Ribosome-inactivating proteins from plants inhibit ribosome activity of Trypanosoma and Leishmania

Ribosomes from Trypanosoma brucei rhodesiense and from Leishmania infantum were isolated and optimal conditions for in vitro translation were established. The effect of ribosome-inactivating proteins extracted from several plants was then assessed in order to identify those suitable for the preparation of immunotoxins against these organisms. Ribosomes from both species were inactivated by some ribosome-inactivating proteins (dianthins, saporins, pokeweed antiviral proteins, and the ribosome-inactivating chain of abrin). The similarity of the effects on the ribosomes from the two species examined indicates that ribosome-inactivating proteins should also be effective in a similar way on ribosomes from other species of Trypanosoma and Leishmania.     

Effects of plant ribosome-inactivating proteins on ribosomes from Musca domestica.

1. Ribosomes from M. domestica larvae were isolated and their susceptibility to the action of several ribosome-inactivating proteins (RIPs) from plants was tested. 2. Ribosome-inactivating proteins inhibited, to different extents, phenylalanine polymerization by ribosomes. 3. Analysis of RNA from RIP-treated ribosomes showed the appearance of an aniline-cleavable rRNA fragment resulting from the N-glycosidase activity of the RIPs. 4. The release of adenine from saporin 6-treated M. domestica ribosomes was demonstrated by h.p.l.c. analysis.     

Studies on proteins of animal ribosomes

Summary Ribosomes from different tissues and species of animals were tested by several different immunochemical methods. With antisera produced in rabbits by injection of intact ribosomes significant species differences in the antigenic properties of the ribosomes could be demonstrated whereas no tissue conditioned properties in the antigenic determinants were found. Abbreviations. RRL: ribosomes of rat liver; RBL: ribosomes of bovine liver; RBK: ribosomes of bovine kidney; anti-RRL: antiserum against RRL; anti-RBL: antiserum against RBL; anti-RBK: antiserum against RBK.     

Ribosome reactivation by replacement of damaged proteins

Ribosomal functions are vital for all organisms. Bacterial ribosomes are stable 2.4 MDa particles composed of three RNAs and over 50 different proteins. Accumulating damage to ribosomal RNA or proteins can disturb ribosome functioning. Organisms could benefit from degrading or possibly repairing inactive or partially active ribosomes. Reactivation of chemically damaged ribosomes by a process of protein replacement was studied in vitro. Ribosomes were inactivated by chemical modification of Cys residues. Incubation of modified ribosomes with total ribosomal proteins led to reactivation of translational activity. Intriguingly, ribosomal proteins extracted by LiCl are equally active in the restoration of ribosome function. Incubation of 70S ribosomes with isotopically labelled r‐proteins followed by separation of ribosomes was used to identify exchangeable proteins. A similar set of proteins was found to be exchanged in vivo under stress conditions in the stationary phase. We propose that repair of damaged ribosomes might be an important mechanism for maintaining protein synthesis activity following chemical damage.     

Wheat ribosome-inactivating proteins: Seed and leaf forms with different specificities and cofactor requirements

Distinct forms of ribosome-inactivating proteins were purified from wheat (Triticum aestivum L.) germ and leaves and termed tritin-S and tritin-L, respectively. These differ in size and charge and are antigenically unrelated. They are both RNA N-glycosidases which act on 26S rRNA in native yeast (Saccharomyces cerevisiae) ribosomes by the removal of A3024 located in a universally conserved sequence in domain VII which has previously been identified as the site of action of ricin A-chain. Tritin-S and tritin-L differ in both their ribosome substrate specificities and cofactor requirements. Tritin-S shows only barely detectable activity on ribosomes from the endosperm, its tissue of synthesis, whereas tritin-L is highly active on leaf ribosomes. Additionally, tritin-S is inactive on wheat germ, tobacco leaf and Escherichia coli ribosomes but active on rabbit reticulocyte and yeast ribosomes. Tritin-L is active on ribosomes from all of the above sources. Tritin-S, unlike tritin-L shows a marked requirement for ATP in its action.Abbreviations CM carboxymethyl - FPLC fast protein liquid chromatography - NEPHGE non-equilibrium pH gradient gel electrophoresis - PAP pokeweed antiviral protein - RIP ribosome-inactivating protein A.J.M. was the recipient of a U.K. Science and Engineering Research Council CASE studentship sponsored by Agricultural Genetics Company Ltd., Cambridge CB4 4GG, UK.     

