天花粉蛋白的核糖体灭活活体部位

目的：天花粉蛋白核糖体灭活活性部位的定位。方法：羟胺特异裂解天花粉蛋白唯一Ａｓｎ－Ｇｌｙ肽键。制备性凝胶电泳获ＨＡＴｆ１和ＨＡＴｆ２二片段。免疫印迹确定天花粉蛋白上不同表位并筛选抗体。兔网织红无细胞系统测定天花粉蛋白及片段对蛋白合成的抑制活性。结果：ＨＡＴｆ１和ＨＡＴｆ２纯度各达９６．６％和８０．５％。ＨＡＴｆ１和ＨＡＴｆ２纯度各达９６．６％和８０．５％。ＨＡＴｆ１保留完整天花粉蛋白的抑制活性。第

Active site of trichosanthin acting as a ribosome-inactivating protein.

AIM: To localize the active site of ribosome inactivation of trichosanthin (Tri), a Chinese herb protein. METHODS: Hydroxylamine was used to specifically cleave the unique Asn-Gly peptide bond of Tri. Preparative SDS-polyacrylamide gel electrophoresis was applied to get 2 cleaved fragments, HATf1 and HATf2. Western blotting was used to determine the different epitopes of Tri and screen the antibodies. A cell-free system, rabbit reticulocyte lysate, was introduced to quantitate the inhibitory activity of Tri and its fragments on protein biosynthesis. RESULTS: HATf1 and HATf2 were separated with the purity of 96.9% and 80.5% respectively. HATf1, like intact Tri, retained the inhibitory activity on protein biosynthesis. The mAb No 14 and No 16 against Tri showed different immunoreactivities with 2 fragments and were selected as representatives in further blocking tests. The mAb No 14 hindered the activities of Tri and HATf1, whereas the mAb No 16 did not. CONCLUSION: The active site of Tri responsible for inhibitory activity on protein biosynthesis was on the HATf1 side near the junction of two portions.