High mobility of N-terminal parts of A and B subunits of ricin

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.     

The specific binding of ricin and its polypeptide chains to rat liver ribosomes and ribosomal subunits

Ricin from Ricinus communis was isolated and the binding of ³H-reductively alkylated or ¹²⁵I-iodinated ricin was studied by incubating the toxic protein with ribosomes and isolating the ricin-ribosome complex by centrifugation. Neither of the labeled ricin derivatives nor ³H-labeled A chain bound Escherichia coli ribosomes, but both bound rat liver ribosomes in a reproducible manner. ³H-labeled ricin bound in a ratio of 1 mol/mol of ribosomes with a dissociation constant of 3 μm as calculated from a Scatchard plot. Similarly, ³H-labeled B chain isolated from ricin also bound in a one-to-one complex with a dissociation constant of 1 μm. The binding of ricin and ricin B chain was sensitive to lactose, while the binding of reduced ricin or ricin A chain was not prevented by lactose. Reduced ¹²⁵I-labeled ricin in the presence of lactose and ³H-labeled A chain bound with a ratio of 2 mol/mol of ribosomes. It was further demonstrated that ³H-labeled ricin A chain bound only to the 60S ribosomal subunit and not to the 40S ribosomal subunit. The dissociation constant for the binding was 2 μm both in the presence and absence of lactose and 2 mol of A chain were bound per mole of 60S ribosomal subunit.     

Homology between ricin and Ricinus communie agglutinin: Amino terminal sequence analysis and protein synthesis inhibition studies

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.     

Blocked and non-blocked ricin immunotoxins against the CD4 antigen exhibit higher cytotoxic potency than a ricin A chain immunotoxin potentiated with ricin B chain or with a ricin B chain immunotoxin

Summary An immunotoxin consisting of ricin A chain linked to the monoclonal antibody M-T151, recognising the CD4 antigen, was weakly toxic to the human T-lymphoblastoid cell line CEM in tissue culture. The incorporation of [³H]leucine by CEM cells was inhibited by 50% at an M-T151-ricin-A-chain concentration (IC₅₀) of 4.6 nM compared with an IC₅₀ of 1.0 pM for ricin. In contrast, immunotoxins made by linking intact ricin to M-T151 in such a way that the galactose-binding sites of the B chain subunit were either blocked sterically by the antibody component or were left unblocked, were both powerfully cytotoxic with IC₅₀ values of 20–30 pM. The addition of ricin B chain to CEM cells treated with M-T151—ricin-A-chain enhanced cytotoxicity by only eight-fold indicating that isolated B chain potentiated the action of the A chain less effectively than it did as an integral component of an intact ricin immunotoxin. Ricin B chain linked to goat anti-(mouse immunoglobulin) also potentiated weakly.Lactose completely inhibited the ability of isolated ricin B chain to potentiate the cytotoxicity of M-T151—ricin-A-chain and partially (3- to 4-fold) inhibited the cytotoxicity of the blocked and non-blocked ricin immunotoxins. Thus, in this system, the galactose-binding sites of the B chain contributed to cell killing regardless of whether isolated B chain was associated with the A chain immunotoxin or was present in blocked or non-blocked form as part of an intact ricin immunotoxin. The findings suggest that the blocked ricin immunotoxin may become unblocked after binding to the target antigen to re-expose the cryptic galactose-binding sites. However, the unblocking cannot be complete because the maximal inhibition of [³H]leucine incorporation by the blocked immunotoxin was only 80% compared with greater than 99% inhibition by the non-blocked immunotoxin.     

The effect of amphotericin B on lectin-induced aggregation of cell surface receptors

The mobility of concanavalin A (ConA) and ricin receptors from NS20 neuroblastoma and C₆ glioma cells was studied using an electrophoretic technique. Cells attached to a solid support were exposed to an electrical field (12V cm⁻¹) at room temperature. The distribution of lectin receptors on the cell surface was revealed by fluorescent conjugates of lectins and microscopic observation of the fixed cells. This technique allowed the estimation of the mobilities of lectin receptors either in free or liganded form, depending on the time at which the cells are labeled with lectins (either after or before electrophoresis). In line with previous observations [1] it is shown that in their free form ConA and ricin receptors are mobile all over the cell surface. Ligand binding induced an apparent receptor immobilization. Immobilization of ricin receptors from C₆ glioma cells could be induced either by the multivalent or the monovalent form of the lectin indicating that cross-linking of receptors by the ligand did not play a predominant role in the process of receptor immobilization. Amphotericin B but not ionophores like valinomycin or gramicidin blocked ligand-induced receptor immobilization. It is concluded from this observation that the effect of amphotericin B is not related to its ionophoretic properties but more likely to its capacity to interact with membrane cholesterol. When cells were incubated at 37 °C extensive patching of lectin receptors could be observed. This process was also inhibited by amphotericin B. A model is proposed to account for a role of cholesterol in ligand-induced receptor immobilization and patching.     

