ESCHERICHIA-COLI HEAT-LABILE ENTEROTOXIN - DNA-DIRECTED INVITRO SYNTHESIS AND STRUCTURE

E. coli heat-labile enterotoxin was synthesized in a cell-free system directed by DNA of the plasmid P307. Synthesis of the toxin, assayed by the elongation induced in Chinese hamster ovary cells, was strongly stimulated by cyclic AMP and occurred at physiological levels of Mg2+ only when the polyamine spermidine was present. Activity was abolished by heat and antisera prepared against the enterotoxins of both E. coli P263 and Vibrio cholera. Tritium-labeled enterotoxin was purified by immunoprecipitation and examined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. When gel slices were assayed for the ability to stimulate adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.] activity in erythrocyte ghosts, 2 peaks were found, 1 at MW 26,000 and frequentyly, but not always, another at MW 23,000. Detection of radiolabeled protein by fluorography and scintillation counting of gel slices revealed 3 prominent polypeptides, 2 corresponding to the peaks having adenylate cyclase-stimulating activity and a further one of MW 11,500, identical to that of the cholera subunit B. The data suggest that the E. coli heat-labile enterotoxin synthesized in the cell-free system has a subunit structure.