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Abrin and ricin are toxic proteins produced by plants. Both proteins are composed of two subunits, an A-chain and a B-chain. The A-chain is responsible for the enzymatic activity, which causes toxicity. The B-chain binds to glycoproteins on the cell surface to direct the A-chain to its target. Both toxins depurinate 28S rRNA, making it impossible to differentiate these toxins based on only their enzymatic activity. We developed an analytical workflow for both ricin and abrin using a single method and sample. We have developed a novel affinity enrichment technique based on the ability of the B-chain to bind a glycoprotein, asialofetuin. After the toxin is extracted with asialofetuin-coated magnetic beads, an RNA substrate is added. Then, depurination is detected by a benchtop matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometer to determine the presence or absence of an active toxin. Next, the beads are subjected to tryptic digest. Toxin fingerprinting is done on a benchtop MALDI-TOF MS. We validated the assay through sensitivity and specificity studies and determined the limit of detection for each toxin as nanogram level for enzymatic activity and μg level for toxin fingerprinting. We examined potential cross-reactivity from proteins that are near neighbors of the toxins and examined potential false results in the presence of white powders.

Abrin is one of the most toxic phytotoxins to date, and is a potential biological warfare agent. A bio-barcode triggered isothermal amplification for fluorometric determination of abrin is described. Free abrin competes with abrin-coated magnetic microparticles (MMP) probes to bind to gold nanoparticle (AuNP) probes modified with abrin antibody and bio-barcoded DNA. Abundant barcodes are released from the MMP-AuNP complex via dithiothreitol treatment. This triggers an exponential amplification reaction (EXPAR) that is monitored by real-time fluorometry, at typical excitation/emission wavelengths of 495/520 nm. The EXPAR assay is easily operated, highly sensitive and specific. It was used to quantify abrin in spiked commercial samples. The detection limit (at S/N = 3; for n = 6) is 5.6 pg·mL -1 which is considerably lower than previous reports. This assay provides a universal sensing platform and has great potential for determination of various analytes, including small molecules, proteins, DNA, and cells. Graphical abstract Schematic representation of the bio-barcode triggered exponential amplification reaction (EXPAR) for a fluorometric competitive immunoassay for abrin. The limit of detection is 5.6 pg mL -1 with a large dynamic range from 10 pg mL -1 to 1 µg mL -1 .

Abrin, an extremely cytotoxic Type II ribosome-inactivating protein (RIP), is a potential bio-warfare agent. Abrin A-chain (ABA) depurinates an adenosine of sarcin-ricin loop (SRL) from eukaryotic 28S rRNA, thereby arresting protein synthesis and leading to cell death. Monoclonal antibody (mAb) D6F10 is the only known antibody that neutralizes ABA's activity in cell-free systems as well as abrin's toxicity in vitro and in vivo. However, how binding of mAb D6F10 to abrin interferes with abrin's catalytic activity at ribosome is still poorly understood. To provide structural basis for mAb D6F10-mediated rescue of ribosome inactivation by abrin, we determined crystal structures of ABA with and without substrate analogs. The structures of ABA-substrate analogs and ribosome were used in an experiment-guided computational protocol, to construct the ABA-Ribosome complex. A homology model of the variable region (F v ) of mAb D6F10 was generated and docked with the apo-ABA structure to construct the ABA-D6F10 F v complex. Structural superposition of ABA common to ABA-D6F10 F v and ABA-Ribosome complexes reveals steric hindrance as the primary mechanism by which mAb D6F10 neutralizes abrin. In contrast to ABA alone, ABA bound to mAb D6F10 is unable to access the SRL on the ribosome owing to steric clashes of mAb D6F10 with the ribosome. Crystal structures of ABA also reveal a catalytic water molecule implicated in hydrolyzing N-glycosidic bond of the susceptible adenosine by RIPs. Furthermore, our strategy provides structural details of steric hindrance important for neutralization of ricin, another RIP, by mAb 6C2 and hence is of wide applicability. ENZYME: EC3.2.2.22. DATABASE: Structural data have been deposited in the Protein Data Bank (PDB) under the accession numbers 5Z37, 5Z3I, and 5Z3J., (© 2018 Federation of European Biochemical Societies.)

Abrin expressed by the tropical plant Abrus precatorius is highly dangerous with an estimated human lethal dose of 0.1-1 μg/kg body weight. Due to the potential misuse as a biothreat agent, abrin is in the focus of surveillance. Fast and reliable methods are therefore of great importance for early identification. Here, we have developed an innovative and rapid multiepitope immuno-mass spectrometry workflow which is capable of unambiguously differentiating abrin and its isoforms in complex matrices. Toxin-containing samples were incubated with magnetic beads coated with multiple abrin-specific antibodies, thereby concentrating and extracting all the isoforms. Using an ultrasonic bath for digestion enhancement, on-bead trypsin digestion was optimized to obtain efficient and reproducible peptide recovery in only 30 min. Improvements made to the workflow reduced total analysis time to less than 3 h. A large panel of common and isoform-specific peptides was monitored by multiplex LC-MS/MS through the parallel reaction monitoring mode on a quadrupole-Orbitrap high resolution mass spectrometer. Additionally, absolute quantification was accomplished by isotope dilution with labeled AQUA peptides. The newly established method was demonstrated as being sensitive and reproducible with quantification limits in the low ng/mL range in various food and clinical matrices for the isoforms of abrin and also the closely related, less toxic Abrus precatorius agglutinin. This method allows for the first time the rapid detection, differentiation, and simultaneous quantification of abrin and its isoforms by mass spectrometry.

