mannosyl residues/erythrocyte. Binding studies with 125I-ConA demonstrated the presence of 66,525 ConA receptors/cell with an average KA = 4.9 X 10(6) M-1 yet the cells failed to aggregate with ConA at concentrations up to 500 microgram ml-1. Treating the cells with 0.1 mM periodate followed by I led to the introduction of 1.42 x 10(6) mannosyl residues/erythrocyte. Binding studies with 125I-ConA indicated the presence of 78,780 binding sites/cell (KA = 5.9 X 10(6) M-1). These cells were readily aggregated by ConA at concentrations greater than or equal to 64 microgram ml-1. We show here that the sugar incorporation technique is random and that no functional differences were detected in the receptors introduced at the different periodate concentrations. Therefore, the ConA-mediated aggregation of these modified erythrocytes is exquisitely sensitive to small changes in functionally identical receptor densities.     

Membrane lipid fluidity as rate limiting in the concanavalin A-mediated agglutination of pyBHK cells.

The initial rate of concanavalin A-mediated agglutination of polyoma transformed Baby Hamster Kidney (pyBHK) cells follows Arrhenius kinetics. There is a smooth decrease in the agglutination rate from 37 degrees C to 22 degrees C with an activation energy of 11.8 +/- 0.2 kcal/mol in this region. There is a sharp decrease in agglutination rate below 22 degrees C. The addition of 0.1 mM 1,3-di-tert-2-hydroxyl-5-methylbenzene, a lipid perturber, increases the agglutination rate by a factor of two and increases the membrane lipid fluidity as determined by the spin label method. The rotational correlation time of the spin label 2N14 (2,2-dimethyl-5-dodecyl-5-methyloxazolidine-N-oxide) was measured. The sum of the enthalpy of activation of rotational diffusion and the enthalpy of activation of translational diffusion is very nearly equal to the enthalpy of activation of agglutination. This is consistent with the rate limiting step of agglutination being receptor diffusion, which is probably limited in pyBHK cells by membrane lipid fluidity.     

Inhibition of intercellular adhesion by concanavalin A is associated with concanavalin A-mediated redistribution of surface receptors

The inhibition of adhesion between aggregates and layers of embryonic retinal cells by concanavalin A (Con A) and Con A-mediated rearrangements of Con A receptors on retinal cells were studied. A short incubation of aggregates and layers with 10 micrograms/ml Con A substantially reduced aggregate-to-layer adhesion in a subsequent assay without soluble lectin present. This effect of Con A was dose-dependent, temperature-sensitive, involved events subsequent to Con A binding, and was reduced by cytochalasin B. The inhibition produced by succinylated Con A was substantially increased by incubation with antibody to Con A. Visualization of ConA- receptor complexes by fluorescence microscopy revealed that binding of Con A induced clearing of Con A receptors from filopodia, flattened regions of growth cones, and the edges of axons. This clearing reaction was prevented by the same agents that reduced Con A's inhibition of cell adhesion: low temperature, succinylation of Con A, or cytochalasin B. Aggregate-layer adhesion was restored by releasing Con A at 37 degrees C. Inhibitors of protein and ATP synthesis did not prevent recovery of ability to make adhesions. However, release of Con A at lowered temperatures did not prevent recovery. The results suggest that intercellular adhesion is inhibited by events associated with redistribution of Con A-receptor complexes on retinal cells.     

Concanavalin A-mediated agglutination and distribution of concanavalin A-binding sites in Acanthamoeba following treatment with colchicine and cytochalasin B

Incubation of Acanthamoeba castellanii (Neff strain) with FITC-ConA (15 micrograms/ml) resulted in the appearance of patches of fluorescence on the amoebae within 2 min of incubation. These patches disappeared following treatment of the amoebae with alpha-MeMan. Pretreatment of the amoebae with colchicine or cytochalasin B or with colchicine and cytochalasin B in combination did not significantly alter the distribution pattern of fluorescence in the amoebae. 2,4-Dinitrophenol and incubation at 4 degrees C on the other hand decreased the degree of patching of the amoebae. Pretreatment with 2,4-dinitrophenol and incubation at 4 degrees C also decreased the ConA-mediated agglutination of the amoebae. No effect on the ConA-mediated agglutination was, however, observed following pretreatment of the amoebae with colchicine and cytochalasin B neither alone nor in combination. Our results indicate that ConA-mediated agglutination and long-range ConA-receptor mobility in the Acanthamoeba are not under the control of structures sensitive to cytochalasin B or colchicine.     

