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1.
Etsuko Taira Naonobu Yoshizuka Gunki Funatsu Masaru Funatsu 《Bioscience, biotechnology, and biochemistry》2013,77(10):1927-1932
Effects of physical and chemical treatments on the cytoagglutinating activity, toxicity and inhibitory activity of cell-free protein synthesis of ricin D or its constituent polypeptide chains were investigated. The results indicated that the isolated polypeptide chains were much less stable than intact ricin D in acidic pH, heating as well as chemicals, and the Ala chain was more unstable than the lie chain.Chemical modifications of ricin D with specific reagents revealed that the tryptophan and tyrosine residues as well as the carboxyl groups participated in the phenomena of cyto- agglutination and toxic action of ricin D, whereas arginine residues were considered not to be directly involved. Trinitrophenylation of free amino groups did not result in a loss of cytoagglutinating activity, whereas caused a loss of toxicity, suggesting that free amino groups in the lie chain were involved in the toxic action of ricin D. 相似文献
2.
Kenji Hara Masatsune Ishiguro Gunki Funatsu Masaru Funatsu 《Bioscience, biotechnology, and biochemistry》2013,77(8):1631-1637
Some physical, chemical and physiological properties of two S-CM subunits (S-CM Ile and S-CM Ala chain) of ricin D were studied. Physical properties of subunits are summerized in Table I. The S-CM subunits consisted of 244 (S-CM Ile chain) and 254 (S-CM Ala chain) amino acid residues. By the specific cleavage of the single intermolecular disulfide bond of ricin D, no remarkable change in conformation of polypeptide chains of ricin D was detected, but the toxicity was markedly decreased. The toxicity of ricin D is due to the quaternary structure of the ricin D molecule. 相似文献
3.
Luisa Montesano Daniel Cawley Harvey R. Herschman 《Biochemical and biophysical research communications》1982,109(1):7-13
Ricin was reacted with disuccinimidyl suberate, to yield a molecule in which the A and B chains were covalently cross-linked through a non-reducible bond. After purification, this cross-linked ricin analog was unable to inhibit protein synthesis in a cell-free translation system from rat liver. In contrast, after modification with the cross-linking agent the isolated ricin A chain maintained its inhibitory activity. These results support the view that ricin must be cleaved into its constituent polypeptide chains to elicit its toxicity, and suggest that reduction of the disulfide bond alone is not sufficient for ribosome inactivation . 相似文献
4.
Two constituent polypeptide chains were isolated from performic acid-oxidized ricin D by DEAE-cellulose column chromatography in phosphate buffer, pH 7.0, containing 8m urea or from reduced-carboxymethylated ricin D by gel filtration on Sephadex G-75 followed by DEAE- cellulose column chromatography in Tris-HCl buffer, pH 8.5. Amino acid analyses of the isolated two chains revealed that they were distinct molecules possessing similar molecular weights of near 30,000 and linked by one disulfide bond in ricin D. 相似文献
5.
Iodinations of two Ricinus communis lectins, ricin D and hemagglutinin (CBH), with potassium iodide at pH 7.0 and 0°C led to inactivation of the cytoagglutinating activity on sarcoma 180 ascites tumor cells as well as the toxicity to HeLa cells of ricin D, whereas the cytoagglutinating activity of CBH was affected slightly. In the presence of lactose, which binds to ricin D, one tyrosyl residue in the B-chain of ricin D was protected from iodination and 40% of the cytoagglutinating activity was retained. This protection against iodination was not observed in the presence of glucose, which does not bind to ricin D. This suggested that the protected tyrosyl residue in the B-chain of ricin D may be situated at or near the saccharide binding site and directly involved in the binding to the saccharide moieties of the cellular receptors.Adsorption of the iodinated ricin D to Sepharose 4B indicated that one of the two saccharide binding sites in ricin D is still intact and participates in the binding to saccharide: ricin D was altered from divalent to monovalent by the iodination.We found from binding experiments with 125I-labeled iodinated ricin D to HeLa cells, that the low toxicity of the iodinated ricin D may be attributed mainly to its decreased internalization into the cells and that the divalent binding of ricin D to the cellular receptors is important for this internalization. 相似文献
6.