The plant-derived toxins ricin and abrin, operate by site-specific depurination of ribosomes, which in turn leads to protein synthesis arrest. The clinical manifestation following pulmonary exposure to these toxins is that of a severe lung inflammation and respiratory insufficiency. Deciphering the pathways mediating between the catalytic activity and the developing lung inflammation, requires a quantitative appreciation of the catalytic activity of the toxins, in-vivo. In the present study, we monitored truncated cDNA molecules which are formed by reverse transcription when a depurinated 28S rRNA serves as template. We found that maximal depurination after intranasal exposure of mice to 2LD50 ricin was reached 48h, where nearly 40% of the ribosomes have been depurinated and that depurination can be halted by post-exposure administration of anti-ricin antibodies. We next demonstrated that the effect of ricin intoxication on different cell types populating the lungs differs greatly, and that outstandingly high levels of damage (80% depurination), were observed in particular for pulmonary epithelial cells. Finally, we found that the magnitude of depurination induced by the related plant-derived toxin abrin, was significantly lower in comparison to ricin, and can be attributed mostly to reduced depurination of pulmonary epithelial cells by abrin. This study provides for the first time vital information regarding the scope and timing of the catalytic performance of ricin and abrin in the lungs of intact animals., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Type 2 ribosome-inactivating protein toxins (RIP-II toxins) were enriched and purified prior to enzymatic digestion and LC-MS analysis. The enrichment of the RIP-II family of plant proteins, such as ricin, abrin, viscumin, and volkensin was based on their affinity for galactosyl moieties. A macroporous chromatographic material was modified with a galactose-terminated substituent and packed into miniaturized columns that were used in a chromatographic system to achieve up to 1000-fold toxin enrichment. The galactose affinity of the RIP-II proteins enabled their selective enrichment from water, beverages, and extracts of powder and wipe samples. The enriched fractions were digested with trypsin and RIP-II peptides were identified based on accurate mass LC-MS data. Their identities were unambiguously confirmed by LC-MS/MS product ion scans of peptides unique to each of the toxins. The LC-MS detection limit achieved for ricin target peptides was 10 amol and the corresponding detection limit for the full method was 10 fmol/mL (0.6 ng/mL). The affinity enrichment method was applied to samples from a forensic investigation into a case involving the illegal production of ricin and abrin toxins.

Abrin, a type II ribosome-inactivating protein, comprises A and B subunits wherein the A subunit harbours toxin activity and the B subunit has a galactose-specific lectin activity. The entry of the protein inside the cell is through the binding of the B chain to cell surface glycoproteins followed by receptor-mediated endocytosis and retrograde transport. A previous study from our laboratory showed that different cell lines exhibited differences of as great as ~200-fold in abrin toxicity, prompting the present study to compare the trafficking of the toxin within cells. Observations made in this regard revealed that the abrin A chain, after being released into the cytosol, is sequestered into the nucleus through interaction with a cellular protein of ~25 kDa, BASP1 (brain acid-soluble protein 1). The nuclear localization of the A chain is seen predominantly in cells that are less sensitive to abrin toxicity and dependent on the levels of BASP1 in cells. The sequestration by BASP1 renders cells increasingly resistant to the inhibition of protein synthesis by abrin and the nucleus act as a sink to overcome cellular stress induced by the toxin.

Abrin-a is the most toxic fraction of lectins isolated from Abrus precatorius seeds and belongs to the family of type 2 ribosome inactivating proteins (RIP). This toxin may act as a defense molecule in plants against viruses, fungi and insects, where attachment of abrin-a to the exposed glycans on the surface of target cells is the crucial and initial step of its cytotoxicity. Although it has been studied for over four decades, the recognition factors involved in abrin-a-carbohydrate interaction remains to be clarified. In this study, roles of mammalian glyco-structural units, ligand clusters and polyvalency in abrin-a recognition were comprehensively analyzed by enzyme-linked lectinosorbent binding and inhibition assays. The results indicate that: (i) this toxin prefers oligosaccharides having alpha-anomer of galactose (Gal) at the non-reducing terminal than the corresponding beta-anomer; (ii) Galalpha1-3Galalpha1- (B(alpha)), Galalpha1-4Gal (E), Galbeta1-3GalNAc (T) and Galbeta1-3/4GlcNAc (I/II) related oligosaccharides were the active glyco-structural units; (iii) tri-antennary II(beta), prepared from N-glycan of asialo fetuin, played a dominant role in recognition; (iv) many high-density polyvalent I(beta)/II(beta) and E(beta) glycotopes enhanced the reactivity; (v) the carbohydrate recognition domain of abrin-a is proposed to be a combination of a small cavity type of Gal as major site and a groove type of additional one to tetrasaccharides as subsites with a preference of alpha1-3/4/6Gal, beta1-3GalNAc, beta1-3/4/6GlcNAc, beta1-4/6Glc, beta1-3DAra and beta1-4Man as subterminal sugars; (vi) size of the carbohydrate recognition domain may be as large enough to accommodate a linear pentasaccharide and complementary to Galalpha1-3Galbeta1-4GlcNAc beta1-3Galbeta1-4Glc (gailipenta) sequence. A comparison of the recognition factors and combining sites of abrin-a with ricin, another highly toxic lectin, was also performed to further understand the differences in recognition factors between these two type 2 RIPs., (2009 Elsevier Masson SAS. All rights reserved.)

The potent toxins ricin, abrin, and other ribosome-inactivating proteins deadenylate a specific base in 28S ribosomal RNA that destroys ribosomes and leads to cell death. We have taken advantage of the fact that reverse transcriptase preferentially inserts an adenine opposite to an abasic site in RNA to create a quantitative polymerase chain reaction (PCR) assay to detect the damage. This assay detects as little as 30pg of ricin. We used the assay to study enzymatic properties of ricin such as pH and temperature optima (pH 4.5-5.0 and 60 degrees C).

In vitro immunostimulatory effect of Abrus lectins derived peptide fractions (AGP and ABP) was investigated in DL bearing mice. Both AGP and ABP were found to activate splenocytes and induced production of cytokines like IL-2, IFN-gamma and TNF-alpha indicating a Th1 type of immune response. Analysis of in vitro treated splenocytes by flow cytometry revealed an increase in percentage of T and B cell with high expression of activation markers (CD25(+) and CD71(+)). At the same time, expression of co-stimulatory markers was significantly high compared to tumor control. The tumor associated macrophages were able to stimulate NO production, IL-1 secretion, increased phagocytosis and decreased expression of mannose receptor. It was also observed that NK cell was activated by AGP and ABP. These results suggest that both AGP and ABP act as immunostimulants in vitro in DL bearing mice.

Abrine is an alkaloid chemical marker and surrogate analyte of abrin, a group of highly toxic glycoproteins. These toxins can be easily isolated from the seed of the rosary pea plant and distributed in a variety of matrices, including food. A procedure for the cleanup of abrine from various beverages, including milk, cola, juice drink, tea, and water, by C18 Strata-X solid-phase extraction (SPE) cartridges is described with comparison to a previously developed liquid-liquid extraction protocol utilizing acetonitrile and water. Analysis was by liquid chromatography/tandem mass spectrometry. Abrine quantitation was based on fragmentation of m/z 219.2 to product ion m/z 188.2. The method detection limit was 0.025 microg/mL, and the quantitation limit was 0.05 microg/mL. Fortifications of the five beverages at 0.5 and 0.05 microg/mL were recovered ranging from 88 to 111% [relative standard deviation (RSD) < 16%] by SPE and from 48 to 101% (RSD < 19%) by liquid-liquid extraction.

