Abstract
Ricin A-chain can inactivate eukaryotic ribosomes, but exhibits no N-glycosidase activity on intact E. coli ribosomes. In the present research, in order to avoid using radiolabeled oligoribonucleotides, two kinds of synthetic 5′-FAM fluorescence-labeled oligoribonucleotide substrates were used to mimic the sarcin/ricin domains of rat 28S rRNA and E. coli 23S rRNA (32mer and 25mer, named as Rat FAM-SRD and E. coli FAM-SRD, respectively). Ricin A-chain was able to specifically release adenine from the first adenosine of the GAGA tetraloop and exhibited specific N-glycosidase activity under neutral and weak acidic conditions with both substrates. However, under more acidic conditions, ricin A-chain was able to release purines from other sites on eukaryotic substrates, but it retained specific depurination activity on prokaryotic substrates. At pH 5.0, the Michaelis constant ( K m) for the reaction with Rat FAM-SRD (4.57 ± 0.28 μM) corresponded to that with E. coli FAM-SRD (4.64 ± 0.26 μM). However, the maximum velocity ( V max) for ricin A-chain with Rat FAM-SRD was 0.5 ± 0.024 μM/min, which is higher than that with E. coli FAM-SRD (0.32 ± 0.011 μM/min).
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