Production of ribosome-inactivating protein from hairy root cultures of Luffa cylindrica (L.) Roem.

Transformed root lines of Luffa cylindrica (L.) Roem. (Cucurbitaceae) were established by inoculation of in vitro grown plantlets with wild type Agrobacterium rhizogenes strain 1855. Cloned lines of hairy roots were tested for the presence of ribosome-inactivating proteins; crude extracts inhibited protein synthesis in a reaction mixture based on rabbit reticulocyte lysate. Inhibitory activity increased during culture period, reaching a maximum value in the stationary phase. No activity could be detected in the culture medium, nor in extracts from callus and/or suspension cultures. A ribosome-inactivating protein having specific activity of 62,100 U mg protein^–1 and a molecular mass of 26–28,000 Da was purified to homogeneity. The protein showed N-glycosidase activity on rat liver ribosomes. The results demonstrate that hairy root cultures can be successfully utilized for the in vitro production of ribosome-inactivating proteins.Abbreviations BAP benzylaminopurine - 2,4D 2,4dichlorophenoxyacetic acid - HPLC high pressure liquid chromatography - MS Murashige and Skoog - NAA naphthaleneacetic acid - NCPPB National Collection of Phytopathogenic Bacteria - Ri root-inducing - RIP ribosome-inactivating protein - UV ultra-violet - YMB yeast mannitol broth     

Altered chloroplast ribosomal proteins in a yellow mutant of Chlamydomonas reinhardii.

Summary Ribosomes and ribosomal proteins from wild-type and a yellow mutant of Chlamydomonas reinhardii were analysed and compared by two-dimensional gel electrophoresis.Mixothrophycally grown yellow-27 mutant differs from wild-type cells in lowered chlorophyll content and grana fromation of the chloroplast.Analytical ultracentrifuge analyses of cell extracts show a reduced amount of free 70S ribosomes and increased level of 50S subunits in the mutant cells. Similar results were obtained by electronmicroscopical method.Two-dimensional gel electrophoresis shows alterations in protein composition of 70S ribosomes of the mutant. Two proteins of 70S ribosomes have been altered. One of them with high molecular weight is practically absent while there is an additional, intensively stained spot in the mutant.Since the mutation is inherited in a non-Mendelian manner it is possible that the protein alterations in 70S ribosome are localized in the chloroplast DNA.     

Biochemical and genetic studies on two different types of erythromycin resistant mutants of Escherichia coli with altered ribosomal proteins

Summary Ribosomes from nine E. coli mutants with high level resistance to the antibiotic erythromycin were isolated and their proteins were compared with those of the parental strains by two-dimensional polyacrylamide gel electrophoresis, by carboxymethylcellulose column chromatography and by immunological techniques. Two 50S proteins were found to be altered in the mutants: either L 4 or L 22.Ribosomes with an altered L4 protein bound erythromycin rather poorly and the formation of N-acetylphenylalanyl puromycin was drastically decreased. On the other handribosomes with an altered L22 protein bound erythromycin as efficiently as wild type ribosomes and their puromycin reaction was at least as good as that of wild type ribosomes.Transduction experiments showed that the mutations affecting both proteins, L4 and L22, are located very close to the str and spc genes, nearer to the spc than to str gene.Paper No. 61 on Ribosomal Proteins. Preceding paper is by Hasenbank et al., Molec. gen. Genet., 127, 1–18 (1973).Communicated by E. Bautz     

