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Genomic clones encoding the mature pokeweed antiviral protein with or without C-terminal extrapeptide (PAPMCand PAPM), which have been reported to be highly toxic to E. colicells, were inserted into the expression vector pMAL-p2. The recombinant PAPs (rPAPMCand rPAPM) were successfully expressed in E. coliat 25°C, being exported to the periplasm as soluble fusions with maltose-binding protein (MBP). The rPAPs were cleaved from MBP by treatment with factor Xa and subsequently purified with final yields of 4.0 mg/liter (rPAPMC) and 5.5 mg/liter (rPAPM). rPAPMwas resistant to protease digestion, but the C-terminal extrapeptide appeared to be susceptible and was partially digested by some protease in E. coli. Both rPAPMCand rPAPMwere as active as the native PAPMfrom pokeweed leaves in depurinating rat liver and E. coliribosomes, while the activities of rPAPMCon both ribosomes were decreased at least 60-fold by fusion with MBP.

Ricin A‐chain, an N‐glycosidase that attacks 28S rRNA at a highly conserved adenine residue, has a unique tryptophan (Trp‐211) in the putative active site cleft. Fluorescence spectroscopy revealed that specific binding of adenine to the A‐chain caused a large enhancement of Trp‐211 fluorescence (70%) and a concomitant red shift of the emission spectrum (8 nm). A Scatchard plot of the fluorescence enhancement data was not linear, indicating that the environment of Trp‐211 was altered by heterogeneous binding of adenines. These results, taken together with the protective effect of adenine on the ribosome‐inactivation by ricin A‐chain, suggest that at least two adenines bind to the active site cleft.

Ricin A-chain, an N-glycosidase that attacks 28S rRNA at a highly conserved adenine residue, has a unique tryptophan (Trp-211) in the putative active site cleft. Fluorescence spectroscopy revealed that specific binding of adenine to the A-chain caused a large enhancement of Trp-211 fluorescence (70%) and a concomitant red shift of the emission spectrum (8 nm). A Scatchard plot of the fluorescence enhancement data was not linear, indicating that the environment of Trp-211 was altered by heterogeneous binding of adenines. These results, taken together with the protective effect of adenine on the ribosome-inactivation by ricin A-chain, suggest that at least two adenines bind to the active site cleft.