There is a heterogeneous group of plant proteins which are able to enzymatically inactivate ribosomes by depurination of an invariant adenine from the 28 S ribosomal RNA. Some of these proteins are heterodimers having a lectin subunit which is joined by disulfide bond to the enzymatic subunit. Ricin and abrin which are among the most toxic substances known belong to the last group. This review focuses on the structure of the heterodimeric plant ribosome-inactivating proteins, the way of their action on ribosome, biosynthesis, intracellular trafficking, and their possible usage in medicine.

Ricin, abrin and related plant toxins have played interesting and important roles in the history of clinical medicine and biomedical research. The use of these proteins in medical treatment since ancient times is reviewed. Later the proteins played important roles in the early days of immunological research and some of the fundamental principles of immunology were discovered with toxic proteins of this group. During the last three decades the mechanism of action of the toxins was elucidated. This led to a major effort to target the toxins to malignant cells. Ricin has been used in bioterrorism. Recently, the toxins have played important roles as experimental models to elucidate the intracellular trafficking of endocytosed proteins.

Abrin-a A chain (ABRaA) is a potent plant toxin, which possesses N-glycosylase activity toward eukaryotic 28S rRNA, and may have potential use in cancer therapy. To improve levels of expression in Escherichia coli, the gene encoding ABRaA was optimized by replacing rare codons with high-frequency ones, and synthesized using two-step PCR. The optimized ABRaA was cloned into the pET-His vector, and highly expressed in cytoplasm of E. coli. The yield of the purified recombinant (r) ABRaA proteins was up to 80 mg/l of induced culture. The rABRaA was one-step purified to homogeneity and its RNA-N-glycosylase ability to inhibit protein biosynthesis in a cell-free system and to depurinate 28S rRNA in rat liver ribosomes was demonstrated in vitro. The MTT assay showed that it also had a killing effect on human hepatoma cell line SMMC-7721 and myeloma cell line Sp2/0. For the first time, ABRaA expressed as soluble form in E. coli from a PCR-synthesized gene is catalytically and functionally active.

Abrin-a consists of A-chain with N-glycosidase activity, which inhibits protein synthesis, and lectin-like B-chain responsible for binding with cell-surface receptors and penetrating of abrin-a molecule into the cells. As a lectin component, the B-chain can also participate in cell signal transduction. It has been reported that abrin induces apoptosis, but the molecular mechanism(s) of this induction have been obscure and several alternative variants have been discussed. The present study demonstrates that abrin-a induces apoptosis in human cultured cell lines, derived from acute lymphoblastic leukemia (ALL) (Jurkat, CCRF-CEM, MOLT-4, HPB-ALL). The apoptosis was estimated by: phosphatidylserine (PSer) exposure at the cell surface, activation of caspase cascade, and DNA fragmentation. The penetrating of abrin-a into the cells was detected by fluorescent confocal microscopy, using fluorescein isothiocyanate (FITC) as a fluorescent marker. It was established that the effect of abrin-a on the apoptosis induction in leukemic cells was dose- and time-dependent. The process was initiated 1 h after abrin-a application (before its penetrating into the cells) and was characterized with PSer translocation from the inner to the outer monolayer of plasma membrane, caspase activation on the first to second hour after beginning of treatment, with maximum on the third to fourth hour, and DNA fragmentation on the fourth to sixth hour, depending of the cell line. The exposure of PSer on the cell surface was detected in Jurkat, CCRF-CEM, and MOLT-4 cells. In HPB-ALL, no significant changes in PSer exposure on the cell surface was observed. Activation of caspase-3, -8, and -9 was detected in Jurkat, MOLT-4, and HPB-ALL. Surprisingly, the activity of caspase-3 increased on the first hour after beginning of treatment, while the activity of caspase-8 and -9 began to increase on the second hour. In CCRF-CEM, activation of caspases was not measured, but the apoptosis progressed to DNA fragmentation in a dose- and time-dependent manner. DNA fragmentation was also detected in Jurkat, but not in MOLT-4 and HPB-ALL cells. It seems that the mechanisms of abrin-a-induced apoptosis are different and the progress of apoptosis depends of the cell line. There was a very good positive correlation between the agglutinating activity of abrin-a and development of apoptosis to DNA fragmentation. The time-dependent effects of abrin-a on apoptosis as well as its time-dependent penetration into the cells suggest that the B-chain probably triggers the apoptosis, while the A-chain and breakage of the disulfide bond are responsible for its progress.

Abrin is a toxic protein obtained from the seeds of Abrus precatorius (jequirity bean), which is similar in structure and properties to ricin. Abrin is highly toxic, with an estimated human fatal dose of 0.1-1 microgram/kg, and has caused death after accidental and intentional poisoning. Abrin can be extracted from jequirity beans using a relatively simple and cheap procedure. This satisfies one criterion of a potential chemical warfare agent, although the lack of large scale production of jequirity seeds means that quantity is unavailable for ready mass production of abrin for weapons. This contrasts with the huge cultivation of Ricinus seeds for castor oil production. At the cellular level, abrin inhibits protein synthesis, thereby causing cell death. Many of the features observed in abrin poisoning can be explained by abrin-induced endothelial cell damage, which causes an increase in capillary permeability with consequent fluid and protein leakage and tissue oedema (the so-called vascular leak syndrome). Most reported cases of human poisoning involve the ingestion of jequirity beans, which predominantly cause gastrointestinal toxicity. Management is symptomatic and supportive. Experimental studies have shown that vaccination with abrin toxoid may offer some protection against a subsequent abrin challenge, although such an approach is unlikely to be of benefit in a civilian population that in all probability would be unprotected.

