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I Shoshani

Publications and source records attributed to I Shoshani.

18 recordsLinked to original sources
PubMed2000

Synthesis and use of 3'-(azidoiodosalicyl) derivatives of 2', 5'-dideoxyadenosine as photoaffinity ligands for adenylyl cyclase.

3'-[(4-Azidosalicyl)glycyl]-2',5'-dideoxyadenosine (1), 3'- [(4-azidosalicyl)-gamma-aminobutyryl]-2',5'-dideoxyadenosine (2), and the (125)I-labeled mono- and diiodinated analogs of 1 were synthesized and tested as photoaffinity probes for adenylyl cyclases. Kinetics for inhibition of purified type I enzyme by 1 was noncompetitive with respect to Mn(*)5'-ATP in the absence of light, implying a P-site mechanism of inhibition. In a UV-dependent manner both 1 and 2 and the iodinated derivative of 1 irreversibly inactivated membrane-bound and purified forms of recombinant type I bovine adenylyl cyclase expressed in ovarian cells of either the fall armyworm (Sf9) or Trichoplasia ni (High Five). Irreversible inactivation was independent of 5'-ATP and was prevented by 2', 5'-dideoxyadenosine. Adenylyl cyclase, whether purified from bovine brain or in membranes from High Five cells expressing type I enzyme, when subjected to UV irradiation in the presence of (125)I-labeled 1 resulted in radioactive incorporation into protein migrating at approximately 116 kDa. The cross-linking of 1 and its iodinated derivative with adenylyl cyclase suggests potential for such compounds to be useful in structural studies of adenylyl cyclases or of other proteins for which adenine nucleosides are substrates or allosteric regulators.

Adenosine↗
PubMed2000

Lys-Ala mutations of type I adenylyl cyclase result in altered susceptibility to inhibition by adenine nucleoside 3'-polyphosphates.

Native and recombinant wild type and mutant forms of type I adenylyl cyclase, expressed in fall army worm ovarian cells (Sf9) cells, with mutations Lys-923-Ala, Lys-921-Ala, and Lys-350-Ala, retained the characteristic noncompetitive inhibition by adenine nucleoside 3'-polyphosphates, but exhibited substantially different sensitivities to inhibition by them. The type I K923A enzyme resulted in increased IC(50) values, e.g., >100-fold for 2'-deoxyadenosine-3'-monophosphate, but the shift diminished as the number of 3'-phosphates increased. The K921A mutation increased IC(50) values approximately 5-fold for all adenine nucleosides tested, whereas the K350A mutation increased IC(50) values approximately 6- to 8-fold for all adenine nucleosides tested except 2'-deoxyadenosine-3'-diphosphate, which was increased >/=2-fold. The data suggest that 3'-phosphates sufficiently increase binding affinity of these ligands to compensate for the reduced coordination of the adenine moiety induced by the K923A mutation. Moreover, the altered structures induced by both K350A and K921A mutations impair ligand binding in general, but paradoxically those resulting from the K350A change minimally affected nucleoside 3'-diphosphate binding, implying that selective changes in ligand binding can be induced by this site-specific mutation.

Adenine Nucleotides↗
PubMed1999

Enzymatic synthesis of unlabeled and beta-(32)P-labeled beta-L-2', 3'-dideoxyadenosine-5'-triphosphate as a potent inhibitor of adenylyl cyclases and its use as reversible binding ligand.

beta-L-2',3'-Dideoxyadenosine-5'-triphosphate (beta-L-2', 3'-dd-5'-ATP) was prepared enzymatically from the corresponding monophosphate by the use of adenylate kinase, creatine phosphate, and creatine kinase in a single step. The beta-(32)P-labeled analog was prepared similarly, but in a two step reaction. beta-L-2', 3'-dd-5'-ATP inhibited adenylyl cyclase from rat brain competitively with respect to substrate (5'-ATP.Mn(2+)) and exhibited an IC(50) approximately 24 nM. The labeled ligand was used in the development of a reversible binding assay for adenylyl cyclases. Binding of beta-L-2',3'-dd-[beta-(32)P]5'-ATP was saturable with increasing concentrations of ligand and increased in proportion to membrane protein, and was enhanced by Mn(2+) to a greater extent than by Mg(2+). Binding was displaced with adenine nucleotides known to be either competitive or noncompetitive inhibitors but not by agents known not to act on the cyclase, or by 3-isobutyl-1-methylxanthine, creatine phosphate, or creatine kinase. Binding was rapid, with a half-time for the on-rate <1.8 min and for the off-rate <0.8 min. The potency and mechanism of the inhibition of this ligand and the pattern of agents that displace binding suggest an interaction with adenylyl cyclase per se and to a configuration of the enzyme consistent with an interaction at the catalytic active site. The data suggest that this is a pretransition state inhibitor and contrasts with the equipotent 2',5'-dd-3'ATP, a post-transition state noncompetitive inhibitor.

