Abstract
In adipocyte membranes, cholera toxin may ADP-ribosylate the islet-activating protein (IAP) substrate, under certain conditions. Covalent modification is maximal in the absence of a guanosine triphosphate; in the presence of 5'-guanylylimidodiphosphate, incorporation of [32P]ADP-ribose is markedly reduced. ADP-ribosylation by cholera toxin has similar functional consequences as does IAP-mediated modification, i.e. the biphasic response of isoproterenol-stimulated adenylate cyclase to GTP and the inhibition by N6-phenylisopropyladenosine is abolished, and only the stimulatory phase remains. In contrast, membranes treated with cholera toxin in the presence of GTP display both the stimulatory and inhibitory responses to GTP. The binding of the adenosine analog [3H]N6-phenylisopropyladenosine is increased in the presence of GTP. Treatment of the membranes with IAP, but not with cholera toxin in the absence of GTP, reverses this GTP effect on [3H]N6-phenylisopropyladenosine binding. However, [3H]N6-phenylisopropyladenosine binding is still sensitive to GTP in membranes treated with cholera toxin in the presence of GTP. In adipocyte and cerebral cortical membranes, the IAP substrate appears as a 39,000/41,000-Da doublet which does not appear to reflect protease activity. On two-dimensional polyacrylamide gels, these two proteins migrate with approximate pI values 6.0 and 5.6, respectively. Although both behave similarly under all conditions explored in this study, it is unknown whether both, or only one, are involved in inhibition of adenylate cyclase activity. These results extend the already striking homology between the adenylate cyclase complex and the visual system. Ni, as well as transducin, may be ADP-ribosylated by cholera toxin and by IAP, and, in all cases, there are functional consequences.
Highlights
In adipocyte membranes, cholera toxin may ADP- thesizing enzyme (C’) is dually regulated, i.e. that some horribosylate the islet-activating protein(IAP) substrate, mone/neurotransmitter receptors stimulate production of undercertain conditions
The present report demonstrates that both cholera toxin and islet activating protein (IAP) modify the aisubunit of adipocyte adenylate cyclase
This extends the observation of Graves et ul. [15] that cholera toxin, in theabsence of added guanosine triphosphates, labels a proteinof approximate molecular weight of 41,000
Summary
With the exception of radioactive compounds, all chemicals were Fig. l A , rat adipocyte plasma membranes were ADP-ribosyobtained from Sigma. IAP was prepared as described [20]. Cholera lated with cholera toxin (lanes 1 and Z), or with IAP (lane 2). All solutions used for suspension of the adipocyte membranes contained 0.1 mM benzamidine, 0.1 mM phenylmethylautoradiography (lane 1). Inclusion of 100 W M Gpp(NH)p increased ADP-ribosylation of the 54,000- and 45,000-Da proteins bultabeling of the 41,000-Daprotein was diminished (lane 2 ). IAP labeled a 41,000-Da protein (lane 3). A 0.1 M quantify the extent of ADP-ribosylation of theseproteins phenylmethylsulfonyl fluoride solution in dimethyl sulfoxide was prepared immediately before use, added to thesolution. The inclusion of Gpp(NH)palso promotes cholera toxin-laheling concentrations were determined by the method of Lowry et al [45]. ToxinLabeling-Membranes that were subsequently to be analyzed tional significance
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