The eye lens small heat shock proteins (sHSP), alphaA- and alphaB-crystallins, have been shown to function like molecular chaperones, both in vitro and in vivo. It is essential to assess the protective effect of alphaA- and alphaB-crystallins under native conditions to extrapolate the results to in vivo conditions. Insulin and alpha-lactalbumin have widely been used to investigate the chaperone mechanism of alpha-crystallin under native conditions. Due to its smaller size, insulin B-chain may not represent the binding of putative physiological substrate proteins. As it stands, the aggregation of alpha-lactalbumin and binding of alpha-crystallin to it varies under different experimental conditions. Abrin, a ribosome inactivating protein isolated from the seeds of Abrus precatorius, consists of a 30 kDa A-chain and a lectin-like B-chain of 33 kDa joined by a single disulfide bond. Reduction of the disulfide link between the two chains of abrin leads to the aggregation of the B-chain. In this study, we demonstrate that dithiothreitol (DTT)-induced aggregation of abrin B-chain could be monitored by light scattering similar to that of insulin. Moreso, this process could be suppressed by recombinant human alphaA- and alphaB-crystallins in a concentration dependent manner, notably by binding to aggregation prone abrin B-chain. SDS-PAGE and HPLC gel filtration analysis indicate that there is a soluble complex formation between alpha-crystallin and abrin B-chain. Interestingly, in contrast to insulin, there is no significant difference between alphaA- and alphaB-crystallin in suppressing the aggregation of abrin B-chain at two different temperatures (25 and 37 degrees C). HSP26, an another small heat shock/alpha-crystallin family protein, was also able to prevent the DTT-induced aggregation of abrin. These results suggest that due to relatively larger size of its B-chain (33 kDa), compared to insulin B-chain (about 3 kDa), abrin may serve as a better model substrate for in vitro chaperone studies of alpha-crystallin and as well as other sHSP.

The coding sequence of abrin-a A-chain (ABRaA) gene was obtained by RT-PCR and cloned into the expression vector pET28b. The mature ABRaA has been highly expressed in the cytoplasm of Escherichia coli by 1 mmol/L IPTG induction, and the yield of the soluble recombinant protein was 4 mg/L of induced culture. The recombinant ABRaA was purified to be homogeneity. The biological activities of expressed ABRaA were demonstrated in vitro. It strongly inhibited the protein biosynthesis of rabbit reticulocyte lysates, with an IC(50) of 0.08 nmol/L. It also depurinated 28 S rRNA through cleaving at the A4324 site in rat liver ribosomes by its N-glycosidase activity. These data suggested that the recombinant ABRaA could be used for the preparation of immunotoxins as a potential cancer chemotherapeutic agent.

To elucidate of the mechanism of intoxication, the affinity of a toxic lectin, abrin A, from the seeds of Abrus precatorius for mammalian carbohydrate ligands, was studied by enzyme linked lectinosorbent assay and by inhibition of abrin A-glycan interaction. From the results, it is concluded that: (1) abrin A reacted well with Gal beta1-->4GlcNAc (II), Gal alpha1-->4Gal (E), and Gal beta1-->3GalNAc (T) containing glycoproteins. But it reacted weakly with sialylated gps and human blood group A,B,H active glycoproteins (gps); (2) the combining site of abrin A lectin should be of a shallow groove type as this lectin is able to recognize from monosaccharides with specific configuration at C-3, C-4, and deoxy C-6 of the (D)Fuc pyranose ring to penta-saccharides and probably internal Gal alpha,beta-->; and (3) its binding affinity toward mammalian structural features can be ranked in decreasing order as follows: cluster forms of II, T, B/E (Gal alpha1-->3/4Gal) > monomeric T > monomeric II > monomeric B/E, Gal > GalNAc > monomeric I >> Man and Glc (inactive). These active glycotopes can be used to explain the possible structural requirements for abrin A toxin attachment.

Abrus agglutinin (AAG), a low-toxicity protein from the plant Abrus precatorius, is less lethal than abrina (ABRa) in mice (LD(50) = 5 mg/kg versus 20 microg/kg of body weight). Nucleotide sequence analysis of a cDNA clone encoding full-length AAG showed an open reading frame with 1641 base pairs, corresponding to a 547-amino acid residue preproprotein containing a signal peptide and a linker region (two amino acid residues) between the AAG-A and AAG-B subunits. AAG had high homology to ABRa (77.8%). The 13 amino acid residues involved in catalytic function, which are highly conserved among abrins and ricins, were also conserved within AAG-A. The protein synthesis inhibitory activity of AAG-A (IC(50) = 3.5 nM) was weaker than that of ABRa-A (0.05 nM). Molecular modeling followed by site-directed mutagenesis showed that Pro(199) of AAG-A, located in amphiphilic helix H and corresponding to Asn(200) of ABRa-A, can induce bending of helix H. This bending would presumably affect the binding of AAG-A to its target sequence, GpApGpAp, in the tetraloop structure of the 28 S rRNA subunit and could be one of the major factors contributing to the relatively weak protein synthesis inhibitory activity and toxicity of AAG.

The effects of pH and ligand binding on the stability of abrin II, a heterodimeric ribosome-inactivating protein, and its subunits have been studied using high-sensitivity differential scanning calorimetry. At pH7.2, the calorimetric scan consists of two transitions, which correspond to the B-subunit [transition temperature (Tm) 319.2K] and the A-subunit (Tm 324.6K) of abrin II, as also confirmed by studies on the isolated A-subunit. The calorimetric enthalpy of the isolated A-subunit of abrin II is similar to that of the higher-temperature transition. However, its Tm is 2.4K lower than that of the higher-temperature peak of intact abrin II. This indicates that there is some interaction between the two subunits. Abrin II displays increased stability as the pH is decreased to 4.5. Lactose increases the Tm values as well as the enthalpies of both transitions. This effect is more pronounced at pH7.2 than at pH4.5. This suggests that ligand binding stabilizes the native conformation of abrin II. Analysis of the B-subunit transition temperature as a function of lactose concentration suggests that two lactose molecules bind to one molecule of abrin II at pH7.2. The presence of two binding sites for lactose on the abrin II molecule is also indicated by isothermal titration calorimetry. Plotting DeltaHm (the molar transition enthalpy at Tm) against Tm yielded values for DeltaCp (change in excess heat capacity) of 27+/-2 kJ.mol-1.K-1 for the B-subunit and 20+/-1 kJ.mol-1.K-1 for the A-subunit. These values have been used to calculate the thermal stability of abrin II and to surmise the mechanism of its transmembrane translocation.

