Ricin Agglutinin Research Articles

Purification of biologically active Epstein-Barr virus by affinity chromatography and non-ionic density gradient centrifugation

Epstein-Barr virus was purified by affinity chromatography on ricin agglutinin Sepharose followed by non-ionic density gradient centrifugation on Nycodenz. The purified virus was highly active in three biological assays: stimulation of immunoglobulin synthesis by B lymphocytes, transformation of B lymphocytes, and superinfection of Raji cells, as well as in an indirect immunofluorescent binding assay. Electron microscopy revealed intact viral particles free of contaminating membranous structures. CsCl density gradient analysis of nick-translated DNA showed material only at the expected viral density with no detectable material at the density of cellular DNA. SDS gel electrophoresis of radioiodinated virus revealed the characteristic viral polypeptides. This method produces highly purified, biologically active virions with an overall recovery of about 30%.

Ricin-like plant toxins are evolutionarily related to single-chain ribosome-inhibiting proteins from Phytolacca.

A comparison has been made of the amino-terminal sequences of a number of ribosome-inhibiting proteins (RIPs) and cytotoxins. These include the monomeric enzymes PAP, PAP-S, PAP-II, and dodecandrin and the enzymatic A chains from the heterodimeric toxins ricin and modeccin. We show that these proteins have all evolved from a single ancestor. A statistical analysis is used to show the likely evolutionary relationship among the proteins. A similar analysis was performed on the amino-terminal sequences of ricin, Ricinus agglutinin, and modeccin B chains. These are galactoside-binding proteins associated with the A-chain enzymes. From the two comparisons we propose a scheme for the development of two major classes of proteins. The RIP and sugar-binding genes probably evolved independently. In some plant lines the genes never fused, although the RIP gene replicated and developed into several proteins expressed at various stages of plant maturation. In another line the RIP gene fused with a sugar binding (B-chain) gene to form the class of heterodimeric toxins. In some species this fused gene appears to have multiplied, one or more of the toxin genes mutating to code for a self-dimerizing agglutinin molecule.

Threshold effects on the lectin-mediated aggregation of liposomes: Influence of the diameter of the liposomes

It had previously been found that small unilamellar liposomes of ca. 0.03 μm diameter which bear synthetic cholesterol-containing glycolipids may be aggregated by an appropriate lectin [8]. Where studied, threshold effects have been observed in that the amount of glycolipid incorporated in the liposomes must exceed a certain minimum concentration in order for aggregation to occur [3, 8, 9, 13, 14]. Threshold effects of this type may be important in mediating cell-cell and virus-cell interactions. However, before studies with small unilamellar liposomes are useful as a model for these recognition and binding phenomena, it must be shown that the observed threshold effects are not associated with the very small radius of curvature of these liposomes. This article reports that larger liposomes of average diameter 0.26 and 0.45 μm which contain the synthetic glycolipidl also show threshold effects when aggregated with the galactose binding lectin ricin agglutinin. Under conditions where more than 1% (mole) glycolipid is required to support the aggregation of the smallest liposomes, those of intermediate size require only 0.18% (mole) while the largest liposomes examined require between 0.095 and 0.15% (mole) depending on the method of preparation.

Chemical modification studies on Ricinus communis (Castor Bean) agglutinin.

Ricinus communis agglutinin was subjected to various chemical treatments and the effect on its hemagglutinating and saccharide-binding properties was studied. Acetylation, succinylation and citraconylation led to a complete loss in the activity of the agglutinin, whereas reductive methylation had no effect on the activity, showing that charged amino groups were involved in the hemagglutinating and saccharide-binding activity of Ricinus agglutinin. Modification of tryptophyl, arginyl and carboxyl-group-containing residues did not lead to any loss in the activity of the agglutinin. Acetylation of tyrosyl groups with N-acetylimidazole strongly reduced the hemagglutinating and saccharide-binding property of Ricinus agglutinin. The loss in activity was restored on deacetylation of the tyrosyl groups. Modification of tyrosyl residues also led to a change in the immunological properties of the agglutinin. The initial rate of modification of tyrosyl and amino groups and the concomitant loss of activity was reduced in the presence of lactose.

