Isolation and characterization of four type-1 ribosome-inactivating proteins, with polynucleotide : adenosine glycosidase activity, from leaves of Phytolacca dioica L.

Four type-1 (single-chain) ribosome-inactivating proteins (RIPs), with isoe lectric points between 9.5 and 9.7, were isolated from leaves of Phytolacca dioica L. The purification procedure furnished the four proteins with an o verall yield of about 16% and separated them from a protein of 29 407 +/- 2 Da, as determined by electrospray mass spectrometry, whose N-terminal amin o acid sequence differed from that of pokeweed (Phytolacca americana L.) le af chitinase (PLC-B) by only one amino acid (R17I). The four RIPs (PD-L1 to PD-L4) inhibited protein synthesis by a rabbit reticulocyte lysate with 50 % inhibition at the picomolar level, and produced the beta-fragment, diagno stic of the specific enzymatic action of RIPs, on yeast ribosomes. Comparis on of their N-terminal sequences, up to residue 45, showed that PD-L1 is id entical to PD-L2 [designated the isoleucine (Ile) form from the N-terminal residue] and PD-L3 is identical to PD-L4 [designated the valine (Val) form from the N-terminal residue] and that there are 35 identical residues betwe en the two forms. Furthermore, the Val form presents the same number of ide ntical residues as PD-S2, an RIP isolated from the seeds of the same plant. With the exception of PD-L4, the purified RIPs gave a positive reaction wh en stained for sugars on SDS-PAGE gels and, when analyzed by electrospray m ass spectrometry, had M-r values of 32 715 +/- 1 (PD-L1), 31 542 +/- 1 (PD- L2), 30 356 +/- 1 (PD-L3) and 29 185 +/- 1 Da (PD-L4). The 1171 kDa differe nce in M-r, within the same RIP form, could be due to glycosylation. Like l eaf saporins and many other RIPs, the four RIPs released several adenines f rom poly(A), herring sperm DNA and rRNA 16S + 23S, thus acting as polynucle otide:adenosine glycosidases. This property was less pronounced in PD-L1 an d PD-L3 than in PD-L2 and PD-L4, respectively. The proteins PD-L1 and PD-L4 showed 3.7% reactivity with the antiserum anti-dianthin 32 and no reactivi ty with antisera to PAP-R saporin-S6, momordin I and even PD-S2, an RIP iso lated from the seeds of the same plant. Protein PD-L4 showed 12.5% cross-re activity with anti-PD-L1, while the opposite cross-reactivity was 100%.