Structure-function relationship of islet-activating protein, pertussis toxin: biological activities of hybrid toxins reconstituted from native and methylated subunits.
Islet-activating protein (IAP), pertussis toxin, is a hexameric protein composed of an A protomer and a B oligomer, the residual pentamer having such a subunit assembly that two different dimers, dimer 1 and dimer 2, are connected with each other by means of the smallest C subunit. Incubation of IAP with formaldehyde and pyridine-borane produced the modified toxin in which most of the free amino groups were dimethylated. The methylated and nonmethylated (native) IAP were disintegrated into their respective constituent components, which were then cross combined to reconstitute hybrid toxins with the original hexameric structure. The binding of the B oligomer to the mammalian cell surface via dimer 2 was, but the binding via dimer 1 was not, seriously impaired by methylation of amino groups in the protein. The binding of the B oligomer allowed the A protomer to enter cells and to catalyze ADP-ribosylation of a membrane Mr 41 000 protein. The diverse biological activities of IAP occurring by this mechanism were mimicked by not only methylated IAP but also all hybrid toxins, indicating that the free amino groups in the protein were not essential for the enzyme activity of the A protomer and that the A protomer was able to enter cells if the B oligomer bound to cells "monovalently" via dimer 1. An additional effect of the B oligomer binding, i.e., the direct stimulation, without the transport of the A protomer, of cells leading to mitosis in lymphocytes in vitro or increases in circulating lymphocytes in vivo, was not mimicked by hybrid toxins containing methylated dimer 2.(ABSTRACT TRUNCATED AT 250 WORDS)
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000273:Adipose Tissue / Q000187:drug effects / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004837:Epinephrine / Q000494:pharmacology; D007700:Kinetics; D008213:Lymphocyte Activation / Q000187:drug effects; D008214:Lymphocytes / Q000187:drug effects; D046911:Macromolecular Substances; D008297:Male; D008745:Methylation; D051379:Mice; D010084:Oxidation-Reduction; D037342:Pertussis Toxin; D055503:Protein Multimerization; D051381:Rats; D013329:Structure-Activity Relationship; D010566:Virulence Factors, Bordetella / Q000302:isolation & purification / Q000494:pharmacology*
D000596:Amino Acids; D046911:Macromolecular Substances; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D037342:Pertussis Toxin; D004837:Epinephrine