Analyses of surface membrane carbohydrates in parasitic flagellates of the order kinetoplastida using lectins.

Crithidia fasciculata, Leishmania donovani, Leishmania major, Leishmania mexicana amazonensis, Leishmania tropica, Leishmania tarentolae, Trypanosoma sp. from Formosan bats (Tb), Trypanosoma lewisi, Trypanosoma musculi, and different strains of Trypanosoma cruzi (Tc) were cultivated at 27 degrees C in a liquid culture medium. Flagellates harvested from log phase culture were analyzed for their lectin agglutinating characteristics with concanavalin A (Con A), Peanut agglutinin, Ricinus communis agglutinin 120, soybean agglutinin (SBA), Ulex europeus agglutinin (UEA) and wheat germ agglutinin (WGA). Results indicated that all these flagellates might have D-galactose and methyl- alpha-D-manopyranoside on their surface. The presence of L-Fucose, which complexes specifically with UEA, could not be demonstrated on the surface of these flagellates. Results from quantitative comparison of surface molecules of Tb and the Tulahuen strain of Tc suggested that Tb may have more WGA-binding molecules while Tc may have more ConA-binding molecules. Pretreatment of the flagellates with 0.05% trypsin at 37 degrees C for 30 minutes caused some reduction of agglutination titers. Cell agglutination with lectins was completely inhibited or reversed in the presence of the specific lectin-binding monosaccharides.     

Inhibition of spontaneous aggregation by wheat germ agglutinin in suspensions of BALB/c-3T3 and BHK21 tissue culture fibroblasts

A quantitative method of measuring cytoaggregation based on the Coulter electronic cell counter has been applied to the agglutination of BALB/c-3T3 and BHK21 tissue culture fibroblasts by wheat germ agglutinin. When agglutinin is added to transformed or trypsinized cell suspensions high aggregation rates are seen, and the results obtained are in close agreement with the well-known sensitivity of transformed cells to agglutination by lectins.In the absence of agglutinin, a small but reproducible amount of spontaneous aggregation can also be detected. It is related in some way to growth, since it is absent in suspension prepared from confluent (density-inhibited) cultures and is induced by either transfer to low density, additional serum, or transformation by viruses. Under conditions favouring growth, both BALB/c-3T3 and BHK21 cells show aggregation indices close to 25, corresponding to 10% of the maximum aggregation rate seen.This spontaneous aggregation is susceptible to inhibition by agglutinin. Inhibition occurs at low concentration (about 10 μg/ml) and aggregation rates thus pass through a minimum as the concentration of agglutinin is increased. Among the four different cell lines studied, sensitivity to inhibition is inversely related to agglutination. Thus 3T3 cells, which are barely agglutinated by 1 mg/ml of agglutinin, show 90% inhibition; polyoma virus-transformed BHK cells, which are agglutinated by 10 μg of agglutinin, show no inhibition at all.It is suggested that the agglutination of transformed cells is a consequence of their failure to respond to an inhibitory effect exerted by lectins upon an intrinsic adhesive property of the cell surface.     

Goat sperm membrane: lectin-binding sites of sperm surface and lectin affinity chromatography of the mature sperm membrane antigens.

The cell surface glycoproteins of goat epididymal maturing spermatozoa have been investigated using lectins as surface probes that interact with specific sugars with high affinity. Concanavalin A (ConA) and wheat-germ agglutinin (WGA) showed high affinity for mature cauda epididymal sperm agglutination, whereas RCA2, kidney beans lectin and peanut agglutinin caused much lower or little agglutination of the cells. The mature sperm exhibited markedly higher efficacy than the immature caput epididymal sperm for binding both ConA and WGA, as evidenced by sperm agglutination and the binding of the fluorescence isothiocyanate (FITC)-labelled lectins. FITC-ConA binds uniformly to the entire mature sperm surface whereas FITC-WGA binds to the acrosomal cap region of the head. The FITC-RCA2 mainly labelled the posterior head of mature cauda sperm. However, no WGA-specific glycoprotein receptors could be detected in sperm plasma membrane (PM) by WGA-Sepharose affinity chromatography. The data implied that the epididymal sperm maturation is associated with a marked increase in the ConA/WGA receptors and that WGA receptors may be glycolipids rather than glycoproteins. Analysis of the ConA receptors of cauda sperm PM identified by ConA-Sepharose affinity chromatography and subsequent resolution in SDS-PAGE demonstrated the presence of five glycopolypeptides of different concentrations (98, 96, 43, 27 and 17 kDa) of goat sperm membrane. The immunoblot of these ConA-specific glycopeptides with anti-sperm membrane antiserum showed that 98- and 96-kDa receptors are immunoresponsive.     