Structure of a New Antioxidative Phenolic Diterpene Isolated from Rosemary (Rosmarinus officinalis L.)

A new antioxidant, phenolic diterpene, named rosmanol, was isolated from the leaves of rosemary (Rosmarinus officinalis L.). Its structure has been determined as 7β,11,12-trihydroxy-6,10-(epoxymethano)abieta-8,11,13-trien-20-one on the basis of chemical and spectroscopic evidence. In addition, all the protons of rosmanol (II) as well as carnosol (I), including the methylene protons of the A ring, were analyzed by 400 MHz ¹-NMR.     

Structure and Dynamics of Ligand-Template Interactions of Topoisomerase Inhibitory Analogs of Hoechst 33258: High Field 1H-NMR and Restrained Molecular Mechanics Studies

Abstract

The binding characteristics of Hoechst 33258 (1), a synthetic bis-benzimidazole, and its structural analog 2, with one of the benzimidazoles replaced by a pyridoimidazole, to the self-complementary decadeoxyribonucleotide sequences d(CGCAATTGCG)² (A) and d-(CATGGCCATG)₂ (B) respectively, were examined using high field ¹H-NMR techniques. Selective complexation induced chemical shift changes, the presence of exchange signals and intermolecular NOE contacts between the ligands and the minor groove protons of the oligonucleotides suggest the preferred binding sites as the centrally located AATT segment for complex Al, and the CCAT segment for complex B2. The B-type conformations of the two DNA duplexes are preserved upon complexation, as confirmed by the 2D-NOESY based sequential connectivities involving DNA base and sugar protons. Close intermolecular NOE based contacts between the ligands and their respective DNA sequences were further refined to model the ligand-DNA complexes starting from the computer generated B-type structures for the oligonucleotides. Force field calculations of ligand-DNA interaction energies indicate a more favorable contribution from the van der Waals energy component in the case of complex Al consistent with its stronger net binding compared with the complex B2.

Overall, the incorporation of a pyridinic nitrogen in Hoechst 33258 structure alters its selectivity for base pair recognition from AT to G.C, resulting largely from the formation of a hydrogen bond between the new basic center and the 2-NH₂ group of a guanosine moiety. The rates for the exchange of ligands between the two equivalent binding sites (AATT for 1, and CCAT for 2) of the self-complementary DNA sequences, are estimated from analyses of coalescence of NMR signals to be 189 _s ^?1 at 301K for A1, and 79_s ^?1 at 297 K for B2; which correspond to ΔG? of 13.8 and 18.6 kcal.mol^?1 respectively.     

The role of calcium ions for the expression of ricin toxicity in cultured macrophages

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.     

Proton NMR studies of myohemerythrin from Themiste zostericola

 One- and two-dimensional NMR experiments have been carried out on different forms of myohemerythrin (MHr), a monomeric 13.9-kDa oxygen carrier, focusing on paramagnetically shifted proton resonances. Compared to the corresponding forms of octameric hemerythrin (Hr), all of the MHr forms exhibit spectra with better resolution and signal-to-noise ratios. The metMHr spectra allow the differentiation of the signals from the N_δ-H protons of the five N_ε-coordinated His ligands and those from the bridging Asp and Glu ligands. The 1D spectra of deoxyMHr exhibit a number of relatively sharp features including three solvent-exchangeable peaks that account for five protons. One of these His N-H protons exchanges more slowly with solvent than the other four and is assigned to His 54, which, by analogy to the crystal structure of deoxyHr, is the only His ligand that is hydrogen-bonded to an amino acid residue, Glu24 in this case. One-dimensional NOE results on the non-exchangeable signals clearly show the connectivities among the α and β protons of the bridging Asp111, and the α, β, and γ protons of the bridging Glu58 ligands. One-dimensional NOE experiments performed on the N-H proton signals of the coordinated His ligands, together with the COSY results, help to identify the geminal β protons of the His ligands. Upon the binding of N₃ ^– to one of the Fe(II) sites in deoxyMHr, the overlapping His N_δ-H proton signals observed in the deoxyMHr spectrum are resolved into individual signals; these have been correlated to the corresponding signals in deoxyMHr by saturation transfer experiments. Similarly, all five His N-H protons are resolved in the ¹H NMR spectrum of the deoxy form of the single point mutant L103N MHr. However, all five N-H protons readily exchange with solvent, indicating that the mutation affects the hydrogen-bonding interaction between His54 and Glu24. Received: 20 May 1996 / Accepted: 24 October 1996     

