Interaction of ADP-ribosylation factor with Escherichia coli enterotoxin that contains an inactivating lysine 112 substitution.
Cholera toxin and Escherichia coli heat-labile enterotoxin (LT) exert their effects on cells through ADP-ribosylation of guanine nucleotide-binding proteins. Both toxins consist of one A subunit, which is an ADP-ribosyltransferase, and five B (or binding) subunits. Their enzymatic activities are latent; activation requires reduction and proteolysis, resulting in a catalytically active A1 protein and a much smaller A2 protein. These ADP-ribosyltransferases are activated by GTP-dependent 20-kDa ADP-ribosylation factors or ARFs. To determine if proteolysis plus reduction is required for appearance of the ARF allosteric site as well as for catalytic activity, an inactive mutant of LT, LT(E112K), with replacement of glutamate by lysine at position 112 of its A subunit, was utilized as a competitor in cholera toxin ADP-ribosyltransferase assays containing limiting amounts of ARF. LT(E112K) required trypsinization and reduction to become a potent, concentration-dependent inhibitor. Inhibition was reversed by increasing concentrations of ARF. Reduction or trypsinization alone did not generate an inhibitory form of LT(E112K). These studies are consistent with the conclusion that the ARF site is not expressed in the latent toxin. Both trypsinization and reduction are required for expression of a functional ARF binding site as well as for catalytic activity.
Moss
J
J
Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Stanley
S J
SJ
Vaughan
M
M
Tsuji
T
T
D020727:ADP-Ribosylation Factors; D000244:Adenosine Diphosphate / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002772:Cholera Toxin / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004768:Enterotoxins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006868:Hydrolysis; D008239:Lysine / Q000378:metabolism; D010084:Oxidation-Reduction; D012266:Ribose / Q000378:metabolism*; D014357:Trypsin
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D000244:Adenosine Diphosphate; D012266:Ribose; D002772:Cholera Toxin; C027538:heat-labile enterotoxin, E coli; D014357:Trypsin; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors; D008239:Lysine; D004229:Dithiothreitol