ACTIVATION OF ESCHERICHIA-COLI HEAT-LABILE ENTEROTOXINS BY NATIVE AND RECOMBINANT ADENOSINE-DIPHOSPHATE RIBOSYLATION FACTORS, 20-KD GUANINE-NUCLEOTIDE BINDING-PROTEINS

Escherichia coli heat-labile enterotoxins (LT) are responsible in part for "traveler's diarrhea" and related diarrheal illnesses. The family of LTs comprises two serogroups termed LT-1 and LT-II; each serogroup includes two or more antigenic variants. The effects of LTs result from ADP ribosylation of G(s-alpha), a stimulatory component of adenylyl cyclase; the mechanism of action is identical to that of cholera toxin (CT). The ADP-ribosyl-transferase activity of CT is enhanced by 20-kD guanine nucleotide-binding proteins, known as ADP-ribosylation factors or ARFs. These proteins directly activate the CTA1 catalytic unit and stimulate its ADP ribosylation of G(s-alpha), other proteins, and simple guanidino compounds (e.g., agmatine). Because of the similarities between CT and LTs, we investigated the effects of purified bovine brain ARF and a recombinant form of bovine ARF synthesized in Escherichia coli on LT activity. ARF enhanced the LT-I-, LT-IIa-, and LT-IIb-catalyzed ADP ribosylation of agmatine, as well as the auto-ADP ribosylation of the toxin catalytic unit. Stimulation of ADP-ribosylagmatine formation by LTs and CT in the presence of ARF was GTP dependent and enhanced by sodium dodecyl sulfate. With agmatine as substrate, LT-IIa and LT-IIb exhibited < 1% the activity of CT and LT-Ih. CT and LTs catalyzed ADP-ribosyl-G(s-alpha) formation in a reaction dependent on ARF, GTP, and dimyristoyl phosphatidylcholine/cholate. With G(s-alpha) as substrate, the ADP-ribosyltransferase activities of the toxins were similar, although CT and LT-Ih appeared to be slightly more active than LT-IIa and LT-IIb. Thus, LT-IIa and LT-IIb appear to differ somewhat from CT and LT-Ih in substrate specificity. Responsiveness to stimulation by ARF, GTP, and phospholipid/detergent as well as the specificity of ADP-ribosyltransferase activity are functions of LTs from serogroups LT-I and LT-II that are shared with CT.