Properties of succinylated wheat-germ agglutinin.

The physicochemical and binding properties of succinylated wheat germ agglutinin are described in comparison with these of unmodified wheat germ agglutinin. Succinylated wheat germ agglutinin is an acidic protein with a pI of 4.0 +/- 0.2 while the native lectin is basic, pI of 8.5. The solubility of succinylated wheat germ agglutinin is about 100 times higher than that of the unmodified lectin at neutral pH. Both lectins are dimeric at pH down to 5, and the dissociation occurs at pH lower than 4.5. The binding of oligosaccharides of N-acetylglucosamine to both lectins is very similar on the basis of fluorescence and phosphorescence studies. The minimal concentration required to agglutinate rabbit red blood cells is about 2 microgram/ml with both lectins and the concentrations of N-acetylglucosamine and di-N-acetylchitobiose which inhibit agglutination are similar with both lectins. The number of succinylated wheat germ agglutinin molecules bound to the surface of mouse thymocytes was ten times lower than that of the unmodified lectin although the apparent binding constant was only slightly different between the two lectins. The dramatic decrease of the apparent number of cell surface receptors upon succinylation of the lectin is discussed on the basis of the decrease of the isoelectric point and of the acidic properties of the cell surface.     

Thermal stability and mode of oligomerization of the tetrameric peanut agglutinin: a differential scanning calorimetry study

Peanut agglutinin is a homotetrameric legume lectin. The crystal structure of peanut agglutinin shows that the four subunits associate in an unusual manner, giving rise to open quaternary structure [Banarjee, R., et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 227-231]. The thermal unfolding of peanut agglutinin has been characterized by differential scanning calorimetry and gel filtration to elucidate its thermal stability and its mode of oligomerization. The unfolding process is reversible and could be described by a three-state model with two transitions occurring at around 331 and 336 K. For the tetramer, the ratio of DeltaHc/DeltaHv for the first transition is close to 4 and for the second transition is close to 0.25, suggesting that 4 and 0.25 cooperative unit(s) of the tetramer are involved in the first and second transitions, respectively. The agreement between the model-independent DeltaHv(S) determined from the values of the temperatures of the peak maximum (Tp1) with the protein concentration with the values of DeltaHv obtained from the fit of the data to the transition confirms that the first peak is associated with the dissociation of peanut agglutinin tetramers (A4) to "folded" monomers (4A), whereas the second peak describes the unfolding (4U) of these monomers. The overall process for the thermal unfolding of peanut agglutinin could therefore be summarized as A4 <==> 4A <==> 4U. Gel filtration studies confirm this process, as peanut agglutinin elutes as a tetramer up to 50 degrees C, and at and above 56 degrees C (Tm of first transition), it elutes at a position commensurate with that of the folded monomer of peanut agglutinin. The unfolding behavior of peanut agglutinin in the presence of saturating amounts of carbohydrate ligands is similar to that observed for the unligated form. The temperature of maximal stability of the peanut agglutinin tetramer at pH 7.4 is calculated to be around 33 degrees C with a maximal free energy of stabilization of 8.70 kcal/mol. The results demonstrate that unfolding of peanut agglutinin goes through two distinct phases with folded monomer being the intermediate.     

Nature of the lectin-induced activation of plasma membrane Mg2+ATPase.