Abrin is a heterodimeric plant protein that occurs in several isoforms (abrin-a, abrin-b, abrin-c and abrin-d), whose B chains are believed to either have (abrin-a and abrin-d) or lack (abrin-b and abrin-c) the ability to bind galactose. The 5' signal sequence and toxin B chain (ATB)-coding region were excised from a preproabrin cDNA [K. A. Wood, J. M. Lord, E. J. Wawrzynczak, and M. Piatak (1991) Eur. J. Biochem. 198, 723-732], tentatively identified as abrin-c, which was predicted to lack lectin activity, and fused in-frame to generate pre-ATB cDNA. Transcripts, synthesized in vitro from pre-ATB cloned into the transcription vector pSP64T, were expressed after microinjection into Xenopus oocytes. The recombinant ATB was shown, using a qualitative sugar-binding assay, to be devoid of lectin activity. Lectin activity could not be restored to this nonbinding ATB by replacing the 2 gamma subdomain with the corresponding galactose-binding 2 gamma subdomain from ricin B chain, but it was restored by replacement with the active galactose-binding 2 gamma subdomain from a different abrin isoform (abrin-a). The putative galactose-binding pocket of the nonbinding ATB 2 gamma subdomain contained a His residue at the position occupied by a residue with an aromatic side chain (Tyr or Trp) in functional 2 gamma subdomains. Mutationally converting this His to either Tyr or Trp restored lectin activity to the nonbinding ATB, emphasizing the contribution of an aromatic side chain in a functional 2 gamma subdomain galactose-binding site for members of this lectin family.

In this work, we found that rotavirus infection induces an early membrane permeabilization of MA104 cells and promotes the coentry of toxins, such as alpha-sarcin, into the cell. This cell permeability was shown to depend on infectious virus and was also shown to be virus dose dependent, with 10 infectious particles per cell being sufficient to achieve maximum permeability; transient, lasting no more than 15 min after virus entry and probably occurring concomitantly with virus penetration; and specific, since cells that are poorly permissive for rotavirus were not permeabilized. The rotavirus-mediated coentry of toxins was not blocked by the endocytosis inhibitors dansylcadaverine and cytochalasin D or by the vacuolar proton-ATPase inhibitor bafilomycin A1, suggesting that neither endocytocis nor an intraendosomal acidic pH or a proton gradient is required for permeabilization of the cells. Compounds that raise the intracellular concentration of calcium ([Ca2+]i) by different mechanisms, such as the calcium ionophores A23187 and ionomycin and the endoplasmic reticulum calcium-ATPase inhibitor thapsigargin, did not block the coentry of alpha-sarcin or affect the onset of viral protein synthesis, suggesting that a low [Ca2+]i is not essential for the initial steps of the virus life cycle. Since the entry of alpha-sarcin correlates with virus penetration in all parameters tested, the assay for permeabilization to toxins might be a useful tool for studying and characterizing the route of entry and the mechanism used by rotaviruses to traverse the cell membrane and initiate a productive replication cycle.

The cytotoxic lectin abrin shows more than 30 variant forms (R. Hegde, T. K. Maiti, and S. K. Podder, 1991, Anal. Biochem. 194, 101-109). The lectin B subunit as cause for variance in abrins I and III was detected by a combination of one- and two-dimensional electrophoresis and Western blotting. Intriguingly, in abrin I but not in abrin III, association of a single A subunit with the variant B subunits shifts the holoprotein pI toward the alkaline side indicating that the subunit association involves neutralization of few negative charges. The B-subunit variants of abrins I and III overlap in their pI, and the A-subunit association gives the holoproteins a distinctness on isoelectric focusing gel. The results were also confirmed by analyzing the pH titration curves. These differences in the subunit association pattern between abrins I and III are in corroboration with the previously observed differences in the kinetics of protein synthesis inactivation and accessibility of the disulfide bridge to reducing agents in the presence or absence of putative receptor (R. Hegde, A. Karande, and S. K. Podder, 1993 Eur. J. Biochem. 215, 411-419). Further, the genetic origin of variance was confirmed by peptide mapping of the individual subunit variants. Considering a theoretical value of 0.1 to 0.2 pI/charge, a 15-17 charge difference could be predicted between the variants of two extreme pIs. The fact that the A subunits are not shared between the groups was taken to interpret that the protein synthesized as prepro form is processed posttranslationally and the processing takes place only after the disulfide bond formation between A and B subunits. The N-terminal 16 amino acids of A subunits of abrins I and III showed 26% dissimilarity. The A subunits of abrins I and III did not react with concanavalin A, indicating that the heterogeneity in the molecular weight is because of differential processing but not because of glycosylation.

Abrin is a toxic protein consisting of two subunits, an enzymatic A chain (ABRaA) and a lectin-active B chain (ABRaB), linked by a disulfide bond. Site-directed mutagenesis was performed using PCR to study how the conserved amino acid residues, Tyr74, Tyr113, Glu164 and Trp198, around the active site of ABRaA are involved in enzyme catalysis, enzyme-substrate recognition and reassociation of ABRaA with ABRaB. The protein biosynthesis inhibitory activities of Y74F, Y113F and W198F were decreased moderately to that of wild type reABRaA, while that of E164Q decreased dramatically. Kinetic analysis showed that the kat of Y74F, Y113F and W198F resembled that of wild type, while the Km increased significantly. W198F did not reassociate with ABRaB to form heterodimers, while Y74F, Y113F and E164Q did. SDS-PAGE analysis of ABRaA treated with trypsin showed that reABRaA, Y74F, Y113F and E164Q survived digestion, whereas W198F was not protected from digestion. CD spectra revealed that W198F showed significant conformational changes. These observations suggest that E164 is directly involved in catalysis, and Tyr74, Tyr113 and Trp198 in substrate binding, while Trp198 also plays an important role in maintaining the conformation of ABRaA required for its reassociation with ABRaB.

An abrin-resistant cell line, CHOR 3-4, was isolated from CHOK1 cells which were resistant to a high concentration of abrin (160 ng/ml), and had a 1,000-fold higher resistance to abrin that of CHOK1 cells. CHOR 3-4 cells were about 25-fold more resistant than CHOK1 cells to the N-glycosidase activity of abrin, which was measured by hydrolyzing the N-glycosidic bond of adenine 4324 nucleotide from 3' end of mammalian 28S rRNA. However, the isolated polysomes of CHOR 3-4 cells had the same sensitivity to abrin as those of CHOK1 cells. On measuring the binding of 125I-abrin to CHOR 3-4 cells, it was decreased to about 20% that of CHOK1 cells. This indicates that the mechanism of the resistance of CHOR 3-4 to abrin is due to the alteration of glycoproteins or glycolipids of cell membrane.

