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L Montanaro

Publications and source records attributed to L Montanaro.

At least 91 records · Page 5Linked to original sources
PubMed1992

Differential requirement of ATP and extra-ribosomal proteins for ribosome inactivation by eight RNA N-glycosidases.

The requirement of ATP and extra-ribosomal proteins for the inactivation of ribosomes by eight plant RNA N-glycosidases [ribosome-inactivating proteins (RIPs)] was investigated. Tritin, pokeweed antiviral protein and barley RIP depend, as gelonin [Sperti, S., Brigotti, M., Zamboni, M., Carnicelli, D. and Montanaro, L. (1991) Biochem. J., 277, 281-284], on the presence of ATP and extra-ribosomal proteins for full inactivation of ribosomes, while bryodin, lychnin, momordin, momorcochin and saporin inactivate isolated Artemia salina ribosomes suspended in buffer saline.

Adenosine Triphosphate↗
PubMed1991

Elongation factor 2 from Artemia salina embryos and its affinity for ribosomes.

Crude extracts from Artemia salina undeveloped embryos do not contain detectable elongation-factor-2 (EF2) kinase and endogenous ADP-ribosylating activities. Accordingly, EF2 purified from this source is an enzyme relatively free from phosphorylated and ADP-ribosylated forms. Endogenous ADP-ribosyltransferase activity appears only after purification of EF2. The affinities of EF2 and of ADP-ribosyl-EF2 for ribosomes from A. salina undeveloped embryos have been calculated by measuring the ability of the factors to inhibit the N-glycosidase activity of ricin on ribosomes.

Adenosine Diphosphate Ribose↗
PubMed1991

Requirements for the inactivation of ribosomes by gelonin.

Inactivation of Artemia salina and rabbit ribosomes by gelonin requires ATP and a high-Mr factor present in the rabbit reticulocyte-lysate post-ribosomal supernatant. The kinetic constants of the gelonin-catalysed release of adenine from A. salina ribosomes are Km = 4.35 microM and Kcat. = 0.1 min-1 in the absence of cofactors, and Km = 1.15 microM and Kcat. = 108 min-1 in their presence. The last two values are similar to those measured for ricin A chain in the absence of cofactors (Km = 2.02 microM and Kcat. = 317 min-1).

Adenine↗
PubMed1991

The Ag-NOR proteins and transcription and duplication of ribosomal genes in mammalian cell nucleoli.

The relationship between the Ag-NOR (silver-stained Nucleolar Organizer Region) proteins and the functional-structural organization of the nucleolar ribosomal chromatin was studied in regenerating and cortisol-stimulated rat hepatocytes. Statistical analysis of Ag-NOR proteins, carried out with an automated image analyzer, indicated that in regenerating rat hepatocytes the quantity of Ag-NOR proteins mainly increased between the 4th and 12th h of regeneration, reaching a level twice that of resting hepatocytes. Also the synthesis of pre-ribosomal RNA (pre-rRNA) was stimulated after the 4th h of regeneration. Cycloheximide administered to rats at a dose of 0.025 mg/100 g body weight (bw) prevented any increase in Ag-NOR proteins but did not hinder the stimulation of pre-rRNA synthesis. In 8 h cortisol-stimulated hepatocytes no significant change in amount of Ag-NOR protein was observed whereas pre-rRNA synthesis was highly increased as in 12 h regenerating hepatocytes. These results indicated that in rat hepatocytes Ag-NOR proteins and stimulation of pre-rRNA synthesis are not related. The relationship between the Ag-NOR proteins and the distribution of the completely extended intranucleolar ribosomal chromatin was also studied in regenerating rat hepatocytes. At 12 h after partial hepatectomy an increased amount of completely extended ribosomal chromatin was observed, contemporaneously with an increased quantity of Ag-NOR proteins. These ribosomal chromatin changes preceded the beginning of DNA synthesis and were prevented by cycloheximide-induced inhibition of protein synthesis.

Animals↗
PubMed1990

Isolation of an inhibitor of cell-free protein synthesis from Salmonella enteritidis.