The Mg2+ATPase activity of liver plasma membranes decreases markedly with increasing temperature above 30 degrees. This negative temperature dependency is counteracted by the binding of wheat germ agglutinin, concanavalin A, or Ricinus communis agglutinin (at concentrations greater than or equal 0.5 mg/ml) to membranes prior to assay of the enzyme. With one of these lectins bound, the enzyme has a single energy of activation between 20 degrees and 45 degrees. The binding of dimeric succinyl concanavalin A, soybean agglutinin, fucose-binding lectin from Lotus tetragonolobus, or the leucoagglutinin from Phaseolus vulgaris does not alter the temperature dependency of the enzyme. The latter two lectins, however, do prevent the concanavalin A-induced activation of the enzyme at 37 degrees. At saturating substrate concentrations, the enzyme is not inhibited by any of the lectins tested over a wide range of concentrations. Cytochalasin B and colchicine separately or in combination have little influence on the lectin-induced enhancement of enzyme activity. Chlorpromazine and vinblastine sulfate each partially prevent the activation and in combination do so completely. Treatment of the membranes with the detergent Lubrol-PX or phospholipase A prevents activation of the enzyme by concanavalin A. The results are consistent with a restriction by the lectin of an environment which is normally too disordered for maximal enzyme activity above 30 degrees.     

Lectin-mediated sperm agglutination of bufo arenarum and leptodactylus chaquensis spermatozoa

Various plant lecins were employed in cell agglutination experiments to ascertain the presence of specific saccharides in the surface of B arenarum and L chaquensis spermatozoa. B arenarum spermatozoa were specifically agglutinated with Concanavalin A (Con A), phytohemagglutinin P (PHA-P), and wheat germ agglutinin (WGA), but not with soybean agglutinin (SBA). In contrast, L chaquensis spermatozoa were strongly agglutinated by SBA, WGA, and PHA-P. L chaquensis spermatozoa did not agglutinate with Con A even at high concentrations. Lectinmediated sperm agglutination was inhibited in the presence of specific lectinbinding sugars. Spermatozoa from both species were agglutinated randomly with all lectins suggesting a uniform distribution in the sperm surface of the lectinbinding saccharide ligands. B arenarum sperm agglutination induced by Con A is sensitive to temperature. B arenarum spermatozoa are more agglutinable at 24°C than at 4°C. These results suggest that lectin-binding site mobility is necessary for sperm agglutination.     

Galactosyl-binding lectins from the tunicate Didemnum candidum. Carbohydrate specificity and characterization of the combining site

The plasma of the ascidian Didemnum candidum possesses lectin activity directed toward galactosyl moieties. We report the characterization of the affinity chromatography-purified galactosyl-binding lectins from the plasma of this protochordate species in terms of their hemagglutination patterns, temperature stability, saccharide specificities, divalent cation requirements, and the comparison of the properties of their combining sites to those of other characterized lectins. The major galactosyl-specific lectin, termed DCL-I, has an apparent mass of 14,500 daltons and a minor lectin (DCL-II) has an apparent subunit mass of 15,500 daltons. The two molecules differed somewhat in their hemagglutination profiles with untreated and enzyme-treated erythrocytes: a 10-fold increase in DCL-II concentration is required to obtain agglutination titers comparable to those of DCL-I. Although both DCL-I and DCL-II will agglutinate neuraminidase-treated erythrocytes from all vertebrate species tested and most Pronase-treated erythrocytes, DCL-I will agglutinate some untreated erythrocytes which are not agglutinated by DCL-II. Both lectins required divalent cations, were inactivated by temperatures above 70 degrees C, and both exhibited optimal agglutinating activity over a wide range of pH (from 5 to 11). The DCL-I molecule was characterized for its saccharide specificity by binding and inhibition assays using characterized sugars and glycoproteins. Galactose and oligosaccharides bearing nonreducing terminal galactose were the best inhibitors. The inhibition analysis indicated that the DCL-I combining site is small, interacts only with hydroxyls on carbons 2, 3, and 4 of galactose, and exhibits moderate steric hindrance for voluminous groups on carbon 6 and the alpha-anomeric linkage. The data suggest that the combining site would be smaller than the peanut lectin combining site for galactose since DCL-I does not interact with the subterminal monosaccharide hydroxyls for C4 and C6 as does peanut agglutinin. To our knowledge, this is the first isolation and detailed characterization of a lectin from a protochordate species.     

Changing levels of uv light and carcinogen-induced unscheduled DNA synthesis in mouse oocytes during meiotic maturation.