Comparison of cooperative and isolated site binding of T4 gene 32 protein to ssDNA by 1H NMR

Deuteriation of all aromatic protons of gene 32 protein (g32P) from phage T4, followed by selective introduction of specific protons, has allowed the precise identification of the number and magnitude of the chemical shift changes induced in the aromatic protons when g32P binds noncooperatively or cooperatively to nucleotides. Signals from five Tyr residues are shifted by binding of g32P to d(pA)8 or d(pA)40-60; however, the change from noncooperative, d(pA)8, to cooperative, d(pA)40-60, binding causes significant increases in the magnitudes of the shifts for only two of these Tyr signals. These two Tyr residues may interact directly with the nucleotide bases, while the shifts associated with the other three Tyr may be due to conformational changes in g32P upon ssDNA binding. Similar conclusions can be drawn for two of the six Phe residues whose protons undergo shifts upon nucleotide binding. Observation of selected proton signals allows for the first time detection by 1H NMR of changes in the proton signals from two Trp residues upon nucleotide binding. The side chains of two Tyr, one or two Phe, and one Trp are probably directly involved in nucleotide base-protein interactions. As assayed by the signals from the H2 and H8 protons of adenine, the bases of a bound nucleotide are undergoing a fast chemical exchange in the noncooperative mode of binding, but shift to slow exchange upon assuming the cooperative mode of ssDNA interaction. When bound to a polynucleotide, the A domain of g32P (residues 254-301) becomes more mobile, as reflected in sharpening of the 1H NMR signals from the A domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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.     

Modifications of 60 S ribosomal subunits induced by the ricin A chain

Incubation of 60 S ribosomal subunits with the ricin A chain reduced their stability during heat treatment. The toxin shifted the thermal denaturation curve of the subunits towards lower temperatures, in a similar way to that produced by the decrease in Mg²⁺ concentration. A brief heating (3 min at 57°C), which did not affect control subunit activity, enhanced protein synthesis inhibition of the toxin-treated subunits that released more 5 S RNA, in the form of nucleoprotein complex(es) with protein L5 and phosphoproteins P1P2 (RNP_H), than did heated control subunits [(1984) Eur. J. Biochem, 143, 303-307]. No nuclease activity tested on 60 S subunits and purified 5 S and 5.8 S RNA was found associated with the toxin. The results suggest that the toxin induced a limited conformational change of the 60 S subunit, which destabilized the interaction between RNP_H and the rest of the subunit.     

Effect of temperature on the uptake, excretion and degradation of abrin and ricin by HeLa cells.

The effect of temperature on the uptake of abrin and ricin and on the subsequent excretion and degradation of the toxins was measured. Uptake was assessed either by monitoring the amount of cell-bound ¹²⁵I-labelled toxin that could not be released with lactose or by measuring the time required for transport of the toxins into a state where they were protected against neutralizing antibodies. The presence of toxin in this state was monitored by measuring inhibition of protein synthesis after a subsequent prolonged incubation period. In the case of abrin, straight lines were found in both cases when the data were plotted according to Arrhenius. The activation energies estimated was 18–21 kcal/mol (75–88 kJ/mol) in the case of uptake of [¹²⁵I]abrin and 15–19 kcal/mol (63–79 kJ/mol) when the indirect method was used.After internalization of [¹²⁵I]abrin and ricin a fraction of the radioactive material is released to the medium. Most of this material can be precipitated by trichloroacetic acid (TCA). There is a rapid release during the first 30 min and then over the next few hours the release occurs at a constant, but lower rate. The release of ricin was not affected by addition of colchicine, cytochalasin B (CB), ammonium chloride, sodium azide or bovine serum albumin, whereas the degradation of ricin was reduced by the above mentioned compounds (except albumin). The release of ricin was strongly temperature-dependent with a sharp transition at about 20 °C. The activation energies for the release above and below 20 °C were found to be 2.5 and 31 kcal/mol (10.5 and 172 kJ/mol), respectively.     