The barley 60 kDa jasmonate-induced protein (JIP60) is a novel ribosome-inactivating protein

The N-terminal region of a 60 kDa, jasmonate-induced protein of barley leaves (JIP60) is shown to be homologous to the catalytic domains of plant ribosome-inactivating proteins (RIP). Western blotting of leaf extracts and in vitro reconstitution experiments indicate that JIP60 is synthesized as a precursor which is processed in vivo. This is in keeping with in vitro translation experiments indicating that a deletion derivative of the N-terminal region, but not the putative precursor, strongly inhibits protein synthesis on reticulocyte ribosomes. The inhibition of ribosome function is associated with depurination of 26S rRNA, characteristic of plant RIPs. This indicates that JIP60 is a novel ribosome-inactivating protein requiring at least two processing events for full activation. JIP60 derivatives do not significantly inhibit in vitro protein synthesis on wheat germ ribosomes. These and other results suggest that JIP60 may be involved in plant defence.     

Characterization of ribosomes of a strain ofStreptomyces aureofaciens producing chlortetracycline

These studies were designated to investigate the effect of chlortetracycline on sedimentation properties of polysomes and ribosomes present in the chlortetracycline producing strain ofStreptomyces aureofaciens. In presence of chlortetracycline polysomes and ribosomes are more stable than the bacterial ones. At lower chlortetracycline concentrations (1–5 μg/ml) dissociation of polysomes into 70 S monomers was not observed. Ribosomes in higher concentration of chlortetracycline (400 μg/ml) form aggregates. A decrease of Mg²⁺ to 0.1mm caused dissociation of ribosomes to two subunits and in this state none of indicated concentrations of chlortetracycline caused aggregation. The exact sedimentation values of ribosomes and ribosomal subunits were calculated from extrapolation to infinite dilution. S_20,w for monomer form was 68.8, and for ribosomal subunits 49.8 and 31.2 respectively. Ribosomal RNA sedimentates as two Schlieren peaks of 16 S and 22 S. It was found that 30 S subunits contain 15 structural proteins, while 21 proteins were resolved from 50 S subunits.     

Ribosomes in Dormant and Germinating Conidia of Aspergillus oryzae

Ribosomes were isolated from dormant and germinating conidia of Asp. oryzae No. 13. The ribosomes which consisted of 80 S were easily dissociated into 40 S and 60 S in low Mg^{+ +} buffer. Polyribosomes were not found in dormant conidia, but were found in germinating conidia. Ribosomes in Aspergillus fungi consisted of almost equal amount of RNA and protein, and the base compositions of RNA were alike, as compared as ribosomal RNA between dormant and germinating conidia.     

Chloramphenicol resistant mutants of Bacillus subtilis

Summary Telve chloramphenicol resistant (CM ^r)-mutants were isolated from B. subtilis ATCC 6633 and were classified into the following six groups. Group I. No 50s ribosomal protein change was detectable. Ribosomes did not show alteration of the binding ability to CM or to erythromycin in vitro. Group II. A 50s protein, 50a, was altered. Ribosomes did not show alteration of the binding ability to CM or to erythromycin in vitro. The genes specifying the 50a protein was in the cysA-str region on B. subtilis chromosome. Group III. A 50s protein, 50b, was altered. Biological properties of the ribosomes were the same as Group I or II so fas as examined. The genes for 50b protein was in the cysA-str region. Group IV. A 50s protein, 50c, was altered. Ribosomes showed a definite decrease in ability to bind to CM in vitro. The binding of erythromycin to the ribosomes was not impaired. The chromosomal locus of the CM ^r (and for 50c protein) was in the cysA-str region. Group V. A 50s protein, 50e, was changed. The ability of the ribosomes to bind in vitro both to CM and to erythromycin was greatly reduced. The genetic locus of the CM ^r (and for 50e protein) was in the cysA-str region. Group VI. A 50s protein, 50f, was altered. Ribosomes showed a decrease in ability to bind in vitro both to CM and to erythromycin. The genes for 50f protein was in the cysA-str region.The results suggest that the ribosomal resistance to CM may be caused by an independent change of at least several 50s ribosomal protein species. The genetic data shown here and those reported previously show that at least two 30s and seven 50s ribosomal protein genes are situated in the cysA-str region on B. subtilis chromosome.     