Concanavalin A-mediated agglutination of plant plastids

Cucumber (Cucumis sativus L.) and pear (Pyrus domestica Medik.) fruit proplastids, and pea (Pisum sativum L., cv. Meteor) leaf chloroplasts, extracted by osmotic rupture of protoplasts isolated after degradation of the cell walls by cellulase and pectinase, agglutinated in the presence of Con A. Agglutination of cucumber proplastids was inhibited by anti-Con A and by methyl D-gluco/manno pyranosides but not by methyl D-galactopyranoside. Fluorescein isothiocyanate-conjugated Con A (FITC-Con A) rendered agglutinated clumps fluorescent. If cellulase was omitted from the macerating medium, Con A-mediated agglutination did not occur even if proplatids were subsequently incubated with cellulase. Proplastids and chloroplasts extracted by conventional mechanical disruption methods were not agglutinated by Con A and did not acquire fluorescence with FITC-Con A. However, cucumber proplastids so extracted could be agglutinated by Con A if incubated with cellulase after preparation.Abbreviation Con A Concanavalin A (Jackbean phytohemagglutinin)     

The role of microvilli in the agglutination of cells by concanavalin A

Morphological correlates of lectin agglutinability were examined in eight cell lines of varying sensitivity to agglutination by concanavalin A (ConA). The number of microvilli on the surface of cells growing in monolayers was positively correlated with agglutinability. However, when cells were brought into suspension, they all developed numerous microvilli which persisted when the cells were treated with ConA regardless of whether or not they were agglutinated by the lectin. Treatment of cells with dibutyryl cyclic AMP (db-cAMP) and theophylline caused a parallel decrease in agglutinability and numbers of microvilli in monolayer cultures, but suspended cells from control and treated cultures were identical in appearance in the absence or presence of ConA. The surface morphology of cells agglutinated by ConA was very similar to that of cells that spontaneously agglutinated in the absence of the lectin, and surface bound ConA was rapidly withdrawn from microvilli on all cell types. Neither the morphology of cells nor the surface distribution of ConA can explain observed differences in agglutinability.     

The effect of diabetes on hepatocyte plasma membrane fluidity and concanavalin A-induced agglutination

The techniques of fluorescence polarization and lectin-induced agglutination have been utilized to investigate the effects of the diabetic state on some of the dynamic properties of cell membranes. Hepatocyte plasma membranes from streptozotocin-induced diabetic rats exhibited a significant decrease in cholesterol and sialic acid with no alteration in phospholipid content. This membrane system also exhibited a decrease in fluorescence polarization, using the fluorescent probe, 1,6-di-phenyl-1, 3,5-hexatriene, suggesting an increase in membrane fluidity over the value observed in normal hepatocytes. When normal hepatocytes were incubated in the presence of the lectin, concanavalin A (ConA), no significant agglutination was observed. In contrast, hepatocytes from diabetic rats which exhibited a slightly decreased lectin-binding capacity underwent extensive agglutination. In addition, normal hepatocytes which were pretreated with 0.1 mM tetracaine also underwent extensive agglutination with no measurable increase in lectin-binding capacity. These results suggest that altered membrane lipid fluidity and/or cytoskeletal organization may have a profound effect on cell surface dynamics and could result in the uncoupling of the insulin receptor complex from the membrane-associated effector system(s), a defect which may play a role in the problem of insulin resistance observed in some forms of diabetes.     

Quantitative agglutination of specific populations of sea urchin embryo cells with concanavalin A

It has been demonstrated that specific changes in carbohydrate-containing cell surface lectin receptor sites occur with differentiation and maturation of sea urchin embryo cells. In this study, evidence is presented, using a quantitative electronic particle counter assay to measure agglutination, which indicates that concanavalin A (Con A) mediated agglutination of dissociated 32/64 cell sea urchin embryos differs dramatically with respect to specific cell populations. The migratory cell type, the micromere, is significantly more agglutinable with Con A than the other cell types and colchicine treatment markedly increases sea urchin embryo cell agglutinability. The results indicate that like many malignant cells which display extensive migratory behavior, specific migratory populations of embryonic cells are agglutinable with Con A. The results are discussed with respect to the possible nature of lectin receptor sites on specific populations of embryonic cells and the possible role of colchicine-sensitive structures in controlling the display patterns of these sites.     

Effects of female sex steroids on concanavalin A-mediated agglutination of hepatocytes from nonregenerating and regenerating rat liver and hepatic tumor marker enzymes

Effect of treatment of female rats with an oral contraceptive agent (OCA), Ovulen-50, for 7 weeks on agglutination of hepatocytes with concanavalin A (con A) and activities of certain tumor marker enzymes were examined to find out if OCA treatment is related to preneoplastic or neoplastic processes. Hepatocytes from regenerating and nonregenerating livers of control female rats showed negligible agglutination with Con A, whereas hepatocytes from non regenerating but not from the regenerating livers of female rats treated with a combination of 5 micrograms ethinyl estradiol and 100 micrograms ethynodiol diacetate showed agglutination. Of the tumor marker enzymes such as hepatic glucose 6-phosphatase, gamma-glutamyl transpeptidase (gamma-GT), and arginase examined in the liver, only gamma-glutamyl transpeptidase showed a significant increase in activity in the steroid-treated rats. Plasma alkaline phosphatase activity was also higher in the treated animals. However, the magnitude of the changes observed was relatively small and perhaps unrelated to the neoplastic process.     

