Showing papers by "Juan Codina published in 1983"

Stimulation and inhibition of adenylyl cyclases mediated by distinct regulatory proteins

Abstract: Adenylyl cyclases are under positive and negative control by guanine nucleotides and hormones1–3. Stimulatory responses are mediated by a guanine nucleotide- and Mg-binding regulatory component4–6 (Ns), a protein that has been purified to homogeneity7–9. Inhibitory responses have been hypothesized1–3,10,11 to be mediated by an analogous regulatory component (Ni) distinct from Ns, but definitive proof for this is lacking and these effects may result from modulation of Ns activity. Recently, Bordetella pertussis toxin has been shown12 to ADP-ribosylate a peptide that is not part of Ns, and this coincides with attenuation of hormonal inhibition of adenylyl cyclase12–17. We show here that cyc− S49 cells contain a substrate for ADP-ribosylation by pertussis toxin and that the toxin alters GTP dependent inhibition of cyc− adenyl cyclase activity18. As cyc− S49 cells do not contain Ns by several criteria5,19–24, we conclude that Ni is a distinct and separate regulatory component of adenylyl cyclase.

Pertussis toxin substrate, the putative Ni component of adenylyl cyclases, is an alpha beta heterodimer regulated by guanine nucleotide and magnesium

Abstract: 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.