Adenosine Triphosphate↗
PubMed1999

Inhibition of adenylyl cyclase by acyclic nucleoside phosphonate antiviral agents.

Acyclic derivatives of adenine, known as highly effective nucleotide analogs with broad spectrum antiviral activity, were evaluated for potential cross-reactivity with adenylyl cyclases, a family of membrane-bound enzymes that share putative topologies at their catalytic sites with oligonucleotide polymerases and reverse transcriptases. A series of derivatives of 9-(2-phosphonylmethoxyethyl)adenine (PMEA) inhibited a preparation of adenylyl cyclase derived from rat brain with IC(50) values that ranged from 66 microM (PMEA) to 175 nM for its diphosphate derivative (PMEApp) and mimics of it. PMEApp mimics included PMEAp(NH)p, PMEAp(CH(2))p, PMEAp(CX(2))p (X = fluorine, chlorine, or bromine), PMEAp(CHX)pp, and PMEAp(C(OH)CH(3)pp. The data suggest that inhibition of adenylyl cyclases may contribute to the therapeutic action of some of these or similar compounds or constitute part of their side effects in therapeutic settings.

Adenine↗
PubMed1999

Adenylyl cyclase P-site ligands accelerate differentiation in Ob1771 preadipocytes.

Differentiation of Ob1771 preadipocytes to adipocytes was characterized by morphological changes and elevated expression of the specific marker enzyme, glycerol-3-phosphate dehydrogenase. A differentiation response substantially more complete and rapid than that obtained with insulin and 3,5,3'-triiodothyronine was observed with established inhibitors of adenylyl cyclases: 2', 5'-dideoxyadenosine (2',5'-dd-Ado), 9-(cyclopentyl)adenine (9-CP-Ade), and 9-(arabinofuranosyl)adenine (9-Ara-Ade), coincident with decreased cellular cAMP levels. These ligands inhibit adenylyl cyclases noncompetitively, via a domain referred to as the P-site because of its requirement for an intact purine moiety. Differentiation was not induced by inosine, a nucleoside known not to act at the P-site, or by N6-(2-phenylisopropyl)adenosine or 1, 3-diethyl-8-phenylxanthine, agonist and antagonist, respectively, for adenosine A1 receptors. Also ineffective were IBMX or forskolin, agents that can raise intracellular cAMP levels. Potency of the differentiation response followed the order 2',5'-dd-Ado (1-20 microM) > 9-CP-Ade (10-100 microM) = 9-Ara-Ade (10-100 microM) >> inosine, consistent with their potencies to inhibit adenylyl cyclases. The data suggest that inhibition of adenylyl cyclase via the P-site and the consequent reduction in cell cAMP levels facilitate the induction of differentiation in Ob1771 cells. The findings raise the question whether the known endogenous P-site ligands participate in the differentiation response induced by hormones.

Adenylyl Cyclases↗
PubMed1997

Isozyme-dependent sensitivity of adenylyl cyclases to P-site-mediated inhibition by adenine nucleosides and nucleoside 3'-polyphosphates.

Recombinant adenylyl cyclase isozyme Types I, II, VI, VII, and three splice variants of Type VIII were compared for their sensitivity to P-site-mediated inhibition by several adenine nucleoside derivatives and by the family of recently synthesized adenine nucleoside 3'-polyphosphates (Désaubry, L., Shoshani, I., and Johnson, R. A. (1996) J. Biol. Chem. 271, 14028-14034). Inhibitory potencies were dependent on isozyme type, the mode of activation of the respective isozymes, and on P-site ligand. For the nucleoside derivatives potency typically followed the order 2',5'-dideoxyadenosine (2',5'-ddAdo) > beta-adenosine > 9-(cyclopentyl)-adenine (9-CP-Ade) >/= 9-(tetrahydrofuryl)-adenine (9-THF-Ade; SQ 22,536), with the exception of Type II adenylyl cyclase, which was essentially insensitive to inhibition by 9-CP-Ade. For the adenine nucleoside 3'-polyphosphates inhibitory potency followed the order Ado < 2'-dAdo < 2',5'-ddAdo and 3'-mono- < 3'-di- < 3'-triphosphate. Differences in potency of these ligands were noted between isozymes. The most potent ligand was 2',5'-dd-3'-ATP with IC50 values of 40-300 nM. The data demonstrate isozyme selectivity for some ligands, suggesting the possibility of isozyme-selective inhibitors to take advantage of differences in P-site domains among adenylyl cyclase isozymes. Differential expression of adenylyl cyclase isozymes may dictate the physiological sensitivity and hence importance of this regulatory mechanism in different cells or tissues.