In 'undifferentiated' PC12 cells, GERL is not segregated from the Golgi apparatus.

The present study examines the endocytosis of conjugates of horseradish peroxidase (HRP) with ricin and wheat germ agglutinin (WGA) in rat adrenal pheochromocytoma cells (PC 12 line) cultured in the absence of nerve growth factor (NGF). In these cells acid phosphatase (ACPase) activity is not confined to a single cisterna and vesicles at the transaspect (mature face) of the Golgi apparatus which correspond to GERL of cultured neurons, neuroblastoma and other cell types. But ACPase is found in several cisternae of the Golgi apparatus as well as in lysosomes. On the other hand, thiamine pyrophosphatase activity, is found in a typical location within two or three cisternae of the Golgi apparatus near its transaspect. Following adsorptive endocytosis of HRP-labelled lectins (ricin-HRP or WGA-HRP) into PC12 cells, a reaction product is seen in dense bodies as well as in small vesicles and tubules throughout the cytoplasm, at the periphery of large vacuoles, in smooth and coated vesicles and tubules near the Golgi apparatus and in anastomosing tubules. The cisternae of the Golgi apparatus are not involved in the endocytosis of lectin-HRP. We conclude that in PC12 cells grown without NGF, unlike the case of cultured neurons and neuroblastoma cells, GERL is not segregated from the Golgi apparatus by either ACPase cytochemistry, or by the functional criterion of endocytosis of lectin-HRP conjugates.

Surface reaction ofLeishmania

A panel of lectins was used to determine surface sugar residues on promastigotes of twelve isolates of Leishmania, grouped on the basis of their excreted factor (EF) serotype. Inhibiting sugars were used to determine the specificity of the lectins. Serotype A reacted with soybean agglutinin, indicating a specificity for N-acetyl-D-galactosamine, while B serotypes lacked this determinant. Subserotype A2 was differentiated by its greater reactivity to Concanavalin A, specific for α-D-glucose and α-D-mannose. Ricinus agglutinin, specific for β-D-galactose and N-acetyl- galactosamine, gave the strongest reactions with the A1 and B1 subserotypes. The mixed sub-serotypes, A2B2 and A3B2, were differentiated by peanut agglutinin, which is specific for galactose. Wheat germ agglutinin and Phytohaemagglutinin-P did not agglutinate the promastigotes of any of the strains tested.

Lectin Derivatives of Methotrexate and Chlorambucil as Chemotherapeutic Agents<xref ref-type="fn" rid="FN2">2</xref>

Methotrexate and chlorambucil, each covalently linked to either abrus agglutinin, abrin, ricinus agglutinin, ricin, or concanavalin A, were prepared. A single dose of the derivative injected ip into sarcoma 180-bearing noninbred N:NIH(S) white mice resulted in prolongation of the survival time and was more effective than an equivalent dose of free drug and lectin. Drug-lectin also showed a higher inhibitory effect on the DNA biosynthesis of the tumor cell than did an equivalent dose of the free drug and lectin.

Fluorescence-polarization studies on binding of 4-methylumbelliferyl beta-D-galactopyranoside to Ricinus communis (castor-bean) agglutinin.

The binding of Ricinus communis (castor-bean) agglutinin 1 to saccharides was studied by equilibrium dialysis and fluorescence polarization by using the fluorescently labelled sugar 4-methylumbelliferyl beta-D-galactopyranoside. No appreciable change in ligand fluorescence of 4-methylumbelliferyl beta-D-galactopyranoside was considerably polarized on its binding to the lectin. The association constants obtained by Scatchard analysis of equilibrium-dialysis and fluorescence-polarization data do not differ much from each other, and at 25 degrees C, Ka = 2.4 (+/- 0.2) X 10(4)M-1. These values agree reasonably well with that reported in the literature for Ricinus agglutinin 1. The number of binding sites obtained by the different experimental procedures is 1.94 +/- 0.1 per molecule of 120 000 daltons and is equal to the reported value of 2. The consistency in the values of Ka and number of binding sites indicate the absence of additional subsites on Ricinus agglutinin 1 for its specific sugars. In addition, the excellent agreement between the binding parameters obtained by equilibrium dialysis and fluorescence polarization indicate the potential of ligand-fluorescence-polarization measurements in the investigation of lectin-sugar interactions.

