TY  - JOUR
TI  - Ribosomal RNA N-glycosylase Activity Assay of Ribosome-inactivating Proteins
AU  - Iglesias, Rosario  
AU  - Citores, Lucía  
AU  - Ferreras, José  M. 
VL  - 7
IS  - 6
PY  - 2017
DA  - 2017/03/20
SP  - e2180
C1  - Bio-protocol 2017;7:e2180
DO  - 10.21769/BioProtoc.2180
UR  - https://doi.org/10.21769/BioProtoc.2180
AB  - Ribosome-inactivating proteins (RIPs) are enzymes that irreversibly inactivate ribosomes as a consequence of their N-glycosylase (EC 3.2.2.22) activity. The enzyme cleaves the N-glycosidic bond between the adenine No. 4324 from the 28S rRNA and its ribose in rat ribosomes (or the equivalent adenine in sensitive ribosomes from other organisms). This adenine is located in the α-sarcin-ricin loop (SRL) that is crucial for anchoring the elongation factor (EF) G and EF2 on the ribosome during mRNA-tRNA translocation in prokaryotes and eukaryotes, respectively. RIPs have been isolated mainly from plants and examples of these proteins are ricin or Pokeweed Antiviral Protein (PAP). These proteins, either alone or as a part of immunotoxins, are useful tools for cancer therapy. The following protocol describes a method to detect the RNA fragment released when the RIP-treated apurinic RNA from rabbit reticulocyte lysate is incubated in the presence of acid aniline by electrophoresis on polyacrylamide gels. The fragment released (Endo’s fragment) is diagnostic of the action of RIPs.
KW  - Ribosome-inactivating protein (RIP)
KW  - rRNA N-glycosylase
KW  - Protein synthesis (inhibition)
KW  - Sarcin-ricin loop
KW  - Polynucleotide:adenosine glycosylase
KW  - Ricin
KW  - Pokeweed Antiviral Protein (PAP)
KW  - Beetin 27
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False