The effects of cytochalasin B (CB) and colchicine on the lectin-mediated agglutination of dissociated cells from chick embryos at the early primitive streak stage were studied. Cells incubated in the absence of the above-mentioned drugs were agglutinable with concanavalin A (ConA), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA). A pre-incubation with neuraminidase was required to render the cells agglutinable with soybean agglutinin (SBA). This treatment had no appreciable effect on the agglutinability of the cells with the other three lectins. Treatment with the drug colchicine had no appreciable effect on the extent of agglutination with any of the above-mentioned lectins. Cells treated with CB dissolved in dimethylsulfoxide (DMSO) in saline, exhibited a reduced lectin-mediated agglutinability. However, a similar decline in agglutinability was observed in controls incubated in saline containing DMSO alone. It is suggested that structures sensitive to colchicine and CB do not play a major role in the control of surface lectin receptors in early embryonic cells.     

Factors affecting hemagglutination by concanavalin A and soybean agglutinin

While investigating the effect of temperature on hemagglutination by concanavalin A, we noted three factors that seriously interfere with the usual microscopic agglutination assay and produce misleading or ambiguous results. (1) Adherence of concanavalin A-treated erythrocytes to surfaces of plastic Petri dishes, especially at (2) commonly used cell densities, effectively prevents determination of agglutination. (3) In addition, incubation times usually used may be insufficient to demonstrate agglutination. Failure to account for these factors may explain the previously reported temperature-sensitive, concanavalin A-mediated agglutination of trypsinized erythrocytes and transformed cells (Vlodavsky, I., Inbar, M. and Sachs, L., (1972) Biochim. Biophys. Acta 274, 364–369). By controlling these factors, we demonstrated that concanavalin A does agglutinate trypsinized, human erythrocytes equally well at 24 and 4 °C.Investigation of the kinetics of erythrocyte agglutination by lectins revealed that the rate of agglutination by concanavalin A is markedly slower at lower temperatures while soybean agglutinin-mediated agglutination is faster at lower temperatures. Ultracentrifugation data indicate that at low temperature concanavalin A exists partially as a dimer (mol. wt 50 000) and at warmer temperatures exists mainly as a tetramer (mol. wt 100 000). The correlation of the effect of temperature on molecular weight with the agglutinating activity of concanavalin A suggests that temperature-dependent forms of concanavalin A may determine the rate of cell agglutination by this lectin. No temperature-dependent change in molecular form was observed with soybean agglutinin.     

Growth and the cell cycle of the yeast Saccharomyces cerevisiae. I. Slowing S phase or nuclear division decreases the G1 cell cycle period

To determine changes in distribution or mobility of cell-surface glycoconjugates during myogenesis the binding of fluorescein-conjugated plant lectins to myoblasts and myotubes of the L6 rat skeletal muscle cell line has been studied. Binding has been carried out at 4 degrees C on either live or glutaraldehyde-fixed cells. Fluorescein conjugates of soybean agglutinin (Fl-SBA), wheat germ agglutinin (Fl-WGA), concanavalin A (Fl-conA) and Lens culinaris agglutinin (Fl-LCA) produced predominantly uniform fluorescence on both live and fixed myoblasts. On fixed myotubes, Fl-LCA, Fl-conA and Fl-SBA again produced predominantly uniform fluorescence, whereas Fl-WGA showed a pattern of diffuse, irregular spots in addition to uniform fluorescence. Fl-conA, Fl-LCA and Fl-WGA binding to live myotubes resulted in patterns quite similar to those on fixed myotubes; the only differences being the presence of weak patterns of diffuse spots with Fl-LCA and Fl-conA and an enhanced pattern of diffuse spots with Fl-WGA. Fl-SBA, however, showed a unique pattern on live myotubes which consisted of discrete, round spots and minimal uniform fluorescence. With shorter labeling times, Fl-SBA produced relatively more prominent uniform fluorescence on live myotubes. It appears, therefore, that the native distribution of SBA, conA and LCA-binding sites is similar and predominantly random on L6 myoblasts and myotubes, whereas some WGA-binding sites may be aggregated on myotubes. The results also suggest that SBA-binding sites readily cluster at 4 degrees C on myotubes but not myoblasts, whereas the other lectin sites undergo little or no redistribution on either cell type. Thus the mobility of SBA-binding sites may increase with differentiation.     

