Cholera toxin activates adenylyl cyclase by catalyzing the ADP- ribosylation of Gs alpha, the stimulatory guanine nucleotide- binding protein of the cyclase system. ADP-ribosylation factor (ARF), was identified as necessary for ADP-ribosylation of Gs alpha by cholera toxin. ARF, in a GTP-dependent manner, directly enhances cholera toxin ADP-ribosyltransferase activity and auto- ADP-ribosylation of the cholera toxin Al subunit (CT-Al). ARF activity is markedly enhanced by 0.003% SDS, and to a lesser extent, by 0.1 to 0.5 % sodium cholate. Under these conditions, however, no GTP binding to ARF is detectable. To determine the basis for this apparent disparity, we investigated, in greater detail, the interaction of guanine nucleotides with ARF. High affinity GTP binding by sARF II (apparent KD of approximately 70 nM) required Mg++, DMPC and sodium cholate. sARF II, in DMPC/cholate, also enhanced CT-A ADP- ribosyltransferase activity with an apparent EC50 for GTP of approximately 5 nM; however, there was a delay of approximately 90 min before achievement of a maximal rate of sARF II-stimulated toxin activity. In contrast, a maximal rate of activation of toxin by sARF II in 0.003% SDS occurred without delay and the apparent EC50 for GTP was approximately 5 muM. Enhancement of cholera toxin ADP-ribosyltransferase activity by ARF, therefore, can occur under conditions in which ARF exhibits either a relatively low or high affinity for GTP. These findings may reflect the way in which subcellular localization could influence the interaction of ARF with GTP and its activation.