Gunki Funatsu Shigetoshi Ueno Masaru Funatsu 《Bioscience, biotechnology, and biochemistry》2013,77(9):1737-1743
Two constituent polypeptide chains of castor bean hemagglutinin (CBH-A) were isolated from the performic acid-oxidized or reduced-carboxymethylated CBH-A by chromatography on DEAE-cellulose or Sepharose 4B. From the analyses of the N-terminal amino acids, the amino acid compositions and the tryptic peptides of each chain, it was found that the larger chain with mol. wt. 34,000 and the smaller chain with mol. wt. 31,000 were homologous with the Ala and He chains of ricin D, respectively, and the subunit structure of CBH-A is represented as (α′/β′)2 in relation to αβ of ricin D. 相似文献
7.
Syed M. Naseem Robert B. Wellner Judith G. Pace 《Journal of biochemical and molecular toxicology》1992,7(2):133-138
Ricin toxin, which consists of two distinct polypeptide moieties, A and B chains, is cytotoxic to the cultured macrophage cell line, J774A.1. Ricin is a protein synthesis inhibitor, and incubating macrophages for 4 hours with ricin (1 pM to 10 nM) in standard medium containing calcium and magnesium inhibited 3H-leucine incorporation into protein (97%, at 1 nM ricin). However, in Ca2+-free medium, protein synthesis was inhibited only 19%. EGTA pretreatment (to deplete intracellular calcium) also partly protected cells from protein synthesis inhibition, in spite of added calcium (2 mM) in the incubation medium. Decreased toxicity in the absence of extracellular calcium resulted from decreased toxin binding. Adding or deleting Mg2+ did not affect protein synthesis or binding of 125I-ricin in cultured macrophages. We conclude that calcium is required for ricin to exert its inhibitory effect on protein synthesis in cultured macrophages. 相似文献
8.
Fate and action of ricin in rat liver in vivo: translocation of endocytosed ricin into cytosol and induction of intrinsic apoptosis by ricin B‐chain
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François Authier Mojgan Djavaheri‐Mergny Séverine Lorin Jean‐Pierre Frénoy Bernard Desbuquois 《Cellular microbiology》2016,18(12):1800-1814
Cytotoxicity of many plant and bacterial toxins requires their endocytosis and retrograde transport from endosomes to the endoplasmic reticulum. Using cell fractionation and immunoblotting procedures, we have assessed the fate and action of the plant toxin ricin in rat liver in vivo, focusing on endosome‐associated events and induction of apoptosis. Injected ricin rapidly accumulated in endosomes as an intact A/B heterodimer (5–90 min) and was later (15–90 min) partially translocated to cytosol as A‐ and B‐chains. Unlike cholera and diphtheria toxins, which also undergo endocytosis in liver, neither in cell‐free endosomes loaded by ricin in vivo nor upon incubation with endosomal lysates did ricin undergo degradation in vitro. A time‐dependent translocation of ricin across the endosomal membrane occurred in cell‐free endosomes. Endosome‐located thioredoxin reductase‐1 was required for translocation as shown by its physical association with ricin chains and effects of its removal and inhibition. Ricin induced in vivo intrinsic apoptosis as judged by increased cytochrome c content, activation of caspase‐9 and caspase‐3, and enrichment of DNA fragments in cytosol. Furthermore, reduced ricin and ricin B‐chain caused cytochrome c release from mitochondria in vivo and in vitro, suggesting that the interaction of ricin B‐chain with mitochondria is involved in ricin‐induced apoptosis. 相似文献
9.
Incubation of 8-anilino-1-naphthalene sulfonic acid with ricin and its isolated A and B polypeptide chains showed an increase in fluorescence at 470 nm. The A chain induced more fluorescence enhancement than either ricin or ricin B chain. The addition of B chain to A chain resulted in decreased fluorescence enhancement which was pH dependent. Sephadex gel filtration showed that A and B chain efficiently reassociated and the reassociation was not dependent on formation of the interchain disulfide bond and could not be prevented by high salt concentration. 相似文献
10.