THE EFFECT OF MORPHINE ADMINISTRATION ON THE INCORPORATION OF [14C]LEUCINE INTO PROTEIN IN CELL-FREE SYSTEMS FROM RAT BRAIN AND LIVER

—Ribosomes isolated from the brains of rats treated with morphine in vivo were less active in promoting the incorporation of [¹⁴C]leucine into protein than ribosomes isolated from untreated rats. This inhibitory phenomenon was studied in relation to dose of morphine, time after drug administration and the pharmacological responses of hypothermia and analgesia. The inhibition of [¹⁴C]leucine incorporation into brain proteins in vitro was transient after a single injection of morphine and dose-dependent, and related to the hypothermic response, but not prevented by keeping the rats at an ambient temperature which prevented hypothermia. The incorporation of [¹⁴C]leucine into protein by liver ribosomes was also inhibited in preparations from morphine treated rats.     

Gel electrophoresis of ribosomal components from seeds of Pisum sativum L

The ribosomes of dry pea seeds were analysed by polyacrylamide gel electrophoresis. Ribosomes, ribosomal subunits, rRNA and ribosomal proteins were separated by variations of this same basic technique. Pea seed ribosomes were shown to have a subunit structure, rRNA complement and ribosomal protein distribution similar to other eukaryotic ribosomes. A total of 52 ribosomal proteins were identified, 24 on the small and 28 on the large RSU. The molecular weights were mostly in the range 10–35 × 10³.     

The ribosomal stalk is required for ribosome binding,depurination of the rRNA and cytotoxicity of ricin A chain in Saccharomyces cerevisiae

Ribosome inactivating proteins (RIPs) like ricin, pokeweed antiviral protein (PAP) and Shiga‐like toxins 1 and 2 (Stx1 and Stx2) share the same substrate, the α‐sarcin/ricin loop, but differ in their specificities towards prokaryotic and eukaryotic ribosomes. Ricin depurinates the eukaryotic ribosomes more efficiently than the prokaryotic ribosomes, while PAP can depurinate both types of ribosomes. Accumulating evidence suggests that different docking sites on the ribosome might be used by different RIPs, providing a basis for understanding the mechanism underlying their kingdom specificity. Our previous results demonstrated that PAP binds to the ribosomal protein L3 to depurinate the α‐sarcin/ricin loop and binding of PAP to L3 was critical for its cytotoxicity. Here, we used surface plasmon resonance to demonstrate that ricin toxin A chain (RTA) binds to the P1 and P2 proteins of the ribosomal stalk in Saccharomyces cerevisiae. Ribosomes from the P protein mutants were depurinated less than the wild‐type ribosomes when treated with RTA in vitro. Ribosome depurination was reduced when RTA was expressed in the ΔP1 and ΔP2 mutants in vivo and these mutants were more resistant to the cytotoxicity of RTA than the wild‐type cells. We further show that while RTA, Stx1 and Stx2 have similar requirements for ribosome depurination, PAP has different requirements, providing evidence that the interaction of RIPs with different ribosomal proteins is responsible for their ribosome specificity.     

Peptide Chain Initiation in Bacteria,Mitochondria and Chloroplasts

Abstract

Ribosomes prepared from the bacterium Bacillus subtilis, from mitochondria of Neurospora crassa and from chloroplasts of Euglena gracilis carry out the ApUpG-dependent reactions for peptide chain initiation demonstrated for Escherichia coli ribosomes. Initiation factors functionally similar to those from E. coli appear to be present on the above reported ribosomal preparations. The activity of ribosomes washed with high concentrations of ammonium chloride may be restored by adding initiation factors prepared from E. coli.     

