Oligomeric structure of cholera toxin: characteristics of the H and L subunits.
Structural analysis of cholera toxin by sodium dodecylsulphate polyacrylamide electrophoresis demonstrated two types of non-covalentyly linked subunits, heavy (H) AND LIGHT (L), with respective molecular weights 28000 and 800 to 9000. The H:L protein ratio was I:2, indicating that the toxin of molecular weights of in ratio was I:2, indicating that the toxin of molecular weight 84000 consists of IH 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 subunit. The specific clevage of the Hsubunit 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 dodecylsulphate polyacrylamide electrophoresis it was demonstrated that the ability of toxin to attach to the apparent receptor ganglioside, Gm1, is similar to that of choleragenoid toxoid, and is due to the Gm1-binding ability of the L subunits. The toxin Hsubunit did not react with the Gm1 ganglisoside. 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.
D000478:Alkylation; D000818:Animals; D002850:Chromatography, Gel; D004229:Dithiothreitol; D004591:Electrophoresis, Polyacrylamide Gel; D005732:Gangliosides; D005779:Immunodiffusion; D007271:Injections, Intradermal; D007457:Iodine Radioisotopes; D007461:Iodoacetates; D008623:Mercaptoethanol; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010455:Peptides / Q000032:analysis; D011485:Protein Binding; D011817:Rabbits; D011863:Radioimmunoassay; D014118:Toxins, Biological / Q000032:analysis* / Q000276:immunology / Q000633:toxicity; D014121:Toxoids / Q000032:analysis; D014734:Vibrio cholerae / Q000032:analysis* / Q000276:immunology
D005732:Gangliosides; D007457:Iodine Radioisotopes; D007461:Iodoacetates; D010455:Peptides; D014118:Toxins, Biological; D014121:Toxoids; D008623:Mercaptoethanol; D004229:Dithiothreitol