From previous studies we have considerably improved our understanding of the mechanisms by which adenylate cyclase is regulated by guanine nucleotides and adenosine, and the effects that these agents have on the enzyme's affinity for divalent cation at a distinct cation binding site. We have described properties of receptor-mediated effects of adenosine on various adenylate cyclases and have demonstrated binding with a labeled, site-specific analog of adenosine as agonist. From studies with Mn2+, phospholipases, and detergents evidence was obtained indicating that mechanisms of coupling of adenosine and catecholamine receptors to adenylate cyclase are distinct. Adenosine inhibition of adenylate cyclase through a separate """"""""P"""""""" site, which requires the activated form of the cyclase, was found to be stable to detergent solubilization and partial purification and is the probably basis of binding of adenylate cyclase to adenosine agarose affinity systems. We will build on these studies and pursue the identification, characterization, and eventual purification of the proteins mediating adenosin's distinct effects. This will include the characterization of the two populations of high affinity adenosine receptors and of the lower affinity inhibitory site. To aid these studies site-specific agonists will be used and from among a large population of available xanthine analogs, site-specific antagonists will be sought. Effective agonists and antagonists may then be used first, for the development of adenosine-analog-or xanthine-analog-agaroses for affinity chromatography and also for the development of photo-affinity analogs for use in identifying the binding sites. These studies will not only aid our understanding of the regulation of adenylate cyclase, a crucial regulatory enzyme, but will also help clarify mechanisms for the physiological actions of adenosine, a neurotransmitter, and may explain the pharmacological actions of methylxanthines.
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| Bushfield, M; Shoshani, I; Cifuentes, M et al. (1990) Inhibition of adenylate cyclase by polyadenylate. Arch Biochem Biophys 278:88-98 |
| Bushfield, M; Johnson, R A (1990) Regulation of adenylate cyclase by adenosine: characterization of the P-site. Biochem Soc Trans 18:150-1 |
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| Yeung, S M; Shoshani, I; Stubner, D et al. (1989) Ammonium ions enhance proteolytic activation of adenylate cyclase and decrease its sensitivity to inhibition by ""P""-site agonists. Arch Biochem Biophys 271:332-45 |