Sonic extracts of Salmonella enteritidis, serotype enteritidis, contain a factor which inhibits eukaryotic cell-free protein synthesis tested on a rabbit reticulocyte lysate system and on a poly(U)-dependent system from Artemia salina embryos. The factor is heat-labile, does not diffuse through dialysis bags and is precipitated by ammonium sulphate. A 78% recovery of the factor was obtained in the 60-80 per cent ammonium sulphate saturation range, with a 3.3-fold purification. The factor binds to DEAE-cellulose. In the fraction eluted at 0.55 M NaCl, SDS-polyacrylamide gel electrophoresis shows only two protein bands (Mr 55,000 and 29,000). While the inhibitory activity on protein synthesis of the crude factor is fairly stable at 4 degrees C, it becomes very labile after DEAE-cellulose chromatography. The ID50 of the partially purified factor was less than 0.55 microgram (20-fold purification).

Ammonium Sulfate↗
PubMed1989

High-pressure-liquid-chromatographic and fluorimetric methods for the determination of adenine released from ribosomes by ricin and gelonin.

The high fluorescence of adenine-containing compounds after reaction with chloroacetaldehyde was used to measure the adenine released from rat liver and Artemia salina ribosomes by the action of ricin A chain and gelonin, two ribosome-inactivating proteins (RIPs) that share the same mechanism of action, consisting in the hydrolysis of the N-glycosidic bond of A-4324 of 28 S rRNA. Two methods were employed: (i) h.p.l.c. of the chloroacetaldehyde-reactive material released by RIPs; h.p.l.c. associated with a fluorescence detector allows the identification of adenine and its dosage at quantities as low as 2 ng; (ii) the direct fluorimetric measurement of the material that had reacted with chloroacetaldehyde. The amount of adenine released increases when ribosomes are pretreated in conditions that lead to their dissociation into subunits. Adenine protects ribosomes from the inhibition by ricin A-chain. When ribosomes were incubated with ricin A-chain in the presence of [14C]adenine no incorporation of radioisotope in ribosomes was observed, indicating that neither exchange nor reversal reactions occurred. A binding of [14C]adenine to ricin A chain was not detected by equilibrium dialysis.

Adenine↗
PubMed1989

Alpha-sarcin impairs the N-glycosidase activity of ricin on ribosomes.

In a recently described method the RNA N-glycosidase activity of ricin is measured by h.p.l.c. determination of the adenine released from ribosomes after conversion of the base into its fluorescent 1-N6-etheno derivative [Zamboni et al. (1989) Biochem. J., 259, in press]. Unlike previously available methods, based on the separation of RNA fragments by gel electrophoresis, the new method allows one to investigate the activity of ricin on ribosomes pretreated with alpha-sarcin, a cytotoxin which cleaves 28S rRNA at one nucleotide distance from the site of attack of ricin. alpha-Sarcin makes ribosomes a poor substrate for ricin, the release of adenine requiring concentrations of ricin 50-times higher than those effective on untreated ribosomes.

Adenine↗
PubMed1989

Effect of the antibiotic purpuromycin on cell-free protein-synthesizing systems.

Purpuromycin, an antibiotic isolated from the culture broth of Actinoplanes ianthinogenes, which is very active against Gram-positive bacteria and fungi, inhibits protein synthesis in both prokaryotic and eukaryotic cell-free systems. The ID50 was 9 microM with the endogenous mRNA-directed rabbit reticulocyte lysate, 17 microM with a poly(U)-directed system from Escherichia coli and 69 microM with a poly(U)-directed system from Artemia salina cysts. Of the three steps of elongation, purpuromycin does not affect the peptidyl-transferase reaction, inhibits the elongation factor 1 (EF-1) dependent binding of phenylalanyl-tRNA and stimulates the GTP-dependent binding of EF-2. When protein synthesis is stopped by the addition of purpuromycin, the nascent peptide chains are found in the puromycin-reactive P site. The results suggest that the mechanism of action of purpuromycin is similar to that of fusidic acid. Both antibiotics would seem to produce a stable guanine nucleotide-ribosome-EF-2 complex which allows one round of translocation but prevents, because of a common or overlapping ribosomal binding site for the two elongation factors, the subsequent EF-1-dependent binding of aminoacyl-tRNA.

