Studies on Ribosome-inactivating Proteins from Saponaria officinalis

The ribosome-inactivating proteins (RIPs) are a family of plant enzymes for which a unique activity has been determined: rRNA N-glycosidase, which removes adenine at a specific universally conserved position (A4324 in the case of rat ribosomes). Here we report that saporin-L1, a RIP from the leaves of Saponaria officinalis, recognizes other substrates, including RNAs from different sources, DNA and poly(A). Saporin-L1 depurinated DNA extensively and released adenine from all adenine-containing polynucleotides tested. Adenine was the only base released from DNA or artificial polynucleotides. The characteristics of the reactions catalysed by saporin-L1 have been determined: optimal pH and temperature, ionic requirements, and the kinetic parameters Km and kcat. The reaction proceeded without cofactors, at low ionic strength, in the absence of Mg2+ and K+. Saporin-L1 had no activity towards various adenine-containing non-polynucleotide compounds (cytokinins, cofactors, nucleotides). This plant protein may now be classified as a polynucleotide:adenosine glycosidase.

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Cellular and subcellular distribution of saporins, type-1 ribosome-inactivating proteins, in soapwort (Saponaria officinalis L.)

Planta ◽

10.1007/bf00714457 ◽

1994 ◽

Vol 194 (4) ◽

pp. 461-470 ◽

Cited By ~ 22

Author(s):

Raffaella Carzaniga ◽

Lesley Sinclair ◽

Anthony P. Fordham-Skelton ◽

Nick Harris ◽

Ronald R. D. Croy

Keyword(s):

Subcellular Distribution ◽

Ribosome Inactivating Proteins ◽

Saponaria Officinalis

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Ribosome-inactivating proteins from the seeds of Saponaria officinalis L. (soapwort), of Agrostemma githago L. (corn cockle) and of Asparagus officinalis L. (asparagus), and from the latex of Hura crepitans L. (sandbox tree)

Biochemical Journal ◽

10.1042/bj2160617 ◽

1983 ◽

Vol 216 (3) ◽

pp. 617-625 ◽

Cited By ~ 164

Author(s):

F Stirpe ◽

A Gasperi-Campani ◽

L Barbieri ◽

A Falasca ◽

A Abbondanza ◽

...

Keyword(s):

Protein Synthesis ◽

Sugar Content ◽

Asparagus Officinalis ◽

Inhibit Protein Synthesis ◽

Nicotiana Glutinosa ◽

Ribosome Inactivating Proteins ◽

Local Lesions ◽

Hura Crepitans ◽

Reticulocyte Lysate ◽

Saponaria Officinalis

Ribosome-inactivating proteins, similar to those already known [Barbieri & Stirpe (1982) Cancer Surveys 1, 489-520] were purified from the seeds of Saponaria officinalis (two proteins), of Agrostemma githago (three proteins), and of Asparagus officinalis (three proteins), and from the latex of Hura crepitans (one protein). The yield ranged from 8 to 400 mg/100 g of starting material. All proteins have an Mr of approx. 30000 and an alkaline isoelectric point. Their sugar content varies from 0 (proteins from S. officinalis) to 40% (protein from H. crepitans). The ribosome-inactivating proteins inhibit protein synthesis by rabbit reticulocyte lysate, the ID50 (concentration giving 50% inhibition) ranging from 1 ng/ml (a protein from S. officinalis) to 18 ng/ml (a protein from A. githago). Those which were tested (the proteins from S. officinalis and from A. githago) also inhibit polymerization of phenylalanine by isolated ribosomes, acting in an apparently catalytic manner. The protein from H. crepitans inhibited protein synthesis by HeLa cells, with an ID50 of 4 micrograms/ml, whereas the proteins from S. officinalis and from A. githago had an ID50 of more than 50-100 micrograms/ml. The ribosome-inactivating proteins from S. officinalis and from A. githago reduced the number of local lesions by tobacco-mosaic virus in the leaves of Nicotiana glutinosa.

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The differential catalytic activity of ribosome-inactivating proteins saporin 5 and 6 is due to a single substitution at position 162

Biochemical Journal ◽

10.1042/bj20060895 ◽

2006 ◽

Vol 400 (1) ◽

pp. 99-104 ◽

Cited By ~ 12

Author(s):

Paroma Ghosh ◽

Janendra K. Batra

Keyword(s):

Catalytic Activity ◽

Amino Acid ◽

Active Sites ◽

Functional Characterization ◽

Cytotoxic Activities ◽

Type I ◽

Ribosome Inactivating Protein ◽

Aspartic Acid Residue ◽

Ribosome Inactivating Proteins ◽

Saponaria Officinalis

Saporin, a type I ribosome-inactivating protein produced by the soapwort plant Saponaria officinalis belongs to a multigene family that encodes its several isoforms. The saporin seed isoform 6 has significantly higher N-glycosidase and cytotoxic activities compared with the seed isoform 5, although the two have identical active sites. In the present study, we have investigated the contribution of non-conservative amino acid changes outside the active sites of these isoforms towards their differential catalytic activity. The saporin 6 residues Lys134, Leu147, Phe149, Asn162, Thr188 and Asp196 were replaced by the corresponding saporin 5 residues, Gln134, Ser147, Ser149, Asp162, Ile188 and Asn196, to generate six variants of saporin 6, K134Q, L147S, F149S, N162D, T188I and D196N. By functional characterization, we show that the change in amino acid Asn162 in saporin 6 to aspartic acid residue of saporin 5 contributes mainly to the lower catalytic activity of saporin 5 compared with saporin 6. The non-involvement of other non-conservative amino acids in the differential catalytic activity of these isoforms was confirmed with the help of the double mutations N162D/K134Q, N162D/L147S, N162D/F149S, N162D/T188I and N162D/D196N.

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