500-MHz1H NMR studies of insulin: Complete assignment of histidine resonances

The two histidines of the insulin monomer play a vital role in the organization of insulin into insulin hexamers. The B10 histidines bind to zinc to form two-zinc insulin hexamer, and both the B5 and B10 histidines are implicated in the formation of four-zinc insulin hexamer. These two histidines are both accessible to solvent in the dimeric form of insulin, the predominant species present at pH 2–3. In the present work we report the first 500-MHz¹H NMR studies of insulin. At this frequency all four proton resonances from the two histidines of each equivalent monomer are resolved. The resonances are assigned to the C(2)- and C(4)-imidazole protons of B5 His and B10 His employing Carr-Purcell pulse sequences to detect singlets and to observe approximateT ₂ relaxation times. Zinc-free bovine insulin at pH 2.9 was examined at temperatures up to 60°C in acetate buffer and in urea of varying concentrations. The environments of B5 His in molecule I and molecule II of the dimer must be the same, with the same being true for B10 His, since a total of only four sharp resonances are seen. Our assignments for the two C(2) protons are consistent with those determined from recent studies of human (B5 Ala) insulin.     

Specific inhibition of proton pumping by the T315V mutation in the K channel of cytochrome ba3 from Thermus thermophilus

Cytochrome ba₃ from Thermus thermophilus belongs to the B family of heme-copper oxidases and pumps protons across the membrane with an as yet unknown mechanism. The K channel of the A family heme-copper oxidases provides delivery of a substrate proton from the internal water phase to the binuclear heme-copper center (BNC) during the reductive phase of the catalytic cycle, while the D channel is responsible for transferring both substrate and pumped protons. By contrast, in the B family oxidases there is no D-channel and the structural equivalent of the K channel seems to be responsible for the transfer of both categories of protons. Here we have studied the effect of the T315V substitution in the K channel on the kinetics of membrane potential generation coupled to the oxidative half-reaction of the catalytic cycle of cytochrome ba₃. The results suggest that the mutated enzyme does not pump protons during the reaction of the fully reduced form with molecular oxygen in a single turnover. Specific inhibition of proton pumping in the T315V mutant appears to be a consequence of inability to provide rapid (τ ~ 100 μs) reprotonation of the internal transient proton donor(s) of the K channel. In contrast to the A family, the K channel of the B-type oxidases is necessary for the electrogenic transfer of both pumped and substrate protons during the oxidative half-reaction of the catalytic cycle.     

The Amino 1H Resonances of Oligonucleotide Helices: d(CGCG)

Abstract

An examination of the ¹H NMR assignments and exchange properties of the amino resonances of the self-complementary tetramer, d(CGCG) was undertaken with regard to buffer effects, transfer of saturation from the water resonance and temperature dependence of amino ¹H line shape and chemical shift. The lack of buffer effect on visible exchangeable proton resonances is evidence for the stringent requirement for nucleo-base protonation at pH values below neutrality, which is greatly reduced in the helical state. For this reason, sharp resonances are observed for both Watson-Crick and non-Watson-Crick cytosine amino protons for base-paired regions. Considerations of monomeric exchange mechanisms for the cytosine and guanine amino protons formed the basis for successful assignment and isolation of their resonances in the helical state by presaturation of the water resonance at selected pH values. Preirradiation of the water resonance at pH <6 would isolate the guanine amino ¹H resonances of any self-complementary oligonucleotide, to exploit its high sensitivity as a useful proble of helix ? coil premelting.     

Expression of Mutant Dynamin Inhibits Toxicity and Transport of Endocytosed Ricin to the Golgi Apparatus

Endocytosis and intracellular transport of ricin were studied in stable transfected HeLa cells where overexpression of wild-type (WT) or mutant dynamin is regulated by tetracycline. Overexpression of the temperature-sensitive mutant dyn^G273D at the nonpermissive temperature or the dyn^K44A mutant inhibits clathrin-dependent endocytosis (Damke, H., T. Baba, A.M. van der Blieck, and S.L. Schmid. 1995. J. Cell Biol. 131: 69–80; Damke, H., T. Baba, D.E. Warnock, and S.L. Schmid. 1994. J. Cell Biol. 127:915–934). Under these conditions, ricin was endocytosed at a normal level. Surprisingly, overexpression of both mutants made the cells less sensitive to ricin. Butyric acid and trichostatin A treatment enhanced dynamin overexpression and increased the difference in toxin sensitivity between cells with normal and mutant dynamin. Intoxication with ricin seems to require toxin transport to the Golgi apparatus (Sandirg, K., and B. van Deurs. 1996. Physiol. Rev. 76:949–966), and this process was monitored by measuring the incorporation of radioactive sulfate into a modified ricin molecule containing a tyrosine sulfation site. The sulfation of ricin was much greater in cells expressing dyn^WT than in cells expressing dyn^K44A. Ultrastructural analysis using a ricin-HRP conjugate confirmed that transport to the Golgi apparatus was severely inhibited in cells expressing dyn^K44A. In contrast, ricin transport to lysosomes as measured by degradation of ¹²⁵I-ricin was essentially unchanged in cells expressing dyn^K44A. These data demonstrate that although ricin is internalized by clathrin-independent endocytosis in cells expressing mutant dynamin, there is a strong and apparently selective inhibition of ricin transport to the Golgi apparatus. Also, in cells with mutant dynamin, there is a redistribution of the mannose-6-phosphate receptor.     