The binding properties of human Tamm-Horsfall Sd(a+) urinary glycoprotein (THGP) and asialo-THGP with Triticum vulgaris agglutinin(WGA) and three toxic lectins (abrin-a, ricin, and Mistletoe toxic lectin-I) were investigated by quantitative precipitin and precipitin inhibition assays. Both glycoproteins reacted strongly with abrin-a, precipitating over 80% of the lectin nitrogen tested. THGP also bound well to mistletoe toxic lectin-I and precipitated 86% of this lectin added, while the precipitability of its asialo product decreased by 28%. The native glycoprotein completely precipitated the WGA added, but its reactivity was reduced dramatically after desialylation. On the contrary, the poor reactivity of THGP with ricin increased substantially after removal of sialic acid and completely precipitated the lectin added. The glycoprotein-lectin interactions were inhibited by one or several of the following haptens, p-NO2-phenyl alpha GalNAc, p-NO2-phenyl beta GalNAc, Gal beta 1-->4GlcNAc, Gal beta 1-->4Glc, GlcNac beta 1-->4GlcNAc and/or GlcNAc. From the above results, it is concluded that native and/or Tamm-Horsfall glycoproteins serve as important receptors for these three toxic lectins and for WGA.

Kinetic data on inhibition of protein synthesis in thymocyte by three abrins and ricin have been obtained. The intrinsic efficiencies of A chains of four toxins to inactivate ribosomes, as analyzed by ki-versus-concentration plots were abrin II, III > ricin > abrin I. The lag times were 90, 66, 75 and 105 min at a 0.0744 nM concentration of each of abrin I, II, III and ricin, respectively. To account for the observed differences in the dose-dependent lag time, functional and structural variables of toxins such as binding efficiency of B chains to receptors and low-pH-induced structural alterations have been analyzed. The association constants obtained by stopped flow studies showed that abrin-I (4.13 x 10(5) M-1 s-1) association with putative receptor (4-methylumbelliferyl-alpha-D-galactoside) is nearly two times more often than abrin III (2.6 x 10(5) M-1 s-1) at 20 degrees C. Equilibrium binding constants of abrin I and II to thymocyte at 37 degrees C were 2.26 x 10(7) M-1 and 2.8 x 107 M-1 respectively. pH-induced structural alterations as studied by a parallel enhancement in 8-anilino-L-naphthalene sulfonate fluorescence revealed a high degree of qualitative similarity. These results taken with a nearly identical concentration-independent lag time (minimum lag of 41-42 min) indicated that the binding efficiencies and internalization efficiencies of these toxins are the same and that the observed difference in the dose-dependent lag time is causally related to the proposed processing event. The rates of reduction of inter-subunit disulfide bond, an obligatory step in the intoxication process, have been measured and compared under a variety of conditions. Intersubunit disulfide reduction of abrin I is fourfold faster than that of abrin II at pH 7.2. The rate of disulfide reduction in abrin I could be decreased 11-fold by adding lactose, compared to that without lactose. The observed differences in the efficiencies of A chains, the dose-dependent lag period, the modulating effect of lactose on the rates of disulfide reduction and similarity in binding properties make the variants a valuable tool to probe the processing events in toxin transport in detail.

The complete 267 amino acid sequence of abrin-a B chain was determined by analysis of peptides obtained by digestion with trypsin, chymotrypsin, lysyl endopeptidase, Staphylococcus aureus V8 protease and thermolysin. The sequence is not identical with that predicted previously by nucleotide sequencing, indicating the presence of isoforms of abrin. Comparison of the amino acid sequence of abrin-a B chain with that of ricin-D B chain reveals a high degree of sequence identity (59%). Abrin-a B chain appears to consist of two domains, each domain with subdomains (alpha, beta, gamma) of about 40 amino acid residues.

This study elucidates some structural and biological features of galactose-binding variants of the cytotoxic proteins ricin and abrin. An isolation procedure is reported for ricin variants from Ricinus communis seeds by using lactamyl-Sepharose affinity matrix, similar to that reported previously for variants of abrin from Abrus precatorius seeds [Hegde, R., Maiti, T. K. & Podder, S. K. (1991) Anal. Biochem. 194, 101-109]. Ricin variants, subfractionated on carboxymethyl-Sepharose CL-6B ion-exchange chromatography, were characterized further by SDS/PAGE, IEF and a binding assay. Based on the immunological cross-reactivity of antibody raised against a single variant of each of ricin and abrin, it was established that all the variants of the corresponding type are immunologically indistinguishable. Analysis of protein titration curves on an immobilized pH gradient indicated that variants of abrin I differ from other abrin variants, mainly in their acidic groups and that variance in ricin is a cause of charge substitution. Detection of subunit variants of proteins by two-dimensional gel electrophoresis showed that there are twice as many subunit variants as there are variants of holoproteins, suggesting that each variant has a set of subunit variants, which, although homologous, are not identical to the subunits of any other variant with respect to pI. Seeds obtained from polymorphic species of R. communis showed no difference in the profile of toxin variants, as analyzed by isoelectric focussing. Toxin variants obtained from red and white varieties of A. precatorius, however, showed some difference in the number of variants as well as in their relative intensities. Furthermore, variants analyzed from several single seeds of A. precatorius red type revealed a controlled distribution of lectin variants in three specific groups, indicating an involvement of at least three genes in the production of Abrus lectins. The complete absence or presence of variants in each group suggested a post-translational differential proteolytic processing, a secondary event in the production of abrin variants.

After binding, the protein toxins ricin, abrin, and modeccin are endocytosed and processed through the cell's vesicular system in a poorly understood fashion, prior to translocation to the cytosol. The role of the Golgi apparatus in toxin processing was studied using brefeldin-A (BFA), a fungal metabolite which blocks Golgi function. At concentrations that inhibit secretion of interleukin-2 (IL-2), BFA blocks ricin, modeccin, and abrin intoxication of a lymphocyte derived cell line (Jurkat). Paradoxically, BFA enhances the toxicity of two ricin A-chain immunotoxins targeted against distinct cell surface determinants. BFA concentrations which are optimal for immunotoxin enhancement are below those needed to affect ricin intoxication or IL-2 secretion. BFA blockade of ricin does not involve effects on ricin endocytosis, toxin translocation to the cytosol, or the enzymatic activity of toxin A-chain. In contrast, BFA has no effect on immunotoxin processing but does enhance the immunotoxin translocation step. It is concluded that: 1) intact Golgi function is required for holotoxin processing. 2) Intact Golgi function is not required for holotoxin translocation. 3) Golgi function is tightly linked to immunotoxin translocation. 4) BFA has effects on vesicular routing in addition to the block of Golgi function in secretion which has been reported.