Adenosine↗
PubMed1996

Inhibition of adenylyl cyclase by a family of newly synthesized adenine nucleoside 3'-polyphosphates.

The synthesis of a number of adenine nucleoside 3'-polyphosphates has been devised via a phosphotriester approach that combines the method of alkoxide activation with the use of 2,2,2-tribromoethyl phosphoromorpholinochloridate as a phosphorylating agent. The family of compounds included 3'ADP, 3'ATP, 2'-deoxy-3'ADP, 2'-deoxy-3'ATP, 2',5'-dideoxy-3'ADP, and 2',5'-dideoxy-3'ATP. Potency as inhibitors of adenylyl cyclases followed the order: 3'-mono- < 3'-di- < 3'-triphosphate and adenosine (Ado) < 2'-d-Ado < 2',5'-dd-Ado derivatives, with 2',5'-dideoxy-3'ATP exhibiting an IC50 of approximately 40 nM. This order was maintained with purified and recombinant forms of the type I enzyme. The nucleoside 3'-phosphates caused noncompetitive inhibition of the type I adenylyl cyclase from bovine brain, consistent with inhibition via the P-site. Inhibition was not due to hydrolytic products because this was minimal and inhibition kinetics by inorganic polyphosphates were inconsistent with those caused by the nucleoside 3'-polyphosphates. Only 3'ATP underwent cation-catalyzed, nonenzymatic hydrolysis, with the primary product being 2':3'-cAMP. Because 3'-ADP and 3'-ATP are naturally occurring, this class of compounds may physiologically regulate adenylyl cyclases and possibly other enzymes, mediating responses that include a reduction in 3':5'-cAMP levels and consequent reductions in protein kinase A-activated pathways.

Adenine Nucleotides↗
PubMed1996

2',5'-Dideoxyadenosine 3'-polyphosphates are potent inhibitors of adenylyl cyclases.

2',5'-Dideoxyadenosine 3'-di- and triphosphates were tested as inhibitors of brain adenylyl cyclases. With an IC50 approximately 40 nM, 2',5'-dideoxy-3'-ATP is the most potent nonprotein synthetic regulator of adenylyl cyclases thus far described. Neither 2',5'-dideoxy-3'-ADP nor 2',5'-dideoxy-3'-ATP inhibited activity by competition with substrate, and the linear noncompetitive inhibition observed was consistent with interaction via a distinct domain. The availability of this ligand will permit the development of a variety of probes that will be extremely useful in investigating adenylyl cyclase structure and the role(s) that this class of compound may play in physiologically regulating cell function.

Adenylyl Cyclase Inhibitors↗
PubMed1995

Azido-iodo-phenyl-analogs of 2',5'-dideoxy-adenosine as photoaffinity ligands for adenylyl cyclase.

Azidoiodophenyl-analogs of 2',5'-dideoxyadenosine were synthesized and tested as potential 'P'-site selective affinity probes for adenylyl cyclases. The 3'-substituted analogs included: 1: 3'-[(4-nitrophenyl)-acetyl]-2',5'-dideoxy-adenosine 2: 3'-[(4-nitrophenyl)-butyryl]-2',5'-dideoxyadenosine 3: 3'-[(4-azido-3-iodophenyl)-acetyl]-2',5'-dideoxyadenosine and 4: 3'-[(4-azido-3-iodophenyl)-butyryl]-2',5'-dideoxyadenosine. The azidoiodo-phenyl-analogs inactivated adenylyl cyclase irreversibly and in a light-dependent manner. This was observed with detergent-dispersed enzyme from rat brain, purified native enzyme from bovine brain, and recombinant Type I bovine adenylyl cyclase expressed in membranes from fall army worm ovarian (Sf9) cells. Inactivation of the recombinant enzyme was inversely dependent on ATP concentration and was not completely prevented by 2',5'-dideoxyadenosine. Inhibition kinetics with the recombinant enzyme in the absence of light suggested two sites of inhibition, whereas with the native Type I enzyme inhibition kinetics exhibited a straightforward noncompetitive mechanism. Occupation of either or both sites by ligand protected the enzyme against denaturation by UV-irradiation per se. The data are consistent with inactivation of the recombinant enzyme occurring both through the 'P'-site and the catalytic active site, but suggest that this is a characteristic of the recombinant enzyme and is not dependent on the probes per se. The data suggest the potential for independent interactions of such ligands with different sites on a given enzyme and also with other enzymes containing adenosine or adenine nucleotide binding domains.