Animals↗
PubMed1989

Effect of alpha-sarcin and ribosome-inactivating proteins on the interaction of elongation factors with ribosomes.

alpha-Sarcin from Aspergillus giganteus and the ribosome-inactivating proteins (RIPs) from higher plants inactivate the 60 S ribosomal subunit. The former is an RNAase, whereas RIPs are N-glycosidases. The site of cleavage of RNA and that of N-glycosidic depurinization are at one nucleotide distance in 28 S rRNA [Endo & Tsurugi (1987) J. Biol. Chem. 262, 8128-8130]. The effect of alpha-sarcin and that of RIPs on the interaction of elongation factors with Artemia salina (brine shrimp) ribosomes have been investigated. alpha-Sarcin inhibits both the EF1 (elongation factor 1)-dependent binding of aminoacyl-tRNA and the GTP-dependent binding of EF2 (elongation factor 2) to ribosomes, whereas two of the RIPs tested, ricin from Ricinus communis (castor bean) and volkensin from Adenia volkensii (kilyambiti), inhibit only the latter reaction. EF2 protects ribosomes from inactivation by both alpha-sarcin and ricin. The EF1-binding site is affected only by alpha-sarcin. The sensitivity of this site to alpha-sarcin is increased by pretreatment of ribosomes with ricin. A. salina ribosomes were highly resistant to the third RIP tested, namely gelonin from Gelonium multiflorum. All four proteins tested have, however, a comparable activity on the rabbit reticulocyte-lysate system.

Aspergillus↗
PubMed1987

Interaction of diphtheria toxin fragment A and of elongation factor 2 with cibacron blue.

Diphtheria toxin fragment A interacts with Cibacron blue in solution, although it is not retained by blue Sepharose columns. Difference spectral titration of fragment A with the dye gives a dissociation constant of the order of 10(-5) M and a 1:1 stoichiometry for the complex. In equilibrium dialysis experiments Cibacron blue behaves as a competitive inhibitor of the binding of NAD to diphtheria toxin fragment A. The dye inhibits in a non-competitive way the fragment A-catalysed transfer of ADP-ribose from NAD to elongation factor 2 (EF2). By affinity chromatography on blue Sepharose a binding of EF2 and of ADP-ribosyl-EF2 with the dye is also demonstrated. GDP, GTP and GDP(CH2)P are able to displace EF2 from blue Sepharose.

Animals↗
PubMed1986

Interaction of alpha-sarcin and gelonin with cibacron blue.

Alpha-sarcin and gelonin, two proteins which inactivate the 60S ribosomal subunit, interact with Cibacron blue and bind to blue dextran-Sepharose, from which they are partially desorbed by nucleoside triphosphates and, more efficiently, by homopolynucleotides. It is further shown that the two proteins bind to poly(U)-Sepharose and that homopolynucleotides protect dilute solutions of both inhibitors from inactivation. It is suggested that the presence of a polynucleotide site on alpha-sarcin is related to its ribonuclease activity. The existence of a similar site on gelonin might be a clue to its yet unknown mechanism of action.

Binding, Competitive↗
PubMed1986

Dye affinity chromatography of ricin subunits.

A rapid and simple method for the isolation of the two chains of ricin is described. The method involves two chromatographic runs, the first on blue-Sepharose and the second on blue dextran-Sepharose. It is moreover shown that the A chain of ricin, but not the B chain, binds to poly(U)-Sepharose.

Chromatography, Affinity↗
PubMed1981

Ribosomal core-particles as the target of ricin.

Core-particles and split-proteins were prepared by treatment with ethanol and NH4Cl of control and ricin-treated Artemia salina ribosomes. No modifications of the ricin-treated split-proteins was detected by polyacrylamide-gel electrophoresis. Moreover, the split-proteins from ricin-treated ribosomes complemented control core-particles in poly(U)-directed phenylalanine polymerization. Conversely, ricin-treated core-particles remained totally inactive when supplemented with control split-proteins.

