Studies on the disulfide bond connecting the two polypeptide chains of ricin are reported. Reduction of this bond in the native protein requires approximately 50-fold more mercaptoethanol than the reduction of the bond in the protein denatured by sodium dodecyl sulfate. An improved procedure for the formation of this disulfide bond from recombined chains is reported. A and B chains spontaneously and rapidly reassociate into a stable complex with a sedimentation velocity similar to that of native oxidized ricin before the disulfide bond reforms. The mixture of both chains also behaves on Bio-Gel P-100 like native oxidized ricin. However, the complex formed by the two chains, assayed before the disulfide bond can reform, and reduced ricin, carboxymethylated to prevent reoxidation, shows a significant decrease in toxicity to mice and a decrease in ability to inhibit protein synthesis in HeLa cells in culture.
Studies on the structure-function relationship of diphtheria toxin are reported. New methods are described for the preparation of pure intact ("unnicked") molecule (or the A chain) to enter the cell (11). The complete amino acid sequence of the A chain has recently become available (12). A recent report by Iglewski and Rittenberg (13) indicated that protein synthesis in Ehrlich-Lettr6 ascites carcinoma cells is much more sensitive to the action of diphtheria toxin than that in normal mouse spleen cells, and that Ehrlich-Lettre tumors regressed in mice following an injection of the affected mice with diphtheria toxin. A difference in sensitivity to diphtheria toxin was also found in human breast carcinoma cells as compared to normal human cells. These observations suggest that diphtheria toxin may prove to be a useful anti-cancer agent, although more recent data indicate that the difference in sensitivity of tumor cells to toxin as compared to normal cells may not be as large as originally thought (14,15).It is therefore of considerable interest to us to investigate the mechanism of action of diphtheria toxin in greater detail. More specifically, we are trying to gain further information concerning the amino acid residues of the toxin that are essential for its activity, and to investigate the mechanism by which diphtheria toxin enters the cell. Some of the results obtained during this investigation are presented in this paper. MATERIALS AND METHODS Materials
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