Leishmania braziliensis: localization of glycoproteins in promastigotes

Two species of glycoproteins from Leishmania braziliensis promastigotes of apparent molecular weights of 53,000 (glycoprotein 53) and 47,000 (glycoprotein 47) were localized. Four lectins with different sugar specificities bound to the blotting sheet to which the electrophoretically separated materials were transferred. Concanavalin A and Ricinus communis agglutinin bound to the band of glycoprotein 53 and the lectin from Dolichos biflorus bound to the band of glycoprotein 47. Wheat germ agglutinin bound to the bands of both glycoproteins. Histochemical examinations using fluorescence labeled lectins demonstrated that the glycoproteins 53 and 47 were located on the cell surface and in the cytoplasm of promastigotes, respectively. The results are consistent with the result of agglutination test.     

Occurrence of natural lectin with bacterial agglutination property in the serum of lepidopteran pest,Parasa lepida

Insects depend on lectins for non‐self recognition and clearance of invading pathogens. Naturally occurring lectin showing specificity for galactose was purified from the serum of lepidopteran pest Parasa lepida by affinity chromatography using Sepharose 6B coupled with galactose as a gel matrix. Preliminary studies on crude serum agglutinin revealed that the agglutinin molecule showed varying degrees of specificity to avian and mammalian red blood cells tested. Among them, the highest titer of 128 was recorded against rabbit red blood cell type. The agglutinin molecule in the crude serum was stable up to 60°C and at pH between 6 and 9. Also, the hemagglutinating activity was neither dependent on divalent cations nor sensitive to ethylenediaminetetraacetic acid treatment. Galactose inhibited the hemagglutinating activity at minimum inhibitory concentration of 12.5 mM and hence it was used as a ligand for affinity chromatography. Native polyacrylamide gel electrophoresis analysis revealed a single band and the molecular weight of the lectin was found to be approximately 90 kDa. Bacterial agglutination activity of the purified lectin with two significant toxin bacteria, namely Salmonella typhi and Bacillus thuringiensis, was observed.     

A hemagglutinating substance in chitin.

Chitin from crustacean shells has often been used to isolate and purify plant lectins that have an affinity for poly-N-acetylglucosamine (poly-GlcNAc). When we used washed chitin from crab shells as an affinity medium to isolate a lectin from Pinus strobus L. (eastern white pine) ovules, we found that a substance having a strong capacity to agglutinate red blood cells was eluted from the chitin during a weak acid desorption step. The chitin agglutinin is a complex structure containing protein and poly-GlcNAc. Chitin samples from four biochemical suppliers were tested; all contained the elutable agglutinin. Acid (0.05 N HCl or 0.1 N acetic acid) appears to hydrolyze the material from the solid chitin. NaOH at 0.5 N does not remove the agglutinin. Since agglutination is the assay used to monitor lectin purification, care must be taken to avoid the native agglutinin if chitin is used as an affinity matrix.     

AGGLUTINATION OF THE CHLOROPHYCEAN FLAGELLATE DUNALIELLA TERTIOLECTA BY TREATMENT WITH LECTINS OR DIVALENT CATIONS AT ALKALINE pH1

Washed cells of Dunaliella tertiolecta Butcher became immobile and agglutinated upon exposure to 100–400 μ/mL lectins in NaCl solution. The agglutinations were strongest with Limulus polyphemus agglutinin and wheat-germ agglutinin, moderate with soybean agglutinin and weakest with Concanavalin A. All lectin-induced agglutinations were inhibited or mitigated by the simultaneous presence of specific lectin-binding sugars. The differential sensitivity of the alga to these lectins suggested that sialic acid and/or N-acetyl-D-glucosamine might be the predominant lectin-receptor sugars in the algal surface coat, with N-acetyl-D-galactosamine likely present as a lesser component. In the absence of lectins, the divalent cations Mg²⁺, Ca²⁺ or Mn²⁺ also caused agglutination, but this process required an alkaline pH of at least ca. 8.6–8.9. Such cation-induced agglutination was reversibly inhibited by the cation complexing agent EDTA as well as by lowering the pH below 8.0. SEM observations of the agglutinations revealed random flagellar attachments as well as direct body contact between agglutinated cells.     