Ricin is regarded as a high terrorist risk for the public due to its high toxicity and ease of production. Currently, there is no therapeutic or vaccine available against ricin. D9, a murine monoclonal antibody developed previously in our laboratory, can strongly neutralize ricin and is therefore a good candidate for humanization. Humanization of D9 variable regions was achieved by a complementarity-determining region grafting approach. The humanized D9 (hD9) variable regions were further grafted onto human heavy and light chain constant regions to assemble the complete antibody gene. A foot-and-mouth-disease virus-derived 2A self-processing sequence was introduced between heavy and light chain DNA sequences to cleave the recombinant protein into a functional full-length antibody molecule from a single open reading frame driven by a single promoter in an adenoviral vector. After expression in mammalian cells and purification, the hD9 was demonstrated to have equimolar expression of the full-length antibody heavy and light chains. More importantly, the hD9 exhibited high affinity to ricin with KD of 1.63 nM, comparable to its parental murine D9 (2.55 nM). In a mouse model, intraperitoneal (i.p.) administration of hD9, at a low dose of 5 µg per mouse, 4 hours after the i.p. challenge with 5×LD50 ricin was found to rescue 100% of the mice. In addition, administered 6 hours post-challenge, hD9 could still rescue 50% of the mice. The hD9 has the potential to be used for prophylactic or therapeutic purposes against ricin poisoning. 相似文献
11.
B M Foxwell D C Blakey A N Brown T A Donovan P E Thorpe 《Biochimica et biophysica acta》1987,923(1):59-65
Deglycosylation of ricin may be necessary to prevent the entrapment of antibody-ricin conjugates in vivo by cells of the reticuloendothelial system which have receptors that recognise the oligosaccharide side chains on the A- and B-chains of the toxin. Carbohydrate-deficient ricin was therefore prepared by recombining the A-chain, which had been treated with alpha-mannosidase, with the B-chain, which had been treated with endoglycosidase H or alpha-mannosidase or both. By recombining treated and untreated chains, a series of ricin preparations was made having different carbohydrate moieties. The removal of carbohydrate from the B-chain did not affect the ability of the toxin to agglutinate erythrocytes, and alpha-mannosidase treatment of the A-chain did not affect its ability to inactivate ribosomes. The toxicity of ricin to cells in culture was only reduced in those preparations containing B-chain that had been treated with alpha-mannosidase, when a 75% decrease in toxicity was observed. The toxicity of the combined ricin preparation to mice varied from double to half that of native ricin, depending on the chain(s) treated and the enzymes used. Removal of carbohydrate greatly reduced the hepatic clearance of the toxin and the levels of toxin in the blood were correspondingly higher. These results suggest that antibody-ricin conjugates prepared from deglycosylated ricin would be cleared more slowly by the liver, inflict less liver damage, and have greater opportunity to reach their target. 相似文献
12.
Identification of the tryptophan residue located at the low-affinity saccharide binding site of ricin D 总被引:1,自引:0,他引:1
The saccharide binding ability of the low affinity (LA-) binding site of ricin D was abrogated by N-bromosuccinimide (NBS)-oxidation, while in the presence of lactose the number of tryptophan residues eventually oxidized decreased by 1 mol/mol and the saccharide binding ability was retained (Hatakeyama et al., (1986) J. Biochem. 99, 1049-1056). Based on these findings, the tryptophan residue located at the LA-binding site of ricin D was identified. Two derivatives of ricin D which were modified with NBS in the presence and absence of lactose were separated into their constituent polypeptide chains (A- and B-chains), respectively. The modified tryptophan residue or residues was/were found to be contained in the B-chain, but not in the A-chain. From lysylendopeptidase and chymotryptic digests, peptides containing oxidized tryptophan residues were isolated by gel filtration on Bio-Gel P-30 and HPLC. Analysis of the peptides containing oxidized tryptophan revealed that three tryptophan residues at positions 37, 93, and 160 on the B-chain were oxidized in the inactive derivative of ricin D, in which the saccharide binding ability of the LA-binding site was abrogated by NBS-oxidation. On the other hand, the modified residues were determined to be tryptophans at positions 93 and 160 in the active derivative of ricin D which was modified in the presence of lactose, indicating that upon binding with lactose, the tryptophan residue at position 37 of the B-chain was protected from NBS-oxidation. From these results, it is suggested that tryptophan at position 37 on the B-chain is the essential residue for saccharide binding at the LA-binding site of ricin D. 相似文献
13.
