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.     

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.     

Lectins as Probes to Pneumocystis carinii Surface Glycocomplexes

The binding characteristics of a panel of commercially available FITC-conjugated lectins to Pneumocystis carinii (Pc) were assessed by fluorescence microscopy and flow cytometry. Rat Pc obtained from infecteding homogenates were incubated with FTTC-conjugated lectins in a series of concentrations, counlerstained with propidium iodide, and analyzed for percent fluorescence and fluorescence intensity. All organisms bound concanavalin A and Wisteria floribunda agglutinin, 2 representatives of the glucose/mannose-binding group. From the lectin group specific for N-acctylglucosamine, Pc reacted more strongly with wheat germ agglutinin than with Solanum tuberosum agglutinin or Griffonia simpiicifolia II lectin. Pneumocystis treated with lectins specific for N-acetyl-D-galactosamine and galactose exhibited much variation; the cells reacted moderately well to soybean agglutinin and less to Bauhinia purpurea, Madura pomifera and Dolichos biflorus agglutinins and Giffonia simpiicifolia Hectin. Arachis hypogaea agglutinin, Viscum album agglutinin and Griffon'ui simpiicifolia I—β Section had not effect. The organisms reacted weakly with Ulex europeus I agglutinin which is specific for fucose and did not react with Limax ftavus lectin, which is specific for sialic acid. Competitive inhibition studies using relevant carbohydrates were performed to indicate that the positive reactions were specific. These studies should help to elucidate the mechanisms of attachment and pathogenesis of this organism.     

Lectins as probes to Pneumocystis carinii surface glycocomplexes

The binding characteristics of a panel of commercially available FITC-conjugated lectins to Pneumocystis carinii (Pc) were assessed by fluorescence microscopy and flow cytometry. Rat Pc obtained from infected lung homogenates were incubated with FITC-conjugated lectins in a series of concentrations, counterstained with propidium iodide, and analyzed for percent fluorescence and fluorescence intensity. All organisms bound concanavalin A and Wisteria floribunda agglutinin, 2 representatives of the glucose/mannose-binding group. From the lectin group specific for N-acetylglucosamine, Pc reacted more strongly with wheat germ agglutinin than with Solanum tuberosum agglutinin or Griffonia simplicifolia II lectin. Pneumocystis treated with lectins specific for N-acetyl-D-galactosamine and galactose exhibited much variation; the cells reacted moderately well to soybean agglutinin and less to Bauhinia purpurea, Maclura pomifera and Dolichos biflorus agglutinins and Griffonia simplicifolia I lectin. Arachis hypogaea agglutinin, Viscum album agglutinin and Griffonia simplicifolia I-beta 4 lectin had not effect. The organisms reacted weakly with Ulex europeus I agglutinin which is specific for fucose and did not react with Limax flavus lectin, which is specific for sialic acid. Competitive inhibition studies using relevant carbohydrates were performed to indicate that the positive reactions were specific. These studies should help to elucidate the mechanisms of attachment and pathogenesis of this organism.     

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.     

Surfactant-associated protein inhibits phospholipid secretion from type II cells

Secretion of [3H]phosphatidylcholine ([3H]PC) from isolated rat pulmonary type II epithelial cells was inhibited by the surfactant-associated protein of Mr = 35,000 (SAP-35) purified from canine lung surfactant. SAP-35 inhibited [3H]PC secretion in a dose-dependent manner and significantly inhibited basal, phorbol ester, beta-adrenergic, and P2-purinergic agonist-induced [3H]PC secretion. SAP-35 significantly inhibited [3H]PC secretion from 1 to 3 h after treatment. The IC50 for inhibition of [3H]PC secretion by canine SAP-35 was 1-5 X 10(-6) g/ml and was similar for inhibition of both basal and secretagogue-stimulated release. Heat denaturation of SAP-35, addition of monoclonal anti-SAP-35 antibody, reduction and alkylation of SAP-35, or association of SAP-35 with phospholipid vesicles reversed the inhibitory effect on secretagogue-induced secretion. Inhibitory effects of SAP-35 were observed 3 h after cells were washed with buffer that did not contain SAP-35. Although SAP-35 enhanced reassociation of surfactant phospholipid with isolated type II cells, its inhibitory effect on secretion of [3H]PC did not result from stimulation of reuptake of secreted [3H]PC by type II cells. The inhibition of phospholipid secretion by SAP-35 was also not due to inhibition of PC or disaturated PC synthesis by SAP-35. SAP-35, the major phospholipid-associated protein in pulmonary surfactant, is a potent inhibitor of surfactant secretion from type II cells in vitro and may play an important role in homeostasis of surfactant in the alveolar space.     

