Radioassay for RNA N-Glycosidase with Tritium-Labeled Sodium Borohydride or Amino Acid

The aldehyde radical at position 4324 in rat 28S rRNA generated by RNA N-glycosidase was either reduced to hydroxyl group by 3H-labeled sodium borohydride or converted into an aldimine through nucleophilic addition using 3H-labeled amino acid as a primary amine. The radioactivity incorporated into the modified ribosome allows a rapid and sensitive determination and quantification of RNA N-glycosidase activity. Among three different substrates, i.e., 80S ribosome, 60S ribosomal subunit, and ribosomal RNA, the 60S ribosomal subunit was found to be optimal and [3H]-leucine turned out to be more effective in the labeling reaction than [3H]-NaBH4. The following three lines of evidence demonstrate a high specificity of the reaction: Radioactivity was exclusively detected in 28S rRNA as revealed by autoradiography, the preincubation of modified ribosomes with cold NaBH4 or amino acid prevented the incorporation of radioactivity; ribosomes modified by α-sarcin could not be labeled with either [3H]-NaBH4 or [3H]-leucine. The apparent Michaelis constant (Km) of ricin A-chain for 80S ribosome was estimated to be 2.8 × 10-6 M, very close to the result reported by Endo et al. In addition to relative convenience and good reproducibility, this method allows the characterization of ribosome-inactivating proteins belonging to a specific RNA N-glycosidase.