Concanavalin A-mediated agglutination of 3T3 cells after exposure to UV-irradiated herpes simplex virus.

Studies were made to determine the effect of UV-irradiation of herpes simplex virus (HSV) on Concanavalin A (Con-A)-mediated agglutination of 3T3 cells. There were three different phases of agglutination by Con-A of cells infected with HSV. The agglutinability began to increase from 3 or 4 hr, or 72 hr after exposure of cells to HSV. The early-appearing agglutinability was further divided into two phases, based on its sensitivity to metabolic inhibitors. These were tentatively called "Early 1 or inhibitor sensitive", "Early 2 or inhibitor insensitive" and "Late" agglutinability. "Early 1" agglutination, detected from 3 hr post infection (pi), was induced by treating cells with HSV, either active or UV-irradiated for less than 5 min and was inhibited when actinomycin D (1 microgram/ml) or cycloheximide (50 microgram/ml) was added to the cultures. "Early 2" agglutination began to increase from 4 hr pi when cells were inoculated with HSV irradiated for 7 to 20 min and was not affected by either inhibitor. HSV irradiated for 6 min failed to induce either agglutinability. "Late" agglutination, observed 72 hr pi, was detected in cultures which had been treated with HSV irradiated for 4 to 15 min. Among those, virus irradiated for 6 to 8 min was most efficient. HSV-transformed cells were also agglutinated without exception by low concentrations of Con-A.     

Cell surface proteoglycans as a negative modulator in concanavalin a-mediated agglutination of hepatoma cells

Proteoglycan (heparan sulfate-protein conjugate) was solubilized with 8 M urea from rat liver plasma membranes after enzymic (RNAase, neuraminidase) treatments and extensively purified by chromatography and gel filtration. The final products gave an average ratio of hexuronate to protein (weight) of approx. 1.5, contained hexosamine equimolar to hexuronate and were sensitive to β-elimination (the molecular weight being reduced from 20 · 10⁴ to 3 · 10⁴ (gel filtration)).The proteoglycan fraction, when added to trypsinized and untrypsinized ascites hepatoma (AH-130F(N)) cells, inhibited the concanavalin A-mediated agglutination of the cells. However, the alkali-treated proteoglycan (β-elimination) or acid mucopolysaccharide fraction prepared from liver plasma membranes by papain digestion were less effective, and a reference preparation of heparan sulfate was almost ineffective. It was confirmed that significant amounts of proteoglycan labelled with ³⁵SO₄^2? were firmly bound to or taken up by the trypsinized ascites hepatoma cells.These results together with the sensitization of lectin-mediated agglutination by mild protease treatment of cells suggest that cell surface proteoglycans may act as a negative modulator in the lectin-mediated agglutination of cells.     

Regulation of concanavalin A agglutination by the extracellular matrix

The agglutinability of rat C₆ glioma cells by concanavalin A (Con A) depends upon cell density. From sparse density to near confluency agglutinability increases as cell density rises. Both the half-maximal concentration and the maximum amplitude of agglutination by Con A are functions of cell density, but are separate cell parameters differing in the extent to which they are affected by density and the point at which they become insensitive to further density increases. Both trypsin and EDTA reduce cell agglutinability. The similarity in recovery kinetics between low density cells and cells dissociated with EDTA or trypsin suggests that low density cells may lose the same surface agglutination component(s) removed by trypsin and EDTA. Density-dependent regulation of Con A agglutinability is anchorage dependent; cells grown in suspension display no such phenomenon. The cooperative cell regulation of agglutinability is mediated by the extracellular matrix, or micro-exudate. The matrix contains two activities: low density cultures produce a matrix inhibitor of Con A agglutinability, while high density cultures produce a matrix promotor.     

Measurement of concanavalin A induced agglutination of follicular, luteal and atretic rat granulosa cells

To determine changes in the surface membrane of granulosa cells related to follicular atresia and luteinization, their agglutination behavior with Concanavalin A has been studied by quantitative spectrophotometry. As compared to the granulosa cells of normal Graafian follicles, the agglutination rate (delta A546/5 min) and final level of agglutination significantly increased in atretic and decreased in luteinized cells indicating the increase of Con A binding sites with atresia and decrease with luteinization. Treatments of granulosa cells with EDTA and trypsin enhanced agglutination of follicular and luteal cells but had no effect on atretic granulosa cells.