1988 Fiscal Year Final Research Report Summary
Study on transmembrane mechanism of ricin, a toxic protein of castor bean seeds
| Project/Area Number |
62560088
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
応用生物化学・栄養化学
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| Research Institution | Kyushu University |
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Principal Investigator |
FUNATSU Gunki Faculty of Agriculture, Kyushu University, 農学部, 教授 (40038196)
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| Project Period (FY) |
1987 – 1988
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| Keywords | ricin / toxic protein / liposome / transmembrane mechanism / 蛋白質ー脂質相互作用 |
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| Research Abstract |
The results obtained in this study are as follows;
1. Effect of lipid composition for binding of ricin A- and B-chains to lipid bilayer isolated A- and B-chains bound to the liposomes consisting of various lipid compositions in the presence and absence of salt, indicating a participation of the hydrophobic interaction for the binding of ricin A- and B-chains to lipid bilayer. These bindings decreased remerkably in the liposomes containing over 10% of cholesterol.
2. Cytotoxicity of the hybrid molecules of ribosome-inactivating protein/B-chain hybrid molecules were prepared by cross-linking two ribosome-inactivating proteins having no surface hydrophobicity to ricin B-chain with SPDP, and examined their cyto-toxicity toward Hela cells. No toxicity of these hybrid molecules suggested an impor-ance of the A-chain structure including surface hydrophobicity for transmembrane of the ricin molecule.
3. Hydrophobicities of ricin and its subunits. From the results obtained by fluorometric method using cis-parnaric acid and by binding with Triton X-100, it was revealed that the A-chain had about 4 times larger hydrophobic regions on its molecular surface than the B-chain, and these surface hydrophobic regions were masked in the ricin molecule.
4. DPPC-liposome-binding/insertion site of ricin A- and B-chains. From the trypsin treated a-chain-liposome complex, four peptides starting from Ile-1, Phe-57, Glu-135 or Phe-181 were obtained. Since the tryptic peptides Phe-57 - Arg-85 and Glu-135 - Arg-166 bound to liposomes, it was inferred that the lipo-some binding/insertion site of ricin A-chain may be C-terminal domain including Gly-35 - Asn-47 region.
Main peptide remained in the B-chain-liposome complex after subtilisin digestion was identified to be Ala-1 - Asn-46. From this result, it was concluded that the liposome-binding/insertion site of ricin B-chain is N-terminal region of the B-chain.
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