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Results for “Ribosome Inactivating Proteins, Type 1”

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At least 19 recordsLinked to original sources

PubMed1992

Mechanism and site of action of a ribosome-inactivating protein type 1 from Dianthus barbatus which inactivates Escherichia coli ribosomes.

A single chain ribosome-inactivating protein with RNA N-glycosidase activity, here named Dianthin 29, was isolated from leaves of Dianthus barbatus L. Incubation of intact Escherichia coli ribosomes with Dianthin 29 and subsequent aniline treatment of the isolated rRNA releases a rRNA fragment of 243 nucleotides from 23 S rRNA. Nucleotide sequence studies showed that the site of N-glycosidic bond cleavage is at A-2660 within the universally conserved sequence 5'-AGUACGAGAGGA-3' near the 3'-end of 23/28 S rRNAs. To our knowledge, Dianthin 29 is the first ribosome-inactivating protein which is shown to inactivate intact prokaryotic ribosomes in the same manner as eukaryotic ribosomes.

Base Sequence↗

PubMed2005

The 1.4 anstroms structure of dianthin 30 indicates a role of surface potential at the active site of type 1 ribosome inactivating proteins.

Ribosome inactivating proteins (RIPs) are plant proteins with enzymatic activity identified as rRNA N-glycosidase (EC 3.2.2.22), which cleaves the N-glycosidic bond of a specific adenine on the ricin/sarcin region of rRNA, thus causing inhibition of protein synthesis. They also depurinate extensively DNA and other polynucleotides. The three-dimensional structure of dianthin 30, a type 1 (single-chain) RIP of Dianthus caryophyllus (leaves), is now described at 1.4 angstroms, a resolution never achieved before for any RIP. The fold typical of RIPs is conserved, despite some differences in the loop regions. The general structure comparison by superimposed alpha-carbon (249 atoms) and the sequence alignment by structure for dianthin 30 and saporin-S6 give a root mean square deviation of 0.625 angstroms. Despite the differences reported for the biological activities of the two RIPs, their structures fit quite well and both show a protein segment containing strands beta7, beta8, and beta9 shorter than other RIPs. However, the surface electrostatic potential in the active site region neatly distinguishes dianthin 30 from saporin-S6. The possible relationship between the charge distribution and the behavior of the proteins toward different substrates is discussed.

Amino Acid Sequence↗

PubMed2001

Comparison of the embryotoxic effects of saporin, agrostin (type 1 ribosome-inactivating proteins) and ricin (a type 2 ribosome-inactivating protein).

The effects of two type I ribosome-inactivating proteins, saporin and agrostin, and the type 2 ribosome-inactivating protein ricin and its constituent A and B chains, on the development of cultured mouse embryos, were investigated. Saporin and agrostin had similar embryotoxicity which was approximately 10(5) times weaker than that of ricin and its A and B chains, and the embryotoxicity of saporin and agrostin increased gradually with the dosage. In contrast, there was an abrupt rise in embryotoxictiy of ricin-A chain and B chain as the dose was raised from 10 to 20 ng x ml(-1). Saporin and agrostin did not affect the heart-beat and otic placode, but at 10 ng x ml(-1) ricin and 40 ng x ml(-1) ricin-A chain and B chain, all of the treated embryos exhibited abnormalities in heart-beat, yolk sac circulation, body axis, optic placode, otic placode, forelimb buds, branchial apparatus and cranial neural tube.

Animals↗

PubMed1996

A simplified procedure for the purification of trichosanthin (a type 1 ribosome inactivating protein) from Trichosanthes kirilowii root tubers.

A one step rapid and simple purification procedure for trichosanthin, a type 1 ribosome inactivating protein, from root tubers of Trichosanthes kirilowii has been developed using cation-exchange perfusion chromatography. The identity of the protein has been confirmed by its size, immunoreactivity, and sequence information. Yields of 0.16% of electrophoretically pure trichosanthin from dried root tuber have been achieved with a single 10-min chromatographic step giving the ability to purify gram quantities of trichosanthin in 1 day.

Abortifacient Agents↗

PubMed1993

Molecular action of the type 1 ribosome-inactivating protein saporin 5 on Vicia sativa ribosomes.

