Activation by thiol of the latent NAD glycohydrolase and ADP-ribosyltransferase activities of Bordetella pertussis toxin (islet-activating protein).
Pertussis toxin (islet-activating protein) activates adenylate cyclase in susceptible cells by ADP-ribosylating an inhibitory component of the cyclase system. This toxin, assayed in a cell-free system in the presence of high concentrations of thiol, catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide. This NAD glycohydrolase activity co-chromatographed on Sephacryl G-200 in 6.5 M urea, pH 3.2, 0.1 M glycine with the ADP-ribosyltransferase activity of the toxin, as monitored by the transfer of [32P]ADP-ribose from [32P]NAD to a 41,000-Da protein in NG108-15 neuroblastoma X glioma hybrid cells. In the absence of thiol, the native holotoxin was enzymatically inactive. Following addition of 250 mM dithiothreitol to the assay, maximal enzymatic activity was evident after a delay of approximately 1 h; with 20 mM thiol, the delay was longer. The Km for NAD with the fully activated enzyme was 25 microM; the Km did not appear to vary with the extent of activation. Thiol was necessary in a cell-free system to demonstrate NAD glycohydrolase activity. When extensively washed membranes were used as a source of 41,000-Da substrate, thiol was necessary to observe ADP-ribosylation in some cases (human erythrocytes) and significantly stimulated activity in others (NG108-15 cells). In contrast to the bacterial toxins choleragen and Escherichia coli heat-labile enterotoxin that ADP-ribosylate stimulatory components of the cyclase system, pertussis toxin did not transfer ADP-ribose to low molecular weight guanidino compounds, such as arginine or agmatine.
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D037361:Adenylate Cyclase Toxin; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D001886:Bordetella pertussis / Q000201:enzymology*; D002460:Cell Line; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation; D004910:Erythrocyte Membrane / Q000378:metabolism; D005910:Glioma; D006822:Hybrid Cells / Q000378:metabolism; D007515:Islets of Langerhans / Q000187:drug effects; D007700:Kinetics; D051379:Mice; D009243:NAD / Q000378:metabolism; D009244:NAD+ Nucleosidase / Q000378:metabolism*; D009447:Neuroblastoma; D009713:Nucleotidyltransferases / Q000378:metabolism*; D037342:Pertussis Toxin; D011065:Poly(ADP-ribose) Polymerases; D051381:Rats; D013438:Sulfhydryl Compounds / Q000494:pharmacology*; D010566:Virulence Factors, Bordetella
D037361:Adenylate Cyclase Toxin; D001426:Bacterial Proteins; D013438:Sulfhydryl Compounds; D010566:Virulence Factors, Bordetella; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D011065:Poly(ADP-ribose) Polymerases; D037342:Pertussis Toxin; D009713:Nucleotidyltransferases; D009244:NAD+ Nucleosidase; D004229:Dithiothreitol