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S Olsnes

Publications and source records attributed to S Olsnes.

At least 181 records · Page 10Linked to original sources
PubMed1977

Studies on the accessability of ribosomes to inactivation by the toxic lectins abrin and ricin.

The rate of protein synthesis in HeLa cells was measured at various periods of time after addition of abrin and ricin to the medium and compared with the concurrent ability of the isolated ribosomes to support poly(U)-stimulated synthesis of polyphenylalanine in a cell-free system. Similarly, the endogenous synthesis in unfractionated cell-free systems from HeLa cells and rabbit reticulocytes was compared with the ability of the isolated ribosomes to support poly(U)-stimulated polymerization of phenylalanine. In the intact cells and the unfractionated cell-free systems protein synthesis decreased progressively with the time after addition of toxins or toxin A chains. In contrast, the ability of the isolated ribosomes to support polyphenylalanine synthesis was only moderately reduced initially and then remained constant or even increased. The activity of isolated monosomes decreased progressively with time after addition of toxin A chain, whereas polysomes were only partly inactivated and the extent of inactivation varied from one experiment to another. The results indicate that the inactivation of one or a few ribosomes per polysome stops the translation of mRNA. It is suggested that the intact ribosomes thus trapped are inaccessible to the toxins and that the isolation of polysomes results in release of functionally intact ribosomes capable of supporting poly(U)-directed polymerization of phenylalanine.

Abrin↗
PubMed1976

Kinetics of binding of the toxic lectins abrin and ricin to surface receptors of human cells.

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.

Abrin↗
PubMed1976

Rates of different steps involved in the inhibition of protein synthesis by the toxic lectins abrin and ricin.

The kinetics of protein synthesis inhibition in a cell-free system from rabbit reticulocyte lysate was studied after addition of abrin and ricin and the isolated A chains. The toxin A chains inhibited protein synthesis at a rate proportional to the amount added. When intact toxins were added to the reticulocyte lysate, the kinetics of protein synthesis inhibition indicated that the A chains must be liberated before ribosome inactivation can take place. The splitting of the toxin in the lysate was directly demonstrated by the use of labeled toxins. The amount of abrin and ricin bound to HeLa cells under different experimental conditions was correlated to the concomitant inhibition of cellular protein synthesis. In the presence of lactose, which inhibits toxin binding, much higher concentrations of toxins were required to inhibit protein synthesis than in the absence of lactose. A linear relationship was found between the lactose concentration in the medium and the toxin concentration required to give 50% reduction in protein synthesis after 3 hours. The amount of toxin bound to the cell surfaces in the presence of lactose was either determined directly or calculated from the apparent association constant between toxins and surface receptors at the various lactose concentrations. Under different conditions involving a 300-fold variation in the concentration of toxin required to reduce protein synthesis by 50% after 3 hours, the amount of toxin bound to the cell surface was found to be the same. The toxicity thus appears to be determined by the number of toxin molecules bound to the cell surface. Purified ricin B chain was used to compete with the toxins for the receptor sites. Only after addition of high amounts of B chain was the toxicity of abrin and ricin appreciably reduced. The data do not support the view that receptors with especially high affinity are involved in the uptake of the toxins. When the time required for 50% inhibition was plotted versus the inverse value of the square root of the number of toxin molecules bound per cell, a straight line was obtained, intercepting at about 30 min. The data indicate that the observed lag time cannot be due entirely to the fact that the A chains must be liberated before they can act.

Abrin↗
PubMed1976

Protective effect of elongation factor 2 on the inactivation of ribosomes by the toxic lectins abrin and ricin.

The inactivation of rabbit reticulocyte ribosomes by abrin and ricin A-chains was studied by incubating ribosomes with the A-chains and testing, after various periods of time, aliquots of the ribosomes for their ability to polymerize phenylalanine. The presence of elongation factor 2 (EF-2) reduced the rate of inactivation of ribosomes by the A-chains. The protective effect of EF-2 was strongly enhanced by GTP and, to a lesser extent, also by GDP or dGTP. Other nucleotides had no demonstrable effect. Much less protection was found after binding of Phe-tRNA to ribosomes in the presence of EF-1 (enzymic binding) or in the presence of high Mg2+ concentration (non-enzymic binding). The data indicate that when EF-2 binds to the ribosomes it completely or partially covers the target site for abrin and ricin A-chains. The possibility that EF-1 also binds to this site is discussed.

Abrin↗
PubMed1976

Toxicity, distribution and elimination of the cancerostatic lectins abrin and ricin after parenteral injection into mice.

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↗
PubMed1975

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

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

Abrin↗
PubMed1975

On the mechanism of protein-synthesis inhibition by abrin and ricin. Inhibition of the GTP-hydrolysis site on the 60-S ribosomal subunit.

