Pertussis toxin substrate, the putative N(i) component of adenylyl cyclases, is an αβ 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 (N(s)) of adenylyl cyclase [adenylate cyclase; ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from human erythrocyte membrane 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 M(r) values of 39,000 and 35,000. The second fraction (HAP II) contains predominantly N(s) formed of two peptides of M(r) 42,000 and 35,000. The HAP I, M(r) 39,000 peptide is shown to be a substrate for the ADP-ribosylating toxin of Bordetella pertussis (pertusssis toxin). Upon sucrose density gradient centrifugation, both the M(r) 35,000 and the M(r) 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 αβ heterodimeric protein composed of an α subunit of M(r) 39,000 and a β subunit of M(r) 35,000. Further, this protein dissociates under the influence of guanine nucleotides and Mg²⁺ into its individual α and β 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 M(r) ≃40,000 peptide, we believe that we have purified a guanine nucleotide- and Mg²⁺-binding inhibitory regulatory component of adenylyl cyclases-i.e., the N(i).