Lectin staining of carbohydrates of haemic cells. II. The cells of normal lymphoid origin, of lymphatic leukaemias and related diseases.

Normal and leukaemic lymphoid cells, both human and murine, were stained for specific carbohydrates with three fluorescein-labelled lectins: Aprotinin for sialyl (or uronyl) groups: Ricinus agglutinin for galactosyl groups; and Concanavalin A for mannosyl (or glucosyl) groups. The method gives permanent preparations of sections from methanol fixed, paraffin embedded tissues, from blood and bone marrow films or touch preparations of lymph nodes that were methanol fixed. Whereas normal lymphocytes and lymphoblasts reacted strongly for sialyl groups, lymphoblasts of acute lymphoblastic leukaemia and lymphocytes of chronic lymphocytic leukaemia gave a much weaker reaction. The same was the case of the lymphocytes of the Sézary variant and the lymphocytes of macroglobulinaemia. The fine processes of the cells of hairy cell leukaemia stained well for sialyl groups. No obvious differences were detected between normal monocytes and the cells of monocytic leukaemia, nor between normal plasma cells and those of myeloma.

Physical studies on three lectins from the seeds of Abrus precatorius

The physical properties of three lectins from the seeds of the Abrus precatorius plant, abrin C, abrin A and the Abrus agglutinin, were studied. All three exhibited similar circular dichroic (CD) spectra in the near-ultraviolet having negative maxima at 286 and 293 nm. In addition, d-galactose induced similar conformational alterations in the three proteins as observed through changes in the near-ultraviolet CD from 280 to 295 nm. The near-ultraviolet CD spectrum of the toxic subunit of abrin C was very different from that of the parent molecule. The fluorescence emission spectra of the three proteins were also studied. All exhibited fluorescence near 335 nm which is quenched 9% by galactose. Iodide quenching of fluorescence using the Stern-Volmer analysis indicated different tryptophan accessibilities in the presence and absence of d-galactose for the Abrus agglutinin. The result suggest that there is a saccharide-induced conformational change which buries several partially exposed tryptophan residues. A comparable analysis of the closely related Ricinus agglutinin revealed that its tryptophan residues are more buried than those of the Abrus agglutinin and, unlike the Abrus agglutinin, there was no saccharide-induced change in tryptophan accessibility.

Formation of a hybrid toxin from ricin agglutinin and a non-toxic mutant protein of diphtheria toxin

CRM45, a non-toxic mutant protein of diphtheria toxin, is treated with glutaraldehyde and conjugated to ricin agglutinin. The hybrid protein thus obtained is purified by gel filtration and affinity chromatography. The toxicity of the purified hybrid toxin is about 8–10 times grater than that of ricin agglutinin when tested in mice and cultured L cells.

35] Ricin and ricinus agglutinin, toxic lectins from castor bean

35] Ricin and ricinus agglutinin, toxic lectins from castor bean

Isolation of IgG3 from Polyclonal Human IgG by Affinity Chromatography with Ricinus Agglutinin

Ricinus agglutinin purified to homogeneity reacts with serum glycoproteins containing terminal nonreducing galactose residues, including about 10% polyclonal IgG. To study the IgG subclass distribution of this reaction, four monoclonal IgG1 proteins, two IgG2, four IgG3, and two IgG4 proteins were tested by affinity chromatography experiments with insolubilized ricinus agglutinin. Only the IgG3 proteins reacted, and the reactive sites were localized to the Fc fragment. Passage of polyclonal human IgG through a ricinus agglutinin–Sepharose column, followed by elution with lactose, permitted the isolation of IgG3 from normal polyclonal IgG.