The mechanism of protein synthesis inhibition by the toxic lectins, abrin and ricin, has been studied in crude and in purified cell-free systems from rabbit reticulocytes and Krebs II ascites cells. In crude systems abrin and ricin strongly inhibited protein synthesis from added aminoacyl-tRNA, demonstrating that the toxins act at some point after the charging of tRNA. Supernatant factors and polysomes washed free of elongation factors were treated separately with the toxins and then neutralizing amounts of anti-toxins were added. Recombination experiments between toxin-treated ribosomes and untreated supernatant factors and vice versa showed that the toxin-treated ribosomes had lost most of their ability to support polyphenylalanine synthesis, whereas treatment of the supernatant factors with the toxins did not inhibit polypeptide synthesis. Recombination experiments between toxin-treated isolated 40-S subunits and untreated 60-S subunits and vice versa showed that only when the 60-S subunits had been treated with the toxins was protein synthesis inhibited in the reconstituted system. The incorporation of [3H]puromycin into nascent peptide chains was unaffected by the toxins, indicating that the peptidyl transferase is not inhibited. Both the EF-1-catalyzed and the EF-2-catalyzed ability of the ribosomes to hydrolyze [gamma-32P]GTP was inhibited by abrin and ricin. An 8-S complex released from the 60-S subunit by EDTA treatment possessed both GTPase and ATPase activity, while the particle remaining after the EDTA treatment had lost most of its GTPase activity. Both enzyme activities of the 8-S complex were inhibited by abrin and ricin. The present data indicate that there is a common site on the 60-S subunits for EF-1- and EF-2- stimulated GTPase activity and they suggest that abrin and ricin inhibit protein synthesis by modifying this site.

Abrin↗
PubMed1975

Studies on the structure and properties of the lectins from Abrus precatorius and Ricinus communis.

The amino acid composition of the isolated A- and B-chains of the toxic lectins abrin and ricin was determined and compared. Even though the two toxins originate from widely different plants, statistical analysis of the amino acid content indicates extensive homologies in the amino acid sequence of the 4 chains. The intact lectins contain no free SH-groups whereas the isolated A- and B-chains contain close to one free SH-group each. The results indicate that in both toxins the A- and B-chains are connected by a single S-S bond. The B-chains of abrin and ricin contain similar amounts of mannose and glucosamine. The A-chain of ricin also contains some carbohydrate, whereas the A-chain of abrin appears not to be a glycoprotein. The non-toxic abrus and ricinus agglutinins contain more carbohydrate than abrin and ricin. The isoelectric points of the different lectin preparations were measured by isoelectrofocusing. The intact lectins are much more resistant to heat, freezing and chemical treatments than the isolated A- and B-chains. The intact lectins are also very resistant to treatment with proteolytic enzymes, whereas the isolated chains are easily digested. Evidence indicating that the toxins and their chains undergo extensive conformational changes upon reduction of the S-S bond is discussed.

Abrin↗
PubMed1975

Interactions between ricinus agglutinin and human IgM and IgG.

Ricinus agglutinin, purified until homogeneous, precipitates serum glycoproteins with terminal nonreducing galactose residues including IgM and IgG. Almost 100% of IgM reacted with anti-IgM and ricinus agglutinin in quantitative precipitin tests. In similar tests, almost 100% of polyclonal IgG was precipitated by excess anti-IgG, whereas only about 10% reacted with ricinus agglutinin. In quantitative precipitin analyses and affinity chromatography experiments with insolubilized ricinus agglutinin and isolated monoclonal IgG1 and IgG3 proteins, only IgG3 proteins reacted with ricinus agglutinin.

Antibodies, Anti-Idiotypic↗
PubMed1975

Stimulation of human lymphocytes by galactose-specific abrus and ricinus lectins.

Human lymphocyte cultures were incubated with the nontoxic abrus agglutinin and with ricin B chain, and the incorporation of 3H thymidine was measured. Abrus agglutinin stimulated strongly the thymidine incorporation whereas ricin B chain had a much lesser effect. When galactose or lactose was added to the cultures together with the lectins, the abrus agglutinin and ricin B chain induced thymidine incorporation was strongly reduced. There was a linear relationship between the concentration of lectin and the concentration of lactose required for inhibition of lymphocyte stimulation. N-acetyl-galactosamine had a much lesser inhibiting effect and alpha-methyl-mannoside did not cause any inhibition. The abrus agglutinin induced thymidine incorporation was not demonstrable before 36 to 40 hr and reached its maximum after 2 to 5 days. If lactose was added within the first 4 hr of incubation with abrus agglutinin no stimulation was observed.

Abrin↗
PubMed1975

Conformation-dependent antigenic determinants in the toxic lectin ricin.

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

Antibodies↗