The type 1 ribosome-inactivating protein (RIP) saporin 5 isolated from seeds of Saponaria officinalis L. strongly inhibited translation carried out by Vicia sativa L. purified ribosomes. The toxin multidepurinated V. sativa rRNA, which upon treatment with acid aniline releases several RNA fragments including an RNA fragment of approximately 370 nucleotides the 5'-end sequence of which was 5'-GAGGAACG-3'.

Animals↗

PubMed1993

Fusidic acid-dependent ribosomal complexes protect Escherichia coli ribosomes from the action of the type 1 ribosome-inactivating protein crotin 2.

The type 1 ribosome-inactivating protein crotin 2 depurinated Escherichia coli ribosomes which, upon treatment of the isolated rRNA with acid aniline, released a fragment of around 240 nucleotides whose 5'-end sequence was 5'-GAGGACCGGAGUGGAC-3'. The formation of fusidic acid-dependent ribosomal complexes completely prevented release of the fragment. Ribosomes from crotin 2-pretreated fusidic acid complexes were insensitive to acid aniline. They released the RNA fragment only after a second treatment with crotin 2 and acid aniline whereas unprotected ribosomes released the fragment directly after acid aniline.

Apurinic Acid↗

PubMed1995

Ebulitins: a new family of type 1 ribosome-inactivating proteins (rRNA N-glycosidases) from leaves of Sambucus ebulus L. that coexist with the type 2 ribosome-inactivating protein ebulin 1.

A new family of single chain (type 1) ribosome-inactivating proteins (RIPs), that we have named ebulitins, have been found in mature leaves of Sambucus ebulus L., a caprifoliaceae plant also known to contain a non-toxic two chain (type 2) RIP named ebulin I in its leaves. Ebulitins are basic proteins of M(r) 32,000, 29,000 and 29,000 for ebulitins alpha, beta and gamma, respectively. The simultaneous presence of different basic type 1 and acidic type 2 RIPs in the same plant and in the same tissue is described here for the first time and opens a new door in research into RIPs.

Amino Acids↗

PubMed1993

Preparation, optimization and characterization of a polyphenylalanine synthetizing system from Agrobacterium tumefaciens.

A very active cell-free translation system was prepared from Agrobacterium tumefaciens, a bacterium broadly used to transfect plant cells to introduce foreign genes and one that produces tumours in plants. Once optimized for Mg2+, NH4+, high speed supernatant S 370, purified ribosomes and time, the system translates polyuridylic acid very efficiently. A. tumefaciens purified ribosomes were inhibited in vitro by several well-known translational inhibitors including some ribosome-inactivating proteins. Treatment of A. tumefaciens purified ribosomes with type 1-RIP crotin 2 lead to the depurination of the 23S rRNA which, upon treatment with acid aniline, released a diagnostic RNA fragment of about 235 nucleotides.

Agrobacterium tumefaciens↗

PubMed2001

Induction of apoptosis by a ribosome-inactivating protein from Agrostemma githago is associated with down-regulation of anti-apoptotic bcl-2 protein expression.

Agrostin is a type 1 ribosome-inactivating protein (RIP) isolated from the seeds of Agrostemma githago. 3[H]-thymidine incorporation in human leukemic HL-60 cells was significantly reduced after incubation with agrostin showing that the RIP was inhibitory on the cell proliferation. Results from flow cytometric studies illustrated that apoptosis was induced by agrostin, which was associated with activation of endonucleases and DNA fragmentation of the leukemic cells. Anti-apoptotic bcl-2 protein level was also reduced by agrostin showing that the associated apoptotic pathway might involve down-regulation of the intracellular bcl-2 protein level.

Annexins↗

PubMed1996

The activity of plant-derived antiretroviral proteins MAP30 and GAP31 against herpes simplex virus in vitro.

We examined the effect on anti-HIV proteins MAP30 and GAP31, from Momordica charantia and Gelonium multiflorum, on the infection and replication of Herpes Simplex Viruses (HSV). Human lung WI-38 fibroblasts cultured in the presence of tenfold dilutions of MAP30 or GAP31 were exposed to HSV and viral yield was measured at 24-48 hours by ELISA. The effective concentrations for 50% inhibitions (EC50) were 0.1-0.2 microM for HSV-2, and 0.3-0.5 microM for HSV-1 for MAP30 and GAP31, respectively. In comparison, the EC(50) for acyclovir (ACV), a commonly used anti-HSV drug, was 0.2 and 1.7 microM for HSV-2 and HSV-1, respectively. The cytotoxicity of all three antivirals was negligible and comparable. However, the antiherpetic activity of the plant proteins against acyclovir-resistant strains was two to three logs more potent than ACV. These results suggest that MAP30 and GAP31, previously shown to be active against HIV, may be useful for the therapy of herpesvirus infections.