Internalization of lectins in neuronal GERL.

Conjugates of ricin agglutinin and phytohemagglutinin with horseradish peroxidase (HRP) were used for a cytochemical study of internalization of their plasma membrane "receptors" in cultured isolated mouse dorsal root ganglion neurons. Labeling of cells with lectin-HRP was done at 4 degrees C, and internalization was performed at 37 degrees C in a culture medium free of lectin-HRP. 15-20 min after incubation at 37 degrees C, lectin-HRP receptor complexes were seen in vesicles or tubules located near the plasma membrane. After 1-3 h at 37 degrees C, lectin-HRP-receptor complexes accumulated in vesicles and tubules corresponding to acid phosphatase-rich vesicles and tubules (GERL) at the trans aspect of the Golgi apparatus. A few coated vesicles and probably some dense bodies contained HRP after 3-6 h of incubation at 37 degrees C. Soluble HRP was not endocytosed under the conditions of this experiment or when it was present in the incubation medium at 37 degrees C. Internalization of lectin-HRP-receptor conjugates was decreased or inhibited by mitochondrial respiration inhibitors but not by cytochalasin B or colchicine. These studies indicate that lectin-labeled plasma membrane moieties of neurons are endocytosed primarily in elements of GERL.

Structure and toxicity of pure ricinus agglutinin

Highly purified ricinus agglutinin was found to inhibit protein synthesis in HeLa cells. This effect could be prevented by the addition of the specific antiricinus agglutinin serum, whereas specific anti-ricin serum did not protect the cells, demonstrating that the toxic effect of ricinus agglutinin is not due to contamination with ricin. After reduction of ricinus agglutinin with 2-mercaptoethanol in the presence of 0.5 M galactose the constituent peptide chains were separated by chromatography on a DE-52 column. The B′-chain passed through the column, whereas the A′-chain bound and was eluted with a salt gradient. The B′-chain was further purified by chromatography on a CM-52 column. The shortest chain, the A′-chain, was found to inhibit cell-free protein synthesis, whereas the B′-chain did not have this ability. On the other hand, the B′-chain was able to induce agglutination of erythrocytes when tested together with anti-ricinus agglutinin serum indicating that the B′-chains bind to the cells. Ouchterlony immunodiffusion tests with crude anti-ricin and anti-ricinus agglutinin sera revealed that the two constituent chains of ricinus agglutinin are immunologically partial identical and that they also show reaction of partial identity with both chains of the toxic lectin ricin. The data indicate that a similar structure-function relationship exists in ricinus agglutinin as in ricin. The reason for the much lower toxicity of ricinus agglutinin than of ricin in living animals is discussed.

Interactions between ricinus agglutinin and human IgA.

By affinity chromatography on Sepharose columns containing insolubilized ricinus agglutinin, all immunoglobulins of the IgA2 subclass and the polymers of IgA1 were retained and could be eluted with lactose. Monomeric IgA1 showed a more heterogeneous reaction with ricinus agglutinin. The major part was unretarded, whereas some of it was weakly bound to the column and could be eluted with lactose.

Lectin Binding to Human Breast Tumor Cells

A human breast carcinoma cell line (BOT-2) was derived from an infiltrating duct carcinoma (1). These cells were shown to have antigens that selectively bound antibodies from breast cancer patient sera (2). Furthermore, these tumor specific antigens could be removed from the living cells by low frequency sonication and have been partially characterized (3). These proteins have been shown to be around 100,000 MW and contain approximately 6% hexose and hexosamines. However, only the hexosamines appear to be available for lectin binding. This study was designed to use Concanavalin A (Con A) and Ricinus Communis (Ricin) agglutinin for the topagraphical localization of D-mannopyranosyl or glucopyranosyl and D-galactopyranosyl or DN- acetyl glactopyranosyl configurations on BOT-2 cell surfaces.

Ribosome inactivation by the toxic lectins abrin and ricin. Kinetics of the enzymic activity of the toxin A-chains.

