Pertussis toxin substrate, the putative Ni component of adenylyl cyclases, is an alpha beta heterodimer regulated by guanine nucleotide and magnesium.
The final step in a scheme for the purification of the guanine nucleotide- and Mg2+-binding stimulatory regulatory component (Ns) of adenylyl cyclase [adenylate cyclase; ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from human erythrocyte membranes involves chromatography over hydroxylapatite (HAP) which yields two fractions. The first fraction (HAP I) contains predominantly two peptides that, upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis, migrate with Mr values of 39,000 and 35,000. The second fraction (HAP II) contains predominantly Ns formed of two peptides of Mr 42,000 and 35,000. The HAP I, Mr 39,000 peptide is shown to be a substrate for the ADP-ribosylating toxin of Bordetella pertussis (pertussis toxin). Upon sucrose density gradient centrifugation, both the Mr 39,000 and the Mr 35,000 peptides of HAP I migrate at about 4 S. Treatment of HAP I with guanine nucleotide and Mg2+ prior to centrifugation results in a coordinated change in the migration of both peptides to 2 S. It is postulated that HAP I contains an alpha beta heterodimeric protein composed of an alpha subunit of Mr 39,000 and a beta subunit of Mr 35,000. Further, this protein dissociates under the influence of guanine nucleotides and Mg2+ into its individual alpha and beta subunits. Because previous studies have shown that treatment of cells and cell membranes with pertussis toxin results in attenuation of the effects of hormones that inhibit adenylyl cyclase activity, and because this effect correlates with the ADP-ribosylation of a Mr approximately equal to 40,000 peptide, we believe that we have purified a guanine nucleotide- and Mg2+-binding inhibitory regulatory component of adenylyl cyclases--i.e., the Ni.
D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000097:blood*; D001427:Bacterial Toxins / Q000097:blood*; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000201:enzymology*; D004912:Erythrocytes / Q000201:enzymology*; D019204:GTP-Binding Proteins; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008970:Molecular Weight; D037342:Pertussis Toxin; D011956:Receptors, Cell Surface / Q000097:blood*; D010566:Virulence Factors, Bordetella
D037361:Adenylate Cyclase Toxin; D001427:Bacterial Toxins; D011956:Receptors, Cell Surface; D010566:Virulence Factors, Bordetella; D006165:Guanylyl Imidodiphosphate; D006160:Guanosine Triphosphate; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D008274:Magnesium