Animals↗
PubMed1980

Inhibition of protein synthesis in vitro by proteins from the seeds of Momordica charantia (bitter pear melon).

1. A haemagglutinating lectin was purified from the seeds of Momordica charantia by affinity chromatography on Sepharose 4B and on acid-treated Sepharose 6B. It has mol.wt. 115 000 and consists of four subunits, of mol.wts. 30 500, 29 000, 28 500 and 27 000. 2. The lectin inhibits protein synthesis by a rabbit reticulocyte lysate with an ID50 (concentration giving 50% inhibition) of approx. 5 micrograms/ml. Protein synthesis by Yoshida ascites cells is partially inhibited by the lectin at a concentration of 100 micrograms/ml. 3. From the same seeds another protein was purified which has mol.wt. 23 000 and is a very potent inhibitor of protein synthesis in the lysate system, with an ID50 of 1.8 ng/ml. This inhibitor has no effect on protein synthesis by Yoshida cells, and has no haemagglutinating properties. 4. Artemia salina ribosomes preincubated with the lectin or with the inhibitor lose their capacity to perform protein synthesis. The proteins seem to act catalytically, since they inactivate a molar excess of ribosomes. 5. The lectin and the inhibitor are somewhat toxic to mice, the LD50 being 316 and 340 micrograms/100 g body wt. respectively.

Animals↗
PubMed1980

Purification and properties of different forms of modeccin, the toxin of Adenia digitata. Separation of subunits with inhibitory and lectin activity.

1. The subunits were isolated of modeccin (subsequently referred to as modeccin 4B), the toxin purified from the roots of Adenia digitata by affinity chromatography on Sepharose 4B [Gasperi-Campani, Barbieri, Lorenzoni, Montanaro, Sperti, Bonetti & Stirpe (1978) Biochem J. 174, 491-496]. They are an A subunit (mol.wt. 26 000), which inhibits protein synthesis, and a B subunit (mol.wt. 31 000), which binds to cells. Both sununits, as well as intact modeccin, gave single bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, but showed some heterogeneity on isoelectric focusing and on polyacrylamide-gel electrophoresis at pH 9.5. 2. A second form of modeccin, not retained by Sepharose 4B, was purified by affinity chromatography on acid-treated Sepharose 6B: this form is subsequently termed modeccin 6B 3. Modeccin 6B has a molecular weight indistinguishable from that of modeccin 4B, and consists of two subunits of mol.wts. 27 000 and 31 000, joined by a disulphide bond. The subunits were not isolated because of their high insolubility in the absence of sodium dodecyl sulphate. 4. As compared with modeccin 4B, modeccin 6B is slightly less toxic to animals, does not agglutinate erythrocytes, and is a more potent inhibitor of protein synthesis in a lysate of rabbit reticulocytes, giving 50% inhibition at the concentration of 0.31 microgram/ml.

Animals↗
PubMed1979

Effect of modeccin on rat liver ribosomes in vivo.

1. Rat liver microsomes isolated at 6 and 12 h of poisoning with 3 x LD50 (0.3 microgram/100 g body wt.) of modeccin, the toxin of Adenia digitata, have a decreased capacity of protein synthesis in vitro. 2. A similar decrease of protein synthesis is observed with polysomes at 6 h of poisoning. Experiments with recombined ribosomal subunits demonstrate that this is due to inactivation of the 60 S ribosomal subunit. 3. At 6 h of poisoning there is a marked vesiculation and degranulation of the hepatocyte rough endoplasmic reticulum, which is completely fragmented at 24 h of poisoning. Hepatocyte mitochondria are swollen at 6 h and shrunk at 24 h of poisoning. 4. It is concluded that modeccin penetrates inside hepatocytes in vivo, and damages ribosomes in the same manner as it does in vitro. However, mitochondrial damage indicates that ribosomes may not be the only target of modeccin in vivo.

Animals↗