The effects of treatment with varying doses of abrin, a D-galactose binding lectin, on DNA and protein synthesis of normal and Epstein Barr virus-transformed lymphocytes have been investigated. Using data on EBV-transformed lymphocyte cell density as a function of time and dose of abrin, one can demonstrate that the mean number of sites bound/EBV-lymphocyte needed to exert a biological influence upon the cell DNA synthesis lies between 59,264 and 370,000 sites/cell. Using a simple packing model, one can demonstrate that a theoretical estimate places the number of binding sites between 57,600 and 360,000 sites/cells.

The nature of binding of abrin to Chinese hamster ovary cells was examined in relation to the ensuing intoxication of the treated cells. Approx. 20% of [125I] abrin bound to CHO cells at 37 degree C was found to be resistant to the addition or presence of 0.1 M lactose. The extent of lactose-resistant binding depended inversely upon the temperature of incubation. Among various proteins, lectins and sugars, only non-labeled abrin could strongly inhibit the lactose-resistant binding of [125I] abrin. Lactose-resistant binding could lead to an inhibition of cellular protein synthesis and to a loss of cell viability. Abrin molecules bound at the lactose-sensitive and lactose-resistant binding sites apparently have an equal probability of being internalized by CHO cells. Binding of approx. 3.10(3) abrin molecules per CHO cell was required to elicit 50% loss of cell viability regardless of whether the binding occurs in the presence or absence of lactose. The result of a cross-linking experiment suggested that a membrane protein with an Mr of about 45 000 may be responsible for the lactose-resistant binding of abrin.

The effects of treatment with varying doses of abrin, a D-galactose binding lectin, on DNA and protein synthesis of normal and Epstein Barr virus-transformed lymphocytes have been investigated. Studies of activation and stimulation indices, as well as two new indices; the metabolic self and cross-coupling indices, lead to the prediction that there are three morphologically distinct subpopulations of EBV-transformed lymphocytes with different abrin binding site numbers. This conclusion is supported by SEM morphological differences.

In the absence of Ca2+ cells were not sensitive to the toxic proteins abrin and modeccin and the sensitivity to ricin and diphtheria toxin was reduced. Calcium deprivation had little effect on the binding and endocytosis of abrin, modeccin, and ricin. The binding of diphtheria toxin to cells was, however, reduced, Verapamil and Co2+ inhibited 45Ca2+ uptake and protected cells against abrin and modeccin at low concentrations of Ca2+. At higher Ca2+ concentrations the protection was overcome. La3+ inhibited strongly 45Ca2+ uptake and protected well against all four toxins, even at high Ca2+ concentrations. Fe3+ also afforded protection although it did not inhibit Ca2+ uptake. The Ca2+ ionophore, A23187, which strongly increases the uptake of 45Ca2+, protected cells well against abrin and modeccin, slightly against diphtheria toxin, but not against ricin. Both Ca2+ deprivation and treatment with A23187 protected well against the hybrid toxin abrin A-chain/ricin B-chain. Such treatment afforded little protection against the hybrid ricin A-chain/abrin B-chain. Apparently the protection against abrin is associated with its A-chain. The calmodulin inhibitor, trifluoperazine, protected strongly against modeccin and diphtheria toxin. The data indicate that Ca2+ is involved in the entry mechanism for abrin, modeccin, and ricin, possibly as a Ca2+ flux together with the toxins.

The effects of treatment with varying doses of abrin, a D-galactose binding lectin, on DNA an protein synthesis of normal and Epstein Barr Virus transformed lymphocytes has been investigated. Activation, stimulation, and relative toxicity factor indices are studied, as well as possible relationships between DNA and protein synthesis rates, as measured by simultaneous tritiated thymidine (3H-TdR) and 14C-leucine uptake. Studies of the two new indices, the metabolic self and cross coupling indices lead to the prediction that there are three morphologically distinct subpopulations of EBV-transformed lymphocytes with different abrin receptor site concentrations. This prediction is supported by SEM morphological differences. Using data on EBV-transformed lymphocyte cell density as a function of time and dose of abrin, one can demonstrate that the mean number of receptors bound-EBV-lymphocyte needed to exert a biological influence lies in the interval 59,264 receptors/cell to 370.040 receptors/cell. Using a simple packing model, one can demonstrate that a theoretical estimate places the number of binding sites between 57,600 receptors/cell and 360,000 receptors/cell.

At neutral pH, NH4Cl and chloroquine protected cells against diphtheria toxin. A brief exposure of the cells to low pH (4.5-5.5) at 37 degrees completely abolished this protection. When, to cells preincubated with diphtheria toxin and NH4Cl, neutralizing amounts of anti-diphtheria toxin were added before the pH was lowered, the toxic effect was considerably reduced, but it was not completely abolished. A much stronger toxic effect was seen when antibodies were added immediately after incubation at low pH. Upon a short incubation with diphtheria toxin at low pH, the rate of protein synthesis in the cells decreased much faster than when the normal pH was maintained. The data suggest that, at low pH, diphtheria toxin (or its A fragment) penetrates directly through the surface membrane of the cell. The possibility is discussed that, when the medium has a neutral pH, the entry of diphtheria toxin involves adsorptive endocytosis and reduction of the pH in the vesicles possibly by fusion with lysosomes. Low pH did not facilitate the entry of the closely related toxins abrin, ricin, and modeccin.

The effects of treatment with varying doses of abrin, a D-galactose binding lectin, on DNA and protein synthesis of normal and Epstein Barr virus-transformed lymphocytes have been investigated. Activation, stimulation and relative toxicity factor indices are studied as well as possible relationships between the DNA and protein synthesis rates, as measured by simultaneous 3H-TdR and 14C-leucine uptake.

The effects of treatment with varying doses of abrin, a D-galactose binding lectin, on DNA and protein synthesis of normal and Epstein-Barr virus (EBV)-transformed lymphocytes have been previously investigated. Using data on EBV-transformed lymphocyte cell density as a function of both time and dose of abrin, the authors introduced the concept of self- and cross-coupling metabolic variables as a means of understanding how abrin affected DNA and protein uptake. In this paper, the self-coupling constant is studied in more detail and the relationship between DNA and protein synthesis is further expanded. We find that there is a significant linear relationship between DNA and protein synthesis in normal lymphocyte culture as measured by abrin interaction in the culture. We further find that there is a much stronger relationship between these variables in EBV-transformed lymphocyte culture. This relationship is further examined, and possible analytic equations are expressed.