Peripheral Nervous System Myelin and Schwann Cell Glycoproteins: Identification by Lectin Binding and Partial Purification of a Peripheral Nervous System Myelin-Specific 170,000 Molecular Weight Glycoprotein

Radioiodinated lectins were used to detect glycoproteins of peripheral nervous system (PNS) myelin (rat, human, bovine) and cultured rat Schwann cells. Proteins were resolved by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis and transferred to nitrocellulose filters. The filters were overlaid with radioiodinated lectins of known saccharide affinities. These included concanavalin A, Helix pomatia, Limulus polyphemus, Maclura pomifera, peanut, soybean, Ulex europaeus, and wheat germ agglutinins. Inclusion of the appropriate monosaccharide in the overlay solution (0.2 M) inhibited lectin binding to the nitrocellulose-fixed proteins. Fluorography permitted identification of 26 myelin glycoproteins and many more in Schwann cells. All lectins labeled a band present in myelin, but not Schwann cells, corresponding to the major PNS myelin protein, P0. Our attention focused on a high-molecular-weight myelin glycoprotein [apparent molecular weight (Mr) 170,000], which appeared abundant by Coomassie Blue staining and which was heavily labeled by all lectins except concanavalin A. A protein with approximately this Mr and lectin-binding pattern was present in human and bovine PNS myelin as well, but not detected in rat Schwann cells, CNS myelin, liver and fibroblast homogenates, or cultured bovine oligodendroglia. Hence this 170,000 Mr glycoprotein is apparently unique to PNS myelin.     

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.     

Flow cytometric analyses of lectin binding to Pneumocystis carinii surface carbohydrates.

Pneumocystis carinii obtained from infected rat lung homogenates was incubated with fluorescein isothiocyanate-conjugated lectins, counterstained with the nuclear stain, propidium iodide (PI), and analyzed by dual parameter histograms for lectin-associated green and PI-associated red fluorescence using a fluorescence-activated cell sorter. The presence of glucose/mannose moieties was evidenced by the binding of all organisms to concanavalin A and Wisteria floribunda. From the lectin group specific for N-acetyl-D-glucosamine, P. carinii reacted strongly with wheat germ agglutinin and less intensely with Solanum tuberosum. Reaction with lectins specific for N-acetyl-D-galactosamine/galactose was variable, probably reflecting the secondary binding affinities of the lectins used. Soybean agglutinin, Bauhinia purpurea agglutinin, and Maclura pomifera agglutinin reacted moderately, whereas Dolichos biflorus agglutinin, and Griffonia simplicifolia I reacted less avidly. The organisms reacted partially with Ulex europaeus agglutinin, a lectin specific for fucose, and did not react well with Arachis hypogaea, Viscum album agglutinin, and Griffonia simplicifolia I beta 4, lectins specific for galactose. A very weak fluorescent signal was detected with Limax flavus agglutinin, suggesting little or no sialic acid was present. All lectin-binding reactions were confirmed for specificity by inhibition with the relevant carbohydrates. Flow cytometric analysis of lung-derived Pneumocystis organisms stained with fluorescent surface and nuclear dyes provides a rapid method for characterization of large parasite populations.     

Active cell aggregation by immature rat Sertoli cells in primary culture: a role for cell surface glycoproteins.

