Comparative analysis of depurination catalyzed by ricin A-chain on synthetic 32mer and 25mer oligoribonucleotides mimicking the sarcin/ricin domain of the rat 28S rRNA and E. coli 23S rRNA

摘要

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 sub(m)) for the reaction with Rat FAM-SRD (4.57+ /-0.28 mu M) corresponded to that with E. coli FAM-SRD (4.64+ /-0.26 mu M).However, the maximum velocity (V sub(m) sub(a) sub(x)) for ricin A-chain with Rat FAM-SRD was 0.5+/-0.024 mu M/min, which is higher than that with E. coli FAM-SRD (0.32+/-0.011 mu M/min). oligoribonucleotide mimicking the sarcin/ricin domains of rat 28SrRNA oligoribonucleotide mimicking the sarcin/ricin domains of E. coli 23S rRNA