Antiviral Agents↗

PubMed2002

Ribosome-inactivating and adenine polynucleotide glycosylase activities in Mirabilis jalapa L. tissues.

Several tissues of Mirabilis jalapa L. (Nyctaginaceae) were assayed for inhibition of translation by a rabbit reticulocyte lysate (as a signal of ribosome-inactivating activity) and for adenine DNA glycosylase activity, activities that are both due to the presence of a class of enzymes called ribosome-inactivating proteins (RIPs), currently classified as rRNA N-glycosylases (EC ). These activities were highest in seed; intermediate in flower bud, immature seed, sepal + gynoecium, leaf, and root; and very low in all other tissues. By cation-exchange chromatography, four protein peaks with inhibitory activity on cell-free translation were identified in extracts from seeds, and two proteins were isolated from peaks 1 and 4, all of which have the properties of single-chain type 1 RIP. One is Mirabilis antiviral protein (MAP), so far purified only from roots. The second is a new protein that we propose to call MAP-4. The distribution of MAP and MAP-4 in several tissues was determined with a novel experimental approach based on liquid chromatography/mass spectrometry. The direct enzymatic activity of MAP on several substrates is described here for the first time. MAP depurinated not only rRNA in intact ribosomes, thus inhibiting protein synthesis, but also other polynucleotides such as poly(A), DNA, and tobacco mosaic virus RNA. Autologous DNA was depurinated more extensively than other polynucleotides. Therefore, the enzymatic activity of this protein may be better described as adenine polynucleotide glycosylase activity rather than rRNA N-glycosylase activity. Finally, MAP does not cross-react immunologically with other commonly utilized RIPs.

Adenine↗

PubMed1999

The antiviral agents, MAP30 and GAP31, are not toxic to human spermatozoa and may be useful in preventing the sexual transmission of human immunodeficiency virus type 1.

OBJECTIVE: To investigate the effects of two virucidal compounds, MAP30 (Momordica anti-human immunodeficiency virus [HIV] protein; molecular weight, 30 kd) and GAP31 (Gelonium anti-HIV protein; molecular weight, 31 kd), obtained from Momordica charantia and Gelonium multiflorum, respectively, on the motility and vitality of human spermatocytes. DESIGN: Prospective, controlled study. SETTING: New York University School of Medicine. PATIENT(S): Ten healthy men undergoing evaluation for infertility provided 10 semen specimens. INTERVENTION(S): Human sperm were treated with the anti-HIV agents, MAP30 and GAP3 1. Nonoxynol-9, a commonly used spermicide, and phosphate-buffered saline were used as the positive and negative controls, respectively. MAIN OUTCOME MEASURE(S): The motility and vitality of human spermatocytes treated with MAP30 and GAP31 at doses that inhibit HIV-1 and herpes simplex virus. RESULT(S): MAP30 and GAP31 did not inhibit the motility or vitality of human sperm cells over a dose range of 100-0.1 microg/mL, whereas nonoxynol-9 demonstrated spermicidal action on all 10 samples over the same dose range. CONCLUSION(S): The antiviral agents, MAP30 and GAP31, were not toxic to human sperm cells at the doses at which they inhibit HIV-1 and herpes simplex virus. They had no effect on the motility of spermatozoa, even at a dose of 1,000 times the maximum effective concentration. These results indicate that MAP30 and GAP31 may be useful as nonspermicidal protection against sexually transmitted diseases.

Anti-HIV Agents↗

PubMed1995

Inhibition of the integrase of human immunodeficiency virus (HIV) type 1 by anti-HIV plant proteins MAP30 and GAP31.