Follicle stimulating hormone (FSH) stimulates “colony formation” by immature rat Sertoli cells in primary culture. “Colony formation” involves cell aggregation. Consequently, the involvement of cell surface glycoproteins in cell aggregation was investigated by treatment of dissociated 10-day rat testis cells with sodium metaperiodate, glucosamine, various lectins, tunicamycin, and puromycin. Treatment of control cultures with 5 μM glucosamine stimulated cell aggregation; however, glucosamine did not affect FSH-stimulated cultures. Treatment of dissociated testis cells with 5 μM sodium metaperiodate, 10 μg/ml castor bean agglutinin (ricin), or 2.5 μg/ml horseshoe crab agglutinin inhibited FSH stimulation of cell aggregation. A similar inhibition of cell aggregation was observed following addition of 10 μg/ml puromycin or tunicamycin to culture media from 0- to 18-hours incubation. Treatment with soybean agglutinin, concanavalin A, or wheat germ agglutinin had no effect. The galactose-specific lectins, Ricin, Ricinus communis agglutinin I, and Bendeirea simplicifolia agglutinin, inhibit the FSH stimulation of ³H-aminoacid incorporation as well as cell aggregation in 24-hour cultres. The inhibition of cell aggregation by sodium metaperiodate treatment was reversed with 5 μM sodium borohydride reduction. Sodium metaperiodate treatment did not alter cell viability (as assayed with trypan blue dye exclusion), did not alter cell attachment, nor significantly decrease ¹²⁵I-FSH binding by cultured testis cells. The results suggest that FSH stimulation of cell aggregation by immature rat Sertoli cells requires cell surface glycoprotein interactions. Furthermore, the specificity of lectin inhibition suggests that glycoproteins with terminal galactose and sialic acid residues are required for the FSH induction of cell aggregation.     

Effect of trypsinization on lectin binding to germ cells from ICR and T/t6 mice

Flow cytometry was used to quantify the binding of fluorescein isothiocyanate (FITC)-labeled lectins to testis cells from ICR and T/t6 mice before and after trypsin treatment. Soybean agglutinin, wheat germ agglutinin, and concanavalin A bound well to testis cells of both mouse strains. Limax flavus agglutinin (LFA) bound very slightly and Ulex europeas agglutinin (UEA) did not bind at all. Trypsinization increased binding of soybean agglutinin and decreased binding of wheat germ agglutinin in both mouse strains, providing evidence for masked carbohydrate-binding sites on the surface of germ cells. It did not affect binding of the other lectins. Trypsin treatment was an attempt to increase lectin binding, particularly the binding of LFA and UEA to the reported T/t-specific carbohydrates, sialic acid, and L-fucose, respectively. These studies indicate that the T/t6 locus alleles do not alter the surface carbohydrate content of testis cells sufficiently to be detected by lectin-binding differences.     

High density of transmembrane glycoproteins on the flagellar surface of boar sperm cells

Membrane halves of boar sperm flagella were produced by freeze-fracture and labeled in situ with concanavalin A and wheat germ agglutinin; the lectins were visualized with protein-gold complexes. Concanavalin A and wheat germ agglutinin binding sites partition with both protoplasmic and exoplasmic halves of the membrane. A high density of lectin marking was found on protoplasmic membrane halves; we conclude that the label corresponds to transmembrane glycoproteins that, on freeze-fracture, are dragged across the outer (exoplasmic) half of the phospholipid bilayer. Our demonstration of numerous transmembrane proteins in sperm flagella offers the structural setting for previous models on flagellar surface motility that postulate accessibility of motile membrane components to the submembranous cytoskeleton.     