Correlation between the activities of five ribosome-inactivating proteins in depurination of tobacco ribosomes and inhibition of tobacco mosaic virus infection

The rRNA depurination activities of five ribosome-inactivating proteins (RIPs) were compared in vitro using yeast and tobacco leaf ribosomes as substrates. All of the RIPs (pokeweed antiviral protein (PAP), dianthin 32, tritin, barley RIP and ricin A-chain) were active on yeast ribosomes. PAP and dianthin 32 were highly active and ricin A-chain weakly active on tobacco ribosomes, whereas tritin and barley RIP were inactive. PAP and dianthin 32 were highly effective in inhibiting the formation of local lesions caused by tobacco mosaic virus (TMV) on tobacco leaves, whereas tritin, barley RIP and ricin A-chain were ineffective. The apparent anomaly between the in vitro rRNA depurination activity, but lack of antiviral activity of ricin A-chain was further investigated by assaying for rRNA depurination in situ following the topical application of the RIP to leaves. No activity was detected, a finding consistent with the apparent lack of antiviral activity of this RIP. Thus, it is concluded that there is a positive correlation between RIP-catalysed depurination of tobacco ribosomes and antiviral activity which gives strong support to the hypothesis that the antiviral activity of RIPs works through ribosome inactivation.     

Characterization of spinach plastid ribosomal proteins by two dimensional gel electrophoresis

Summary Ribosomes are isolated from spinach plastids using conventional sucrose gradients. Their subunits are prepared by dissociation using low Mg²⁺ concentration.It is shown that plastid ribosomes are able to bind f-met-tRNA in the presence of the initiation factors from E. coli.The characterization of ribosomal proteins is carried out using the four two-dimensional gel electrophoretic systems of Madjar et al. (1979). The 30 S and 50 S subunits contain 24 and 34 ribosomal poteins, respectively. These proteins are found in the 70S monosomes which also contain most often nine additional faintly stained proteins.     

The ribosomal proteins of Drosophila melanogaster. I. Characterization in polyacrylamide gel of proteins from larval, adult, and ammonium chloride-treated ribosomes

Summary Ribosomes were isolated from larvae and adult flies, and the purity of the preparation was checked by electron microscopy. The ribosomal proteins were extracted with cold dilute hydrochloric acid, and precipitated with cold acetone. The proteins were characterized by polyacrylamide gel electrophoresis. At pH 3.0 at least 25 bands of different color intensities were resolved, forming a complex pattern.On the basis of electrophoretic mobilities, it was shown that some ribosomal proteins are species-specific, and that larval ribosomes have three protein components more than ribosomes from adult flies.Incubation of the ribosomes with 0.75 M NH₄Cl at a low Mg⁺⁺ concentration lead to detachment of 64% of the ribosomal protein. This detachment of protein molecules was considerably reduced by a five-fold increase of Mg⁺⁺ ions.     

Isolation of 80S ribosomes from spores of Aspergillus fumigatus and their antigenicity in rabbits

Ribosomes were obtained from spores of Aspergillus fumigatus by mechanical disruption and differential centrifugation. The initial preparation (crude ribosomes) contained spore components which appeared to be broken fragments of plasmalemma with or without organelles. Purified ribosomes free of membranous material were prepared by gel filtration chromatography on Sepharose CL-4B. Monomeric 80S ribosomes consisting of 40% protein and 60% RNA, and morphologically characteristic of fungal ribosomes were isolated.Serological reactions in sera from rabbits injected with crude or purified ribosomes were similar indicating that the purification process did not change or eliminate antigens that stimulated antibody detectable either by indirect hemagglutination (IHA) or in gel precipitin tests.