1887

Abstract

SUMMARY: Structural analysis of cholera toxin by sodium dodecylsulphate polyacrylamide electrophoresis demonstrated two types of non-covalently linked subunits, heavy (H) and light (L), with respective molecular weights of 28000 and 8000 to 9000. The H:L protein ratio was 1:2, indicating that the toxin of molecular weight 84000 consists of 1 H and 6 or 7 L subunits, linked into an aggregate with non-covalent bonds. Choleragenoid toxoid, a natural toxin derivative, contained only the L subunits of the toxin. Reduction and alkylation cleaved the H but not the L sub-unit. The specific cleavage of the H subunit by reduction appeared to yield identical half-molecules; the smaller peptide seemed to originate from non-specific degradation. The H subunit also differed from L subunits by having a higher affinity for labelling with radioactive iodine and by precipitating below pH 3·5.

In immunodiffusion studies the toxin possessed antigenic determinants shared with the toxoid as well as toxin-specific determinants. Comparative analyses with purified subunit preparations revealed that the toxoid-shared determinants reside in the L-type of subunit and the toxin-specific ones in the H subunit.

By precipitation-in-gel, binding to ganglioside-coated tubes, and sodium dodecyl sulphate polyacrylamide electrophoresis it was demonstrated that the ability of toxin to attach to the apparent receptor ganglioside, G is similar to that of choleragenoid toxoid, and is due to the G-binding ability of the L subunits. The toxin H subunit did not react with the G ganglioside.

The results support our previous structural model for cholera toxin, and explain the antigenic and receptor-binding properties of the toxin in terms of component subunits.

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1975-01-01
2021-05-07
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