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


CONCLUSIONS
The active site of Tri responsible for inhibitory activity on protein biosynthesis was on the HATf1 side near the junction of two portions.
D000818:Animals; D001798:Blood Proteins / Q000096:biosynthesis*; D002474:Cell-Free System; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D015978:Trichosanthin / Q000737:chemistry / Q000494:pharmacology*