title: Cation and structural requirements for P site-mediated inhibition of adenylate cyclase.
abstract: The cation and structural requirements of the intracellular inhibitory "P" site of adenylate cyclase were investigated in human platelet membranes, bovine sperm particles, and detergent-solubilized and purified preparations from rat and bovine brain. Sensitivity of adenylate cyclase to P site-mediated inhibition was enhanced by reversible and irreversible activators of the enzyme. The most effective sensitization of the platelet and brain adenylate cyclases was observed with Mn2+ and upon proteolysis with inhibin in the presence of guanosine 5'-O-(3-thiotriphosphate). These resulted in IC50 values for (2',5'dideoxy-adenosine (2',5'-dd-Ado) and 2'-deoxy-3'-AMP of approximately 1-2 microM. The data were consistent with the ideas that P site-mediated inhibition of adenylate cyclase is dependent on divalent cation and is a function of enzyme activity. A number of nucleosides and nucleotides were synthesized and used to define structural requirements for P site-mediated inhibition of a detergent-solubilized adenylate cyclase from rat brain. The data suggest a strict requirement for an intact adenine moiety and a beta-glycosidic linkage for the ribosyl moiety. 2'-Deoxy-and especially 2',5'-dideoxy-ribosyl moieties enhanced sensitivity and a strong preference for phosphate at the 3'-position was exhibited. Substitutions at the 5'-ribose position impaired sensitivity. The order of potency and IC50 values of the more potent adenosine analogs were 2',5'-dideoxy-3'-AMP (congruent to 0.1 microM) greater than 2'-deoxy-3'-AMP (congruent to 1 microM) greater than 2',5'-dd-Ado (congruent to 3 microM) greater than 3'-AMP (congruent to 9 microM) greater than 2'-deoxy-adenosine (congruent to 15 microM) greater than adenosine (congruent to 80 microM). Large substitutions at the 3'-ribose position were tolerated, e.g., dApdN di- and dAp(dN)4 penta-nucleotides and succinyl- and p-fluoro-sulfonyl-benzoyl- moieties. The purified adenylate cyclase from bovine brain was inhibited by P site agonists with IC50 values of 34 and 45 microM for 2'-deoxy-3'-AMP and 2',5'-dd-Ado, respectively. The data imply, first, that the locus of the P site is the catalytic subunit of adenylate cyclase and, second, that the increased sensitivity observed with Mn2+ is due to an effect of the cation on the catalytic subunit. In contrast with adenylate cyclases from other mammalian tissues, the enzyme from bovine sperm exhibited only weak sensitivity to P site agonists; 2'-deoxy-3'-AMP congruent to 2',5'-dd-Ado greater than adenosine, each with IC50 values greater than 1000 microM.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Physiology and Biophysics, State University of New York, Stony Brook 11794-8661.
authors.forename: R A
authors.initials: RA
authors.lastname: Johnson
authors.forename: S M
authors.initials: SM
authors.lastname: Yeung
authors.forename: D
authors.initials: D
authors.lastname: Stübner
authors.forename: M
authors.initials: M
authors.lastname: Bushfield
authors.forename: I
authors.initials: I
authors.lastname: Shoshani
mesh_terms: D000241:Adenosine / Q000494:pharmacology; D000067956:Adenylyl Cyclase Inhibitors; D001921:Brain / Q000201:enzymology; D005576:Colforsin / Q000494:pharmacology; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology; D016048:Dideoxyadenosine / Q000031:analogs & derivatives*; D015224:Dideoxynucleosides / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D011983:Receptors, Purinergic / Q000187:drug effects / Q000502:physiology*; D013329:Structure-Activity Relationship; D013873:Thionucleotides / Q000494:pharmacology
chemical_list: D000067956:Adenylyl Cyclase Inhibitors; D003838:Deoxyadenine Nucleotides; D015224:Dideoxynucleosides; D011983:Receptors, Purinergic; D013873:Thionucleotides; C028191:2'-deoxy-3'-adenosine monophosphate; D005576:Colforsin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D008345:Manganese; D016048:Dideoxyadenosine; D004533:Egtazic Acid; D006160:Guanosine Triphosphate; C021008:2',5'-dideoxyadenosine; D008274:Magnesium; D000241:Adenosine