MAP30 (Momordica anti-HIV protein of 30 kDa) and GAP31 (Gelonium anti-HIV protein of 31 kDa) are anti-HIV plant proteins that we have identified, purified, and cloned from the medicinal plants Momordica charantia and Gelonium multiflorum. These antiviral agents are capable of inhibiting infection of HIV type 1 (HIV-1) in T lymphocytes and monocytes as well as replication of the virus in already-infected cells. They are not toxic to normal uninfected cells because they are unable to enter healthy cells. MAP30 and GAP31 also possess an N-glycosidase activity on 28S ribosomal RNA and a topological activity on plasmid and viral DNAs including HIV-1 long terminal repeats (LTRs). LTRs are essential sites for integration of viral DNA into the host genome by viral integrase. We therefore investigated the effect of MAP30 and GAP31 on HIV-1 integrase. We report that both of these antiviral agents exhibit dose-dependent inhibition of HIV-1 integrase. Inhibition was observed in all of the three specific reactions catalyzed by the integrase, namely, 3' processing (specific cleavage of the dinucleotide GT from the viral substrate), strand transfer (integration), and "disintegration" (the reversal of strand transfer). Inhibition was studied by using oligonucleotide substrates with sequences corresponding to the U3 and U5 regions of HIV LTR. In the presence of 20 ng of viral substrate, 50 ng of target substrate, and 4 microM integrase, total inhibition was achieved at equimolar concentrations of the integrase and the antiviral proteins, with EC50 values of about 1 microM. Integration of viral DNA into the host chromosome is a vital step in the replicative cycle of retroviruses, including the AIDS virus. The inhibition of HIV-1 integrase by MAP30 and GAP31 suggests that impediment of viral DNA integration may play a key role in the anti-HIV activity of these plant proteins.

Antiviral Agents↗

PubMed1988

Selective cytotoxic activity of immunotoxins composed of a monoclonal anti-Thy 1.1 antibody and the ribosome-inactivating proteins bryodin and momordin.

The ribosome-inactivating proteins, bryodin, from Bryonia dioica, and momordin, from Momordica charantia, were coupled by a disulphide bond to a monoclonal anti-Thy 1.1 antibody (OX7). Both immunotoxins were specifically cytotoxic to the Thy 1.1-expressing mouse lymphoma cell line AKR-A in vitro. The OX7-bryodin immunotoxins were the more powerfully toxic and reduced protein synthesis in AKR-A cells by 50% at a concentration of 1-4 x 10(-11) M as compared with 1 x 10(-9) M for the OX7-momordin immunotoxins. Neither of the immunotoxins was toxic to mouse lymphoma EL4 cells, which lack the Thy 1.1 antigen, at concentrations up to 3 x 10(-8) M. Further, bryodin and momordin immunotoxins made from an antibody (R10) of irrelevant specificity were without effect on AKR-A cells.

Animals↗

PubMed1990

Blood clearance and organ distribution and tissue concentration of native, homopolymerized and IgG-conjugated ribosome-inactivating proteins.

1. The blood clearance, organ distribution and tissue concentration of several native, homopolymerized, and IgG-conjugated 125I-labelled ribosome-inactivating proteins (RIPs) were determined in mice. 2. Native RIPs were cleared rapidly from blood, with half-lives of 4-8 min, and were concentrated mainly in the kidneys. 3. After conjugation to IgG the three RIPs studied showed increased blood half-lives and decreased concentrations in the kidneys. 4. The two homopolymers studied had blood half-lives and kidney concentrations intermediate to those of free and conjugated RIPs. 5. These results indicate that after IgG-conjugation the increased half-lives of the RIPs studied were at least in part due to the larger molecular size of the conjugates and to their lower renal excretion.

Animals↗

PubMed1993

Differential effects of suicide transport lesions of the striatonigral or striatopallidal pathways on subsets of striatal neurons.