A sensitive test system for toxin-treated ribosomes was worked out by treating rabbit reticulocyte ribosomes with abrin A-chain, ricin A-chain or ricinus agglutinin A-chain, adding neutralizing amounts of specific antitoxins and testing for polyphenylalanine-synthesizing activity in a system where the concentration of elongation factors and ribosomes were varied. The strongest inhibition was obtained in the presence of low concentrations of elongation factor (EF-2). The activity of the ribosomes decreased with time of incubation with the toxin A-chains. Addition of anti-toxins stopped further inactivation. In systems containing untreated and toxin-treated ribosomes the ability to polymerize phenylalanine was proportional to the concentration of untreated ribosomes. There was a linear relationship between toxin A-chain concentration and the number of ribosomes inactivated per minute. The inactivation rate increased with temperature, and the estimated activation energy was 10.6 kcal (44.3 kJ). Linewaver-Burk plots of the data obtained by incubating various ribosome concentrations with toxins indicated a molecular activity of about 1500 ribosomes/minute for abrin and ricin A-chains and 100 ribosomes/minute for ricinus agglutinin A-chain. The apparent Michaelis constant was 0.1-0.2 muM for all three A-chains. The activity of the A-chains in the intact cell is discussed.

Interactions between Ricinus Agglutinin and Human Plasma Proteins

Ricinus agglutinin purified to homogeneity precipitates certain serum glyco‐proteins containing terminal non‐reducing galactose residues. Immunoelec‐trophoresis of human sera in agarose gel with ricinus agglutinin in the antibody trough gave 2 fusing precipitin lines due to reaction with IgM and haptoglobin. All of 50 monoclonal IgM proteins precipitated with ricinus agglutinin, and the reactive sites were localized to the Fc μ fragment. By affinity chromatography on Sepharose columns containing insolubilized ricinus agglutinin prealbumin, albumin, α1‐lipoprotein, α1‐antitrypsin, α1‐acid glycoprotein, α1‐antichymotrypsin, α2HS glycoprotein, Ge‐globulin, caerulo‐plasmin, β‐lipoprotein, β1 C‐globulin, and transferrin were not retained, whereas α2‐macroglobulin, haptoglobin, IgM, IgA, and IgG were retained and could be eluted with lactose. Fused rocket immunoelectrophoresis revealed varying degrees of microheterogeneity among the latter proteins; almost all of IgM but only a small fraction of polyclonal IgG was retained on the ricinus column.

Conformation-Dependent Antigenic Determinants in the Toxic Lectin Ricin

The major part of the ricin-precipitable antibodies in sera produced by immunizing rabbits with formaldehyde-treated ricin is precipitated also by the isolated ricin A and B chains. In contrast, in antisera produced by immunizing with formaldehyde-treated ricinus agglutinin only a small part of the antibodies cross-reacting with ricin can be precipitated by the isolated A and B chains, or bound to immunoabsorbents containing the isolated ricin chains. In immunodiffusion studies with anti-ricinus agglutinin sera, a star-shaped precipitate was formed when isolated A and B chains recombined to form intact ricin. Both anti-ricin and anti-ricinus agglutinin sera neutralized effectively the ability of ricin to inhibit protein synthesis in HeLa cells. Anti-ricin serum also neutralized the inhibitory effect of the isolated A chain on protein synthesis in a cell-free system and the ability of the isolated B chain to induce indirect hemagglutination. In contrast, antiricinus agglutinin serum did not neutralize the biologic activities of the isolated ricin A and B chains. Anti-ricinus agglutinin serum formed a precipitate with the hybrid ricin A chain/abrin B chain, and protected against the toxic effect on HeLa cells of this hybrid, indicating conformational changes of ricin A chain upon binding to the B chain. It is concluded that the anti-ricinus agglutinin serum contains antibodies directed against conformational determinants present on intact ricin, but not present or exposed in the isolated A and B chains. At least part of these conformational determinants appears to be carried by the A chain.