Daniel B. Cawley Mary L. Hedblom L.L. Houston 《Archives of biochemistry and biophysics》1978,190(2):744-755
The existence of three forms of ricin and two forms of the Ricinus communis agglutinin (RCA) was established using cation exchange chromatography, isoelectric focusing, and polyacrylamide gel electrophoresis. The preparation of the RCA we obtained was 60–75 times more potent than ricin in the agglutination of erythrocytes, but was about 4% as effective as an inhibitor of cell-free protein synthesis. When reduced with 2-mercaptoethanol, the RCA was activated 3000-fold as an inhibitor of cell-free protein synthesis, whereas ricin was activated about 600-fold by the same treatment. A mixture of the RCA A chains was about one-fifth as effective as the ricin A chain in the inhibition of cell-free protein synthesis. The purified polypeptide subunits of the castor bean lectins were subjected to automated Edman degradation. The sequence for 17 of the first 19 residues of the agglutinin A chain was determined. The first seven residues of the ricin A chain were determined and they are identical with those of the RCA A chain. Nineteen turns of Edman degradation on the RCA B chain resulted in the identification of 18 amino acids. The sequence determined for the first 17 residues of the ricin B chain was identical with that of the RCA B chain. It is likely that the identity of the ricin/RCA A and B chain sequences extends further along the polypeptide chains than the sequences we have determined. The similar structural and catalytic potentials of the RCA and ricin suggest that they bear a precursor-product relationship. 相似文献
14.
Takeshi Sassa 《Bioscience, biotechnology, and biochemistry》2013,77(10):2041-2043
A toxic protein, distinct from ricin D, was isolated from castor beans produced in Japan and referred to as ricin E. Ricin E was extracted from defatted meal of castor beans and purified by gel filtration through Sephadex G-75 column followed by DEAE- and CM-cellulose column chromatographies. Ricin E was found to be a glycoprotein and had two N-terminal amino acids, Ile and Ala, which were identical to those of ricin D. The molecular weight of ricin E was 64,000 by SDS-polyacrylamide gel electrophoresis and its sedimentation coefficient was 4.45 S. The isoelectric point of ricin E, estimated to be 8.8 by ampholine electrophoresis, was higher than that of ricin D. Ricin E had equal toxicity for mice and equal cytoagglutinating activity for Sarcoma 180 ascites tumor cells to those of ricin D, and this cytoagglutination was inhibited by galactose. 相似文献
15.
Mannose receptor-mediated uptake of ricin toxin and ricin A chain by macrophages. Multiple intracellular pathways for a chain translocation 总被引:4,自引:0,他引:4
The role of the high mannose carbohydrate chains in the mechanism of action of ricin toxin was investigated. Ricin is taken up by two routes in macrophages, by binding to cell surface mannose receptors, or by binding of the ricin galactose receptor to cell surface glycoproteins. Removal of carbohydrate from ricin by periodate oxidation led to a large loss in toxicity via both routes of uptake by an effect on the B chain not due to a loss of galactose binding affinity. These data suggest that the carbohydrate chains of ricin B chain may be required for full toxicity. The pathway of uptake of ricin by the macrophage mannose receptor was found to differ in several respects from uptake via the galactose-specific pathway. Analysis of intoxication of macrophages by ricin in the presence of ammonium chloride suggested that mannose receptor bound ligand passes through acidic vesicles prior to translocation, unlike galactose bound ligand. Intoxication by ricin via galactose-specific uptake was potentiated by swainsonine but not by castanospermine, suggesting that ricin may be attacked by an endogenous mannosidase within the cell, and that ricin passes through either a lysosomal or a Golgi compartment prior to translocation. 相似文献
16.
R J Fulton D C Blakey P P Knowles J W Uhr P E Thorpe E S Vitetta 《The Journal of biological chemistry》1986,261(12):5314-5319
This paper describes a protocol for the preparation of highly purified A (A1 and A2) and B chains of the plant toxin, ricin, and biochemical and biological characterization of these proteins. Intact ricin was bound to acid-treated Sepharose 4B and was split on the column into A and B chains with 2-mercaptoethanol. The A chains were eluted with borate buffer containing 2-mercaptoethanol. A1 and A2 were then partially separated by cation exchange chromatography and the contaminating B chain was removed by affinity chromatography on Sepharose-asialofetuin and Sepharose-monoclonal anti-B chain. The B chain was eluted from the Sepharose 4B column by treatment with galactose and was further purified by cation and anion exchange chromatography; contaminating A chains were removed by affinity chromatography on Sepharose-monoclonal anti-A chain. The purified A and B chains were active as determined by their ability to inhibit protein synthesis in a cell-free assay and their binding to asialofetuin, respectively. Furthermore, by polyacrylamide gel electrophoresis, toxicity in mice, and toxicity on several different cell types, both A and B chains were shown to be minimally cross-contaminated. Finally, it was shown that ammonium chloride significantly enhanced the nonspecific toxicity of B chains for cells in vitro. In contrast, ammonium chloride did not enhance either the nonspecific toxicity of A chains in vitro or the specific toxicity of A chain-containing immunotoxins prepared with the highly purified A1, A2 chains. 相似文献
17.
