J. Biochem, 1990, Vol. 107, No. 5 769-775
© 1990 Japanese Biochemical Society
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Immunochemical Identification of the ADP-Ribosyltransferase in Botulinum C1 Neurotoxin as C3 Exoenzyme—Like Molecule1
*Department of Pharmacology, Kyoto University Faculty of Medicine, Kyoto University Sakyo-ku, Kyoto, Kyoto 606
**Department of Veterinary Science, College of Agriculture, University of Osaka Prefecture Sakai, Osaka 591
2To whom correspondence and reprint requests should be addressed.
Botulinum C1 neurotoxin and C3 exoenzyme were purified to apparent homogeneity from the culture filtrate of Clostridium botulinum type C strain 003–9. Both preparations catalyzed ADP-ribosylation of the same substrate, the Mr 22, 000 rho gene product (Gb). When the light and heavy chains of C1 toxin were separated, ADP-ribosyltransferase activity in the toxin was quantitatively recovered in the light chain fraction. Anti-C1 toxin antiserum precipitated the ADP-ribosyltransferase activity and the neurotoxicity of C1 toxin in parallel, whereas it had no effect on C3 exoenzyme. On the other hand, anti-C3 exoenzyme anti serum precipitated the ADP-ribosyltransferase activities of both C3 exoenzyme and C1 toxin. This antibody, however, did not precipitate the neurotoxicity of C1 toxin. The ADP-ribosyltransferase in C1 toxin was quantitatively adsorbed onto the anti-C3 antibody column and separated from the majority of C1 toxin protein. The enzyme was then eluted with acidic urea and Western blotting analysis of this eluate revealed the appearance of a protein band positively stained with anti-C3 antibody at a position similar to that of C3 exoenzyme. Quantitative determination by enzyme-linked immunosorbent assay showed that the C3-like immunoreactivity is present in the C1 toxin molecules at the molecular ratio of 1 to 1, 000. These results suggest that the ADP-ribosyltransferase activity in Cl toxin is expressed by a C3-like molecule which is present in a small amount in the toxin preparation and appears to bind to the toxin components). The above results also indicate that the ADP-ribosyltransferase in C1 toxin is not related to its neurotoxin action.
1This work was supported in part by Grant-in-Aid for Scientific Research (Nos. 01480147, 01641515 and 01658507) from the Ministry of Education, Science and Culture of Japan, and grants from the Cell Science Research Foundation, the C1BA-GEIGY Foundation for Promotion of Science, the Japanese Foundation on Metabolism and Diseases, and the Watanabe Memorial Fund.
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