天花粉蛋白作为核糖体失活蛋白的活性位点。

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

作者信息

Xi Z D, Ma B L, Yang L M, Cao H N, Wang M

机构信息

Shanghai Institute of Immunology, Shanghai Second Medical University, China.

出版信息

Zhongguo Yao Li Xue Bao. 1997 Sep;18(5):447-51.

PMID:10322939

Abstract

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.

摘要

目的

定位中药天花粉蛋白（Tri）核糖体失活的活性位点。

方法

用羟胺特异性切割天花粉蛋白独特的天冬酰胺 - 甘氨酸肽键。采用制备性十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳获得两个切割片段，即HATf1和HATf2。利用蛋白质免疫印迹法确定天花粉蛋白的不同表位并筛选抗体。引入无细胞系统，即兔网织红细胞裂解液，以定量天花粉蛋白及其片段对蛋白质生物合成的抑制活性。

结果

分离得到HATf1和HATf2，纯度分别为96.9%和80.5%。HATf1与完整的天花粉蛋白一样，保留了对蛋白质生物合成的抑制活性。抗天花粉蛋白的单克隆抗体14号和16号与两个片段表现出不同的免疫反应性，并在进一步的阻断试验中被选作代表。单克隆抗体14号阻碍了天花粉蛋白和HATf1的活性，而单克隆抗体16号则没有。