Pertussis toxin substrate is a guanosine 5'-[beta-thio]diphosphate-, N-ethylmaleimide-, Mg2+- and temperature-sensitive GTP-binding protein.
We compared the effects of guanine nucleotides and Mg2+ on ADP-ribosylation of rat brain and liver membrane proteins catalysed by Bordetella pertussis toxin (IAP) and cholera toxin (CT). Labelling of proteins in the presence of [alpha-32P]NAD+, ATP and CT required GTP or guanosine 5'-[gamma-thio]triphosphate (GTP [S]). In contrast, labelling of one (liver) or two (brain) polypeptides by IAP was enhanced by guanosine 5'-[beta-thio]diphosphate (GDP[S]) or GTP, but was blocked by GTP[S] or guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG). The order of labelling intensity was GDP[S] greater than GTP greater than no addition greater than GTP[S] = p [NH]ppG. Mg2+ increased labelling by CT, but decreased labelling by IAP. In addition, Mg2+ potentiated the effects of the guanine nucleotides, increasing the inhibitory effects of GTP[S] and the activatory effects of GDP[S] or GTP. Preincubating liver membranes at 30 degrees C in the presence of 10 mm-MgCl2 inhibited labelling by IAP irreversibly. Pretreatment of liver membranes with 4.95 mM-N-ethylmaleimide decreased labelling by CT by approximately 15%, but almost completely blocked labelling by IAP. These results suggest that the undissociated, GDP-bound, conformation of Ni, the inhibitory GTP-binding protein of adenylate cyclase, is the preferred substrate for ADP-ribosylation by IAP. This conformation, which is prevalent in native membranes, is sensitive to temperature, Mg2+ ions and alkylating agents such as N-ethylmaleimide. At 30 degrees C, Mg2+ may cause dissociation and denaturation of Ni in native membranes.
D037361:Adenylate Cyclase Toxin; D000818:Animals; D001921:Brain / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects; D002772:Cholera Toxin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D008099:Liver / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D037342:Pertussis Toxin; D011487:Protein Conformation; D051381:Rats; D013696:Temperature; D013873:Thionucleotides / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000378:metabolism*
D037361:Adenylate Cyclase Toxin; D008565:Membrane Proteins; D013873:Thionucleotides; D010566:Virulence Factors, Bordetella; D006153:Guanosine Diphosphate; C023427:guanosine 5'-O-(2-thiodiphosphate); D002772:Cholera Toxin; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D008274:Magnesium; D005033:Ethylmaleimide