Abstract 1H-NMR spectra (500 MHz) of ricin and its isolated A and B subunits have been analyzed in this study. It is shown that together with tight packing of the polypeptide chain there also exist flexible highly mobile segments in the structure of the proteins. Examination of the line-widths of resonances and the identification of sharp signals with protons of amino acids indicates that N-terminal peptide segments of A and B subunits are free and undergo rapid segmental motions. Spin-echo sequence (90-τ-180-τ) was used to suppress an intensive unresolved background signal from protons involved in the globular part of ricin, thus permitting selective identification of the unusually sharp signals from mobile side chains. 相似文献
18.
Synergy between immunotoxins prepared with native ricin A chains and chemically-modified ricin B chains 总被引:3,自引:0,他引:3
E S Vitetta 《Journal of immunology (Baltimore, Md. : 1950)》1986,136(5):1880-1887
Ricin B chains treated with chloramine-T in the presence or absence of NaI show a 100-fold to 200-fold reduction in their ability to bind to the galactose-containing protein asialofetuin. Such treated B chains do not form covalently associated homodimers with treated B chains or heterodimers with native ricin A chains. Furthermore, they cannot enhance the toxicity of a ricin A chain-containing rabbit anti-human immunoglobulin (RAHIg-A) for Daudi cells. However, when such B chains are coupled to goat anti-rabbit Ig (GARIg), they potentiate the killing of RAHIg-A-treated Daudi cells only slightly less effectively than GARIg coupled to native B chains. Furthermore, if GARIg-B chain conjugates are treated with chloramine-T after coupling, they fail to bind to asialofetuin but enhance the killing of Daudi cells treated with RAHIg-A. These results demonstrate that the ability of ricin B chains to bind to galactose and to enhance the toxicity of ricin A chains (in the form of an antibody-A chain) can be operationally separated. Thus, the two functions of the B chain may reside on separate domains of the molecule. 相似文献
19.
High mobility of N-terminal parts of A and B subunits of ricin 总被引:1,自引:0,他引:1
1H-NMR spectra (500 MHz) of ricin and its isolated A and B subunits have been analyzed in this study. It is shown that together with tight packing of the polypeptide chain there also exist flexible highly mobile segments in the structure of the proteins. Examination of the line-widths of resonances and the identification of sharp signals with protons of amino acids indicates that N-terminal peptide segments of A and B subunits are free and undergo rapid segmental motions. Spin-echo sequence (90-tau-180-tau) was used to suppress an intensive unresolved background signal from protons involved in the globular part of ricin, thus permitting selective identification of the unusually sharp signals from mobile side chains. 相似文献
20.
The toxicity of ricin in susceptible cells is well characterized biochemically, but the pathophysiological implications of its toxicity and the immune response to ricin challenge in the lung are unknown. Incubating macrophage cell line with ricin (1 pM-10 nM) for 4 hours markedly inhibited 3H-leucine incorporation (acid insoluble) into protein (>95%, at 1 nM) without affecting the acid-soluble radioactivity. In spite of increased uptake of total thymidine (141×13.5%) and total uridine (135×17.2%), DNA synthesis in ricin-treated cells was progressively inhibited although RNA synthesis was not affected. Fluocinolone (an anti-inflammatory glucocorticoid) pretreatment increased the ricin-induced inhibition of protein synthesis. The synergistic effect of fluocinolone on ricin-induced protein synthesis inhibition was due to an increased binding (167%, p < 0.01) and internalization (134×12%, p < 0.025) of ricin. Partial protection from ricin-induced inhibition of protein synthesis by indomethacin (nonsteroidal, anti-inflammatory agent) was due to decreased binding and internalization of ricin. These results show that macrophages are sensitive to ricin and that pharmacologically active drugs may regulate ricin's toxicity, perhaps by controlling synthesis and release of certain mediators of fast death. 相似文献