Lectin affinity chromatography of cell surface proteins of novikoff tumor cells

Novikoff hepatocellular carcinoma cells were radioiodinated by a cell surface-specific method using lactoperoxid ase/¹²⁵I. The iodinated proteins were solubilized in 0.5% Nonidet P-40 and subjected to affinity chromatography on Sepharose-conjugated lectins (Ricinus communis agglutinins I or II, soybean agglutinin, concanavalin A, or wheat germ agglutinin) and analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Almost all the iodinated proteins bound to one or more of the Sepharose-conjugated lectins, presumptive evidence that these peptides are glycosylated. Lectin affinity chromatography resolved defined subsets of iodinated glycoproteins and suggested that certain glycoproteins could be fractionated on the basis of heterogeneity of their heterosaccharide moieties. Incubation of the iodinated cells with neuraminidase resulted in increased binding of iodinated proteins to Sepharose-conjugated Ricinus communis agglutinins I and II and soybean agglutinin and decreased binding to Sepharose-conjugated wheat germ agglutinin. Binding of iodinated proteins to concanavalin A was unaffected by neuraminidase treatment of the cells. These studies demonstrate the utility of lectins for the multicomponent analysis of plasma membrane proteins.     

Distinguishing benign from malignant pleural effusions by lectin immunocytochemistry

Since distinguishing malignant from benign cells in pleural effusions can be difficult, with reactive mesothelial cells simulating adenocarcinoma cells, the binding patterns of a battery of lectins on cells in eight benign and eight malignant effusions were studied using the avidin-biotin peroxidase complex method. The following lectins were used: concanavalin A, Dolichos biflorus agglutinin, peanut agglutinin, Phaseolus vulgaris agglutinin, Ricinus communis germ agglutinin, soybean agglutinin, Ulex europeaus agglutinin (UEA) and wheat germ agglutinin. Several patterns of staining were seen with the lectins, but only UEA was helpful in distinguishing between benign and malignant effusions. Sixty percent of the adenocarcinomas stained with UEA, whereas none of the cells in the benign effusions did. These results imply that UEA positivity is indicative of carcinoma and can be useful in separating reactive or atypical mesothelial cells from adenocarcinoma 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.     

Lectin binding to newt epidermis: fluorescent localization and effects on motility

The ability of seven lectins to bind to newt epidermal cells and influence their motility was examined. Of the seven fluoresceinated lectins applied to frozen sections containing intact newt skin and migrating epidermis (wound epithelium), only Con A (concanavalin A), WGA (wheat germ agglutinin), and PNA (peanut agglutinin) produced detectable epidermal fluorescence. Con A and WGA each heavily labeled all layers of intact epidermis, but PNA bound only to the more superficial layers. In contrast to a single population of labeled cells in migrating epidermal sheets after treatment with Con A, there were both labeled and unlabeled cells after exposure to either WGA or PNA. The wound bed was labeled by both Con A and WGA, but not by PNA. DBA (Dolichos bifloris agglutinin), RCA I (Ricinus communis agglutinin), and UEA (Ulex europaeus agglutinin), did not produce significant fluorescence with either migrating or intact epidermis. In general, inhibitory effects on epidermal motility correlated with the binding studies. Thus, Con A, WGA, and PNA, the lectins which clearly bound to the epidermis, all produced a concentration-dependent depression in the rate of epidermal wound closure. RCA was somewhat paradoxical in that it was moderately inhibitory despite showing essentially no binding. The effects of SBA and UEA were equivocal. DBA had no effect. These results indicate that the inhibition of motility produced by Con A that we have described previously is not peculiar to this mannose-binding lectin, but is shared by at least one lectin with an affinity for D-GlcNAc (WGA), and one with an affinity for B-D-Gal(1-3)-D-GalNAc (PNA).(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of lectins with human platelets. Effects on platelet stimulation by thrombin and ristocetin.