In the basal ganglia, centrally active suicide transport agents produce apparently selective lesions of the striatopallidal and striatonigral pathways based on receptor binding and neuropeptide mRNA studies. In the present study we sought to determine the selectivity of suicide transport lesions for specific subsets of striatal neurons. Using immunohistochemical methods, the neostriata of adult rats were examined 10 days after an injection of volkensin into the substantia nigra or an injection of OX7-saporin into the globus pallidus. Ricin, a suicide transport agent active in the peripheral but not the central nervous system, was injected into each target as a control. Adjacent sections were processed for (1) Nissl stain to assess neuronal density, both overall and for large interneurons, (2) NADPH-diaphorase (NADPH-d) histochemistry, to mark medium-sized aspiny interneurons, (3) enkephalin immunocytochemistry, to label striatopallidal neurons, or (4) substance P immunocytochemistry, to label striatonigral neurons. Ricin injections produced no change in the densities of these subsets of striatal cells. In animals receiving volkensin or OX7-saporin injections, analyses of Nissl material revealed that the striata ipsilateral to the toxin injections appeared normal and did not exhibit shrinkage or gliosis; however, a quantitation analysis revealed a moderate decrease in cell density (12-16% loss, P < 0.01). The densities of both large and NADPH-d-containing striatal interneurons were unchanged after lesions in either target. Following nigral injections with volkensin, the density of striatal substance P-labeled cells decreased (26% loss, P < 0.01), while the density of enkephalin-labeled cells did not decrease significantly (11% decrease, P > 0.1). After pallidal injections with OX7-saporin, the density of striatal enkephalin-labeled cells decreased (20% loss, P < 0.01), while that of substance P-labeled cells remained unchanged. These data show that nigral volkensin and pallidal OX7-saporin injections differentially lesion striatonigral and striatopallidal projection neurons and spare striatal interneurons. This study provides further evidence for the selectivity, specificity, and utility of suicide transport agents to study brain structure and function.

Animals↗

PubMed1990

Pharmacokinetics in the rat of a panel of immunotoxins made with abrin A chain, ricin A chain, gelonin, and momordin.

A panel of immunotoxins was constructed by chemically attaching the ribosome-inactivating proteins abrin A chain, ricin A chain, gelonin, and momordin to the monoclonal mouse IgG2a antibody Fib75 by means of a disulfide linkage. All the immunotoxins were toxic in tissue culture to the EJ human bladder carcinoma cell line expressing the antigen recognized by Fib75, inhibiting the incorporation of [3H]leucine by 50% at concentrations between 1 x 10(-10) M and 8 x 10(-10) M. The pharmacokinetics of the immunotoxins in the normal Wistar rat was determined following i.v. administration. The concentrations of intact immunotoxin in serum samples taken at various intervals after injection for up to 24 h were measured by solid-phase enzyme-linked immunosorbent assays specific for each of the four different ribosome-inactivating proteins. The Fib75 immunotoxins were cleared from the circulation with comparable, but not identical, biphasic kinetics best described by a two compartment open pharmacokinetic model. The alpha-phase half-lives of the panel, between 0.35 and 0.71 h, were similar. The beta-phase half-life of Fib75-abrin A chain, 13.3 h, was significantly longer than the beta-phase half-lives of Fib75-ricin A chain, Fib75-gelonin, and Fib75-momordin, between 7.5 and 8.6 h. Fib75-abrin A chain was found to be about 3- to 4-fold more resistant than the other immunotoxins to breakdown by reduction of the disulfide linkage between the A chain and the antibody with glutathione in vitro. This suggests that the longer serum half-life of Fib75-abrin A chain may have been due to greater stability against reduction in vivo. Analysis of serum samples obtained up to 24 h after injection of Fib75-abrin A chain revealed that the chemically intact immunotoxin present in the circulation retained full cytotoxic activity. An abrin A chain immunotoxin made with a different monoclonal mouse IgG2a antibody was also found to be more stable against reduction by glutathione in vitro than an analogous ricin A chain immunotoxin. Thus, abrin A chain may posses unique molecular properties that endow immunotoxins made with this A chain with greater stability in vivo than immunotoxins made with ricin A chain or other ribosome-inactivating proteins.

Abrin↗

PubMed1996

Induction of apoptosis by ribosome-inactivating proteins and related immunotoxins.

Immunotoxins have been prepared with 3 ribosome-inactivating proteins (RIPs), namely, momordin, pokeweed anti-viral protein from seeds (PAP-S) and saporin, linked to the Ber-H2 monoclonal antibody directed against the CD30 antigen of human lymphocytes. Either the RIPs or the immunotoxins induced apoptosis in the CD30+ L540 cell line, as shown by the morphological aspects of the cells, by the DNA fragmentation visible at the electrophoresis, and by the formation of DNA breaks evidenced by 2 cytofluorometric techniques (propidium-iodide staining and fluoresceine-isothiocyanate conjugate dUTP incorporation). The AC50 (concentration causing apoptosis in 50% of the cells) is in the range 10(-8) to 10(-7) M in the case of RIPs, and 10(-11) to 10(-10) M in the case of the immunotoxins.

Antibodies, Monoclonal↗