Di Maro A, Valbonesi P, Bolognesi A, Stirpe F, De Luca P, Siniscalco Gigliano G, Gaudio L, Delli Bovi P, Ferranti P, Malorni A, Parente A
Dipartimento di Scienze della Vita, Seconda Università di Napoli, Caserta, Italy.
Planta. 1999 Mar;208(1):125-31. doi: 10.1007/s004250050542.
Four type-1 (single-chain) ribosome-inactivating proteins (RIPs), with isoelectric points between 9.5 and 9.7, were isolated from leaves of Phytolacca dioica L. The purification procedure furnished the four proteins with an overall yield of about 16% and separated them from a protein of 29,407 +/- 2 Da, as determined by electrospray mass spectrometry, whose N-terminal amino acid sequence differed from that of pokeweed (Phytolacca americana L.) leaf 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, diagnostic of the specific enzymatic action of RIPs, on yeast ribosomes. Comparison of their N-terminal sequences, up to residue 45, showed that PD-L1 is identical 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 between the two forms. Furthermore, the Val form presents the same number of identical 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 when stained for sugars on SDS-PAGE gels and, when analyzed by electrospray mass 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 difference in M(r), within the same RIP form, could be due to glycosylation. Like leaf saporins and many other RIPs, the four RIPs released several adenines from poly(A), herring sperm DNA and rRNA 16S + 23S, thus acting as polynucleotide:adenosine glycosidases. This property was less pronounced in PD-L1 and 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 reactivity with antisera to PAP-R saporin-S6, momordin 1 and even PD-S2, an RIP isolated from the seeds of the same plant. Protein PD-L4 showed 12.5% cross-reactivity with anti-PD-L1, while the opposite cross-reactivity was 100%.
从商陆（Phytolacca dioica L.）叶片中分离出四种1型（单链）核糖体失活蛋白（RIP），其等电点在9.5至9.7之间。纯化过程得到了这四种蛋白，总产率约为16%，并将它们与一种分子量为29,407 +/- 2 Da的蛋白分离，通过电喷雾质谱测定，该蛋白的N端氨基酸序列与美洲商陆（Phytolacca americana L.）叶片几丁质酶（PLC-B）仅相差一个氨基酸（R17I）。这四种RIP（PD-L1至PD-L4）在皮摩尔水平上就能抑制兔网织红细胞裂解物中的蛋白质合成，半数抑制浓度为50%，并且在酵母核糖体上产生了β片段，这是RIP特异性酶促作用的诊断标志。对它们N端序列直至第45位残基进行比较，结果显示PD-L1与PD-L2相同[从N端残基起称为异亮氨酸（Ile）形式]，PD-L3与PD-L4相同[从N端残基起称为缬氨酸（Val）形式]，并且两种形式之间有35个相同残基。此外，Val形式与从同一植物种子中分离出的RIP PD-S2具有相同数量的相同残基。除PD-L4外，纯化后的RIP在SDS-PAGE凝胶上进行糖染色时呈阳性反应，通过电喷雾质谱分析，其分子量（M(r)）值分别为32,715 +/- 1（PD-L1）、31,542 +/- 1（PD-L2）、30,356 +/- 1（PD-L3）和29,185 +/- 1 Da（PD-L4）。在同一RIP形式内，M(r)相差1171 Da可能是由于糖基化所致。与叶片皂草素和许多其他RIP一样，这四种RIP从聚（A）、鲱鱼精DNA和16S + 23S rRNA中释放出多个腺嘌呤，因此可作为多核苷酸：腺苷糖苷酶发挥作用。该特性在PD-L1和PD-L3中不如在PD-L2和PD-L4中明显。蛋白PD-L1和PD-L4与抗洋地黄毒苷32抗血清的反应性为3.7%，与抗PAP-R皂草素-S6、苦瓜素1甚至与从同一植物种子中分离出的RIP PD-S2的抗血清均无反应。蛋白PD-L4与抗PD-L1的交叉反应性为12.5%，而相反的交叉反应性为100%。
