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