四种蕈菌凝集素的筛选及活性检测

以长刺猴、白平菇、毛尖蘑、滑菇四种蕈菌为材料,经硫酸铵沉淀、透析,得到蛋白质提取液,用不同类型的红细胞检测凝集活性(人的A型、B型、AB型、O型血,兔血、鸡血、蛤蟆血)。结果表明,长刺猴、白平菇、滑菇3种蕈菌的提取物中均含有凝集素,桦树蘑对兔红细胞凝集性最强。凝集活性可分别被一种或多种类型糖所抑制。同时它们均表现出较好的热稳定性及pH耐受性,金属离子对凝集素的影响也相当大。     

Titration of the phase transition of phosphatidylserine bilayer membranes. Effects of pH, surface electrostatics, ion binding, and head-group hydration

The dependence of the gel-to-fluid phase transition temperature of dimyristoyl- and dipalmitoylphosphatidylserine bilayers on pH, NaCl concentration, and degree of hydration has been studied with differential scanning calorimetry and with spin-labels. On protonation of the carboxyl group (pK2app = 5.5), the transition temperature increases from 36 to 44 degrees C in the fully hydrated state of dimyristoylphosphatidylserine (from 54 to 62 degrees C for dipalmitoylphosphatidylserine), at ionic strength J = 0.1. In addition, at least two less hydrated states, differing progressively by 1 H2O/PS, are observed at low pH with transition temperatures of 48 and 52 degrees C for dimyristoyl- and 65 and 68.5 degrees C for dipalmitoylphosphatidylserine. On deprotonation of the amino group (pK3app = 11.55) the transition temperature decreases to approximately 15 degrees C for dimyristoyl- and 32 degrees C for dipalmitoylphosphatidylserine, and a pretransition is observed at approximately 6 degrees C (dimyristoylphosphatidylserine) and 21.5 degrees C (dipalmitoylphosphatidylserine), at J = 0.1. No titration of the transition is observed for the fully hydrated phosphate group down to pH less than or equal to 0.5, but it affinity for water binding decreases steeply at pH greater than or equal to 2.6. Increasing the NaCl concentration from 0.1 to 2.0 M increases the transition temperature of dimyristoyphosphatidylserine by approximately 8 degrees C at pH 7, by approximately 5 degrees at pH 13, and by approximately 0 degrees C at pH 1. These increases are attributed to the screening of the electrostatic titration-induced shifts in transition temperature. On a further increase of the NaCl concentration to 5.5 M, the transition temperature increases by an additional 9 degree C at pH 7, 13 degree C at pH 13, approximately 7 degree C in the fully hydrated state at pH 1, and approximately 4 and approximately 0 degree C in the two less hydrated states. These shifts are attributed to displacement of water of hydration by ion binding. From the salt dependence it is deduced that the transition temperature shift at the carboxyl titration can be accounted for completely by the surface charge and change in hydration of approximately 1 H2O/lipid, whereas that of the amino group titration arises mostly from other sources, probably hydrogen bonding. The shifts in pK (delta pK2 = 2.85, delta pK3 = 1.56) are consistent with a reduced polarity in the head-group region, corresponding to an effective dielectric constant epsilon approximately or equal to 30, together with surface potentials of psi congruent to -100 and -150 mV at the carboxyl and amino group pKs, respectively. The transition temperature of dimyristoylphosphatidylserine-water mixtures decreases by approximately 4 degree C each water/lipid molecule added, reaching a limiting value at a water content of approximately 9-10 H2O/lipid molecule.     

Trypanosoma rhodesiense bloodstream trypomastigote and culture procyclic cell surface carbohydrates

