Toxicity Of Plant Ribosome-Inactivating Protein To Silkworm (Bombyx.mori) And Its Molecular Mechanism

A method for a large-scale preparation of cinnamomin and ricin,two kinds of type II RIPs from the seed of cinnamomun camphora and castor bean,has been developed . Affinity chromatography on acid-treated Sepharose 4B was employed to isolated cinnamomin and ricin. The yield and scale of cinnamomin and ricin according to this method were much higher than those according to the previous method. The 620 mg of cinnamomin and 850 mg of ricin were extacted respectively from 500 g of fresh mature seed. The cinnamomin and ricin purified by the above were homogenous characterized by SDS-polyacylamide gel electrophoresis. The assay of the enzymatic activity of cinnamomin and ricin showed both of them had RNA N-glycosidase activities. The toxicities of cinnamomin and ricin to the silkworm larvae were tested in the third,forth,fifth instars. Cinnamomin and ricin exhibited different toxicities to the silkworm larvae by oral feeding bioassay. The LCs,of ricin to the third instar silkworm larvae was 496. 4 ppm. While the LCS)of cinnamomin was high as 16596. 4 ppm. The silkworm larvae during different developmental periods showed different sensitivities to the toxic proteins. They could be hydrolyzed in the same pattern by the liquid extracted from midgut of silkworm. About 48. 5% of ricin and 52. 3% of cinnamomin were degraded after incubation with silkworm midgut juice for 1 hour. A new method has been developed to isolated ribosome of silkworm. The susceptibilities to the actions of several RIPs including cinnamomin,ricin,cinnamomin A chain,ricin A chain,trichosanthin,a-sarcin were tested. Analysis on RNA from RIP-treated ribosomes showed the appearance of a aniline-cleavable RNA fragment,which was very similar to the R-fragment released from rat ribosome treated with RIPs. The R-fragment disappeared after rRNA extracted from cinnamomin-treated silkworm ribosome incubating with 100 mM of KBtifor 1 hour. So we could conclude that RIPs acting on silkworm ribosome were situated at the S/R domain. We compared the primary and secondary structures of S/R domains between rat and silkworm.The molecular mechanism of RIPs killing silkworm was elucidated.