NMR Studies of Tris-Intercalation: Solution Structure and Interaction of d(CTTCGCGCGAAG) with an Acridine Trimer

Abstract

Tris-intercalation of an acridine trimer into the self-complementary dodecanucleotide d(CTTCGCGCGAAG) has been studied, in solution, by means of ¹H and ³¹P nuclear magnetic resonance. In a first step all the non-exchangeable protons (except H₅', H_5”), the imino protons and seven of the eleven phosphorus have been assigned. The dodecanucleotide is shown to adopt a double helical B-type structure. Most of the sugar puckers are in the O_1′ endo range, those of the internal guanosines being closer to C_2′endo. Deviations from the canonical B structure are observed in the base stacking and the phosphodiester torsional angles at the ³T⁴C⁵G stretch. The addition of an acridine trimer to the base-paired dodecanucleotide leads to the conclusion that the trimer, which is in slow exchange at the NMR time scale, tris-intercalates into the three C(3′-5′)G sites of the central core, according to the excluded site model. This is evidenced by the large (1.4 ppm) upfield shift experienced by the imino protons of the three internal guanines and the shielding undergone by the acridine ring protons. Tris-intercalation is also supported by the downfield shift experienced by 6 out of the 22 phosphorus. Two of them are shifted by nearly 2 ppm, a shift range reported for oligonucleotides complexed to actinomycin D; this suggests that the structure of the backbone of the dodecanucleotide is altered.     

SECO C-NUCLEOSIDE ANALOGS OF THE 1,2,4-TRIAZOLE

The seco C-nucleosides 3-(1,2,3,4,5-penta-O-acetyl-D-gluco- and D-galacto-pentitol-1-yl)-1H-1,2,4-triazoles (6 and 7) were obtained in one pot by deamination and dethiolation of 4-amino-3-(D-gluco- and D-galacto-pentitol-1-yl)-5-mercapto-1,2,4-triazoles (1 and 2), respectively, using sodium nitrite in orthophosphoric acid and subsequent acetylation. The structures were confirmed by using ¹H, ¹³C and 2D NMR spectra, DQFCOSY, HMQC and HMBC experiments. The favored conformational structures were deduced from the vicinal coupling constants of the protons.     

Binding of ricin A chain to rat liver ribosomes: Relationship to ribosome inactivation

Ricin A chain was radioactively labeled using reductive alkylation, lactoperoxidase catalyzed iodination, and reaction with iodoacetamide or N-ethylmaleimide (NEM). The inhibition of cell-free rat liver protein synthesis by the modified A chains and the ribosome binding characteristics of each of the labeled derivatives was examined. [³H] NEM was found to quantitatively react with the A chain sulfhydryl group normally involved in a disulfide bond with the B chain in intact ricin. Labeling the protein with [³H] NEM had no effect on the in vitro inhibition of protein synthesis by the A chain. [³H] NEM-labeled A chain binds to rat liver ribosomes in a manner which is dependent on the concentrations of NaCl and Mg²⁺. At optimal Mg²⁺ concentration (5.5 mM), A chain binding to ribosomes is saturable and fully reversible either by dilution of the reaction mixture or by addition of unlabeled A chain. At 5.5 mM Mg²⁺, A chain was found to bind to a single site on rat liver ribosomes with a dissociation constant of 6.2 X 10^?8 M. [³H] NEM-labeled A chain did not bind to isolated 40S ribosomal subunits and bound to 60S ribosomal subunits with a 1 : 1 molar stoichiometry and a dissociation constant of 2.2 X 10^?7 M. The relationship between ribosome binding and A chain inhibition of eucaryotic protein synthesis is discussed.