The effects of the plant toxin abrin on normal mouse embryonic fibroblasts (MEF), an untransformed mouse cell line (NIH 3T3), and two mouse tumor cell lines (LMTK- and S-180) were studied. Measurements of cell growth and colony formation showed that MEF and S-180 cells were more sensitive to abrin intoxication than NIH 3T3 and LMTK- cells. Also, the effects of abrin on the inhibition of [3H]leucine and [3H]thymidine incorporation were more evident in MEF and S-180 cells. The basis for these varying responses to abrin by the four different cells was examined. The number of abrin binding sites per cell was determined from [125I]abrin binding studies: NIH 3T3 and LMTK- cells had significantly fewer abrin binding sites than MEF and S-180 cells. The fate of the [125I]abrin after internalization was examined by gel electrophoresis and autoradiography. A pattern of time-dependent degradation was observed, degradation being more rapid in NIH 3T3 and S-180 cells than in LMTK- and MEF cells. We conclude that the varying responses of different cells to the toxin abrin may be due to several factors, including the relative number of abrin binding sites on the cell surface and the rate of degradation of the toxin once internalized. The results also show that the sensitivities of the cells to abrin do not necessarily correlate with their normal or neoplastic state.

Kinetic parameters of the interaction of the toxic lectins abrin and ricin with human erythrocytes and HeLa cells have been measured. The binding of 125I-labeled abrin and ricin to human erythrocytes and to HeLa cells at 37 degrees was maximal around pH 7, whereas at 0 degrees the binding was similar over a broad pH range. The binding occurred at similar rates at 0 degrees and 37 degrees with rate constants in the range 0.9 to 3.0 X 10(5) M-1 s-1. The dissociation was strongly temperature-dependent with rate constants in the range 3.4 to 45 X 10(-4) s-1 at 0 degrees and 3.9 to 18 X 10(-3) s-1 at 37 degrees. The presence of unlabeled lectins as well as lactose increased the rate of dissociation. The association constants measured at equilibrium or calculated from the rate constants were between 0.64 X 10(8) M-1 and 8.2 X 10(8) M-1 for abrus lectins, and between 8.0 X 10(6) M-1 and 4.2 X 10(8) M-1 for ricinus lectins. The association constants for the toxins were lower at 37 degrees than at 0 degrees. Isolated ricin B chain appeared to bind with similar affinity as intact ricin. The number of binding sites was estimated to be 2 to 3 X 10(6) per erythrocyte and 1 to 3 X 10(7) per HeLa cell. The binding sites of HeLa cells all displayed a uniform affinity towards abrin and ricin, both at 0 degrees and at 37 degrees. The same was the case with the binding sites of erythrocytes at 0 degrees. However, the data indicated that at 20 degrees erythrocytes possessed binding sites with two different affinities. Only a fraction of the cell-bound toxin appeared to be irreversibly bound and could not be removed by washing with 0.1 M lactose. The fraction of the total amount of bound toxin which became irreversibly bound to HeLa cells was for both toxins about 2 X 10(-3)/min at 37 degrees, whereas no toxin was irreversibly bound at 0 degrees. In the case of erythrocytes no toxin became irreversibly bound, either at 0 degrees or 37 degrees, indicating that the toxins are unable to penetrate into these cells.

The toxicity of abrin, modeccin, and ricin to Vero cells was maximal at neutral and slightly alkaline pH, and it was strongly reduced at pH 6.0 and below. Diphtheria toxin was most toxic at low pH. Binding and endocytosis of abrin, modeccin, and ricin did not vary much within the pH range tested. High concentrations of the carboxylic ionophore Br-X-537A, protected against all four toxins. Combined treatment of cells with an inhibitor of glycolysis and an uncoupler of oxidative phosphorylation strongly inhibited endocytosis of toxins and protected against intoxication. The protective effect of Ca2+ deprivation, of pH 6.0, and of metabolic inhibitors disappeared soon after transfer of the cells to normal medium, whereas the protective effect of Br-X-537A and of trifluoperazine disappeared slowly. The decay rate of the protection by NH4Cl and by the ionophore A23187 differed with the different toxins. Cells exposed to abrin, modeccin, and ricin under protective conditions which did not inhibit endocytosis of the toxins (Ca2+ deprivation, pH 6.0, Br-X-537A), and then treated with antitoxins to inactivate extracellular toxin, were intoxicated when the protection was released. In contrast, cells exposed to toxins while endocytosis was arrested by treatment with metabolic inhibitors were not intoxicated when antitoxins were added and the metabolic inhibitors removed. Modeccin and diphtheria toxin endocytosed in the presence of trifluoperazine and NH4Cl were unable to intoxicate cells. The possibility that endocytosis is a step in the normal entry route of the toxins is discussed.

The survival time of mice after i.v. injection of the cancerostatic lectins, abrin and ricin was recorded. The LD50 dose was found to be 10-13 ng and 55-65 ng per mouse for abrin and ricin, respectively. Increasing amounts of toxin reduced the survival time, reaching a minimum of about 10 h. Lactose injected with ricin, provided partial protection against ricin, as measured by the survival time. Abrin and ricin labelled with 125I, and shown to retain their full toxic activity, were injected into mice. Most of the radioactivity found in the organs was present in the form of intact toxins, at least up to 5 h after injection. After i.v. injection the highest concentration/g tissue was found in spleen, followed by kidneys, heart, liver and thymus. The relative concentration in liver was considerably higher for ricin than for abrin. Similar results were found after i.p. injection. When lactose was administered together with ricin, almost 80% of the ricin injected was found in the liver after 30 min, compared to 48% without lactose, and the amount in other organs was concurrently reduced. The elimination of total radioactivity was much faster for ricin than abrin. The radioactivity found in the urine was largely present in non-trichloroacetic acid precipitable form, indicating that the toxins were extensively degraded before excretion.

Abrin, a potent cytotoxin, was utilized as a probe to elucidate the mechanism by which external proteins are delivered to the cytoplasm of mammalian cells. Abrin bound rapidly to the surface receptors of the Chinese hamster cells (line CHO) and appeared to be internalized immediately without any significant lag. The maximum level of abrin internalization was achieved within eight minutes, based on both biochemical and electron microscopic autoradiographic studies with [125I]abrin. About 10% of the silver grains of internalized [125I] abrin were associated with vesicular structures, irrespective of the incubation time. Inhibition of protein synthesis began 30 minutes postincubation, and this latent period was not dependent on extracellular toxin concentration. SDS-polyacrylamide gel electrophoresis of the internalized [125I]abrin indicated that internalized abrin molecules remained intact even after two hours of incubation.