Wheat germ agglutinin induced aggregation and secretion of fresh platelets. Aggregation, but not secretion of serotonin by platelets in plasma, by the lectin was inhibited by 5 mM EDTA. Further, the lectin-induced stimulation of fresh platelets was blocked by prostaglandin E1. Thus, this lectin stimulates platelets by a mechanism which closely mimics thrombin activation and is independent of intercellular crosslinking. Lentil lectin did not stimulate platelets. Each platelet contained about 6 . 10(-5) binding sites for the lectins with an apparent dissociation constant of 3.0 . 10(-7) M. Wheat germ agglutinin, which binds mainly to glycoprotein I (Mr 150 000), increased the subsequent binding of thrombin to fixed platelets while lentil lectin was without effect. It appears that thrombin and wheat germ agglutinin bind to independent but interacting sites. Wheat germ agglutinin, but neither thrombin nor lentil lectin, inhibited the agglutination of platelets by ristocetin. Further, rat platelets were not aggregated by either ristocetin or wheat germ agglutinin. It appears that the interaction sites of ristocetin and wheat germ agglutinin on platelets are overlapping.     

Isolation of lectins of different specificities on a single affinity adsorbent.

Affinity chromatography on Sepharose-fetuin columns was used in a single step procedure to isolate the lectins concanavalin A, Favin, phytohemagglutinin, wheat germ agglutinin, and Limulus hemagglutinin. New lectins with unknown binding specificities were also purified by the same procedure from extracts of small California white beans, Idaho red beans, and white pea beans. The purified lectins exhibited different cell surface mapping properties on erythrocytes, lymphocytes, and sperm cells. It was particularly striking that neither 131I-labeled concanavalin nor 125I-labeled wheat germ agglutinin had any effect on the binding of the other to mouse spleen cells. In accord with this observation, gel electrophoretic analysis of radiolabeled lymphocyte receptors for these two lecithins yielded different patterns. These results indicate that highly purified lectins prepared by affinity chromatography on the same adsorbent can possess strikingly different binding specificities for cell surface receptors.     

Characterization of the Carbohydrate Chain on the Substance P Receptor in the Rat Brain Cortex: Effect of Lectins on [3H]Substance P Binding

The characteristics of the carbohydrate chain on the rat cerebral cortical substance P (SP) receptor were studied. We examined the effects of pretreatment with three lectins (concanavalin A, wheat germ agglutinin, lens culinaris agglutinin) on the [3H]SP binding activities. Each lectin can bind to the specific carbohydrate chain. Among these lectins, only concanavalin A inhibited specific [3H]SP binding by reducing the affinity of the binding sites. The inhibitory action of concanavalin A was dose-dependent and diminished by the addition of alpha-methyl-D-mannoside. The present results suggest that the rat cortical SP receptor has either a biantennary complex-type or a high mannose-type of carbohydrate chain, and that the carbohydrate chain is implicated in the SP binding activity of the SP receptor system.     

Conjugation in Tetrahymena pyriformis. The effect of polylysine, concanavalin A, and bivalent metals on the conjugation process

The polycation polylysine, at different degrees of polymerization, was found to cause a marked inhibition of the conjugation process. Inhibition of conjugation by polylysine was highly dependent on the molecular weight of the polymer. When polylysine of a mol wt of 1,250 (degree of polymerization=6) was used, a concentration of 1.6 X 10(-5) M was required for a complete inhibition of conjugation, while only 2 X 10(-7) M of polylysine of a mol wt of 71,000 (degree of polymerization=340) was needed for the same effect. Polyaspartic acid prevented the inhibition of conjugation by polylysein. Chelators of bivalent metals such as O-phenanthroline (10(-3) M), EDTA (10(-3) M), and EGTA (5 X 10(-3) M) strongly inhibit the conjugation process in Tetrahymena pyriformis. The inhibition was partially prevented when bivalent metals such as Zn++, Fe++, and Ca++ were added together with the chelators. The lectin concanavalin A (25 mug/ml) completely prevented the conjugation process, while other lectins, such as phytohemagglutinin (500 mug/ml), soybean agglutinin (75 mug/ml) and wheat germ agglutinin (250 mug/ml) had no effect. Inhibition of conjugation by concanavalin A is completely reversible by 40 mM of alpha-methyl-D-mannoside.