Bloodstream trypomastigote and culture procyclic (insect midgut) forms of a cloned T. rhodesiense variant (WRAT at 1) were tested for agglutination with the lectins concanavalin A (Con A), phytohemagglutinin P (PP), soybean agglutinin (SBA), fucose binding protein (FBP), wheat germ agglutinin (WGA), and castor bean lectin (RCA). Fluorescence-microscopic localization of lectin binding to both formalin-fixed trypomastigotes and red cells was determined with fluorescein isothiocyanate (FITC)-conjugated Con A, SBA, FBP, WGA, RCA, PNA (peanut agglutinin), DBA (Dolichos bifloris), and UEA (Ulex europaeus) lectins. Electron microscopic localization of lectin binding sites on bloodstream trypomastigotes was accomplished by the Con A-horseradish peroxidase-diamino-benzidine (HRP-DAB) technique, and by a Con A-biotin/avidin-ferritin method. Trypomastigotes, isolated by centrifugation or filtration through DEAE-cellulose or thawed after cryopreservation, were agglutinated by the lectins Con A and PP with agglutination strength scored as Con A greater than PP. No agglutination was observed in control preparations or with the lectins WGA, FBA or SBA. Red cells were agglutinated by all the lectins tested. Formalin-fixed bloodstream trypomastigotes bound FITC-Con A and FITC-RCA but not FITC-WAG, -SBA, -PNA, -UEA or -DBA lectins. All FITC-labeled lectins bound to red cells. Con A receptors, visualized by Con A-HRP-DAB and Con A-biotin/avidin-ferritin techniques, were distributed uniformly on T. rhodesiense bloodstream forms. No lectin receptors were visualized on control preparations. Culture procyclics lacked a cell surface coat and were agglutinated by Con A and WGA but not RCA, SBA, PP and FBP. Procyclics were not agglutinated by lectins in the presence of competing sugar at 0.25 M.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative lectin-binding and agglutination properties of the strain-specific,TA3-St,and the non-strain-specific,TA3-Ha,murine mammary carcinoma ascites sublines. Further studies of receptors in epiglycanin

The complex carbohydrates at the cell surfaces of two TA3, murine mammary carcinoma ascites sublines (the strain-specific, TA3-St subline and the nonstrain-specific, TA3-Ha line) were compared by binding studies with ¹²⁵I-labelled concanavalin A (con A), Ricinis communis agglutinin (RCA), and eel-serum agglutinin (ESA). The TA3-Ha cell bound equal amounts of con A, 1.5-fold more RCA, and 4-fold more ESA than the TA3-St cell. Binding-inhibition studies by these lectins and two others [wheat-germ agglutinin (WGA) and potato lectin (STA)] suggest complementary binding-sites between con A and both RCA and ESA. Quantitative agglutination studies with the five lectins, and inhibition determinations by both neuraminidase-treated and untreated epiglycanin revealed that TA3-St, but not TA3-Ha, cells were agglutinated by con A, and that epiglycanin inhibited this agglutination, as well as the agglutination of rabbit erythrocytes by con A. The presence of a con A receptor on epiglycanin was also suggested by the binding of epiglycanin to con A-Sepharose, and its specific elution with methyl α-d-manno-pyranoside. TA3-St cells were agglutinated at a 10-15-fold lower concentration of either STA or RCA than TA3-Ha cells, but both cells were agglutinated by the same concentration of WGA and ESA. Inhibition by epiglycanin of agglutination of TA3-St cells by either STA or ESA occurred at a concentration lower than that of TA3-Ha cells, but epiglycanin inhibited RCA agglutination of TA3-Ha cells at a concentration     

Effect of temperature, pH, and metal ion binding on the secondary structure of bacteriorhodopsin: FT-IR study of the melting and premelting transition temperatures

We have measured the temperature dependence of the FT-IR spectra of bacteriorhodopsin (bR) as a function of the pH and of the divalent cation regeneration with Ca(2+) and Mg(2+). It has been found that although the irreversible melting transition shows a strong dependence on the pH of the native bR, the premelting reversible transition at 78-80 degrees C shows very little variation over the pH range studied. It is further shown that the acid blue bR shows a red-shifted amide I spectrum at physiological temperature, which shows a more typical alpha-helical frequency component at 1652 cm(-)(1) and could be the reason for the observed reduction of its melting temperature and lack of an observed premelting transition. Furthermore, the thermal transitions for Ca(2+)- and Mg(2+)-regenerated bR (Ca-bR and Mg-bR, respectively) each show a premelting transition at the same 78-80 degrees C temperature as the native purple membrane, but the irreversible melting transition has a slight dependence on the cation identity. The pH dependence of the Ca(2+)-regenerated bR is studied, and neither transition varies over the pH range studied. These results are discussed in terms of the cation contribution to the secondary structural stability in bR.