Adenosine Triphosphate↗
PubMed1995

Purification, characterization, and N-terminal amino acid sequence of the adenylyl cyclase-activating protease from bovine sperm.

We previously reported the extraction of a factor from bovine sperm that activated adenylyl cyclases of rat brain and human platelets, and identified it as a trypsin-like protease that was referred to as "ninhibin." This proteolytic activity was purified to near homogeneity from an alkaline extract of washed sperm particles by sequential chromatography on p-aminobenzamidine agarose and CM-Sephadex. Purification was greater than 100-fold with nearly 30% recovery of protease activity exhibiting a major band of approximately 40 kDa. An approximately 45-kDa form of the protease was also evident in crude extracts and was preferentially isolated when the enzyme was prepared in the presence of a mixture of protease inhibitors. The larger form of the protease was substantially less effective in stimulating adenylyl cyclase than was the smaller form; it is likely to be a zymogen form from which the smaller, more active form is derived. Purified forms of acrosin and ninhibin exhibited similar mobilities on PAGE, similar capacities for activating adenylyl cyclase, similar patterns of proteolytic fragmentation, and similar immunoblot patterns obtained with an antibody against purified bovine acrosin. More importantly, the N-terminal amino acid sequence of bovine ninhibin was found to be identical with that of bovine acrosin and caprine acrosin and more than 75% identical with porcine acrosin. The data support the conclusion that the adenylyl cyclase-activating protease previously referred to as ninhibin is, in fact, acrosin.

Acrosin↗
PubMed1990

Inhibition of Bordetella pertussis and Bacillus anthracis adenylyl cyclases by polyadenylate and "P"-site agonists.

Inhibition of adenylyl cyclases from Bacillus anthrasis and Bordetella pertussis by polyadenylate and by the most potent "P"-site agonists was investigated. These bacterial adenylyl cyclases differed in their sensitivity to inhibition by nominal "P"-site agents and in the effect of divalent cations on this inhibition. The enzyme from Bordetella pertussis was relatively insensitive to inhibition by "P"-site agonists, exhibiting a rank order of potency of 2'd3'AMP greater than 3'-AMP greater than 2',5'-ddAdo approximately Ado approximately 2'-dAdo, with IC50 values for 2'd3'AMP and 3'-AMP of 1-3 mM. Inhibition by 2'd3'AMP, however, was not affected by divalent cation, making it distinct from "P"-site-mediated inhibition of most mammalian adenylyl cyclases. The sensitivity to these nucleosides was comparable with potency for inhibition of bovine sperm adenylyl cyclase but was 3 orders of magnitude less potent than for activated enzyme from bovine or rat brain. The Bordetella pertussis enzyme was similarly insensitive to inhibition by polyadenylate, with 16 microM inhibiting less than 20%. By comparison, Bacillus anthrasis adenylyl cyclase was more potently inhibited by 2'd3'AMP (IC50 approximately 85 microM) but not by the other nucleosides (less than 15% inhibition at 1 mM), and inhibition by 2'd3'AMP was optimally enhanced by 5-10 mM Mg2+ or Mn2+, as is typical for inhibition by "P"-site agonists. The Bacillus anthrasis enzyme was potently inhibited by polyadenylate (IC50 approximately 0.3 microM), comparable to inhibition of brain adenylyl cyclases. Sensitivity of Bacillus anthrasis adenylyl cyclase to poly(A) was diminished somewhat by Ca2+/calmodulin (to IC50 approximately 1 microM) although Ca2+/calmodulin was without effect on inhibition by 2'd3'AMP. In contrast to inhibition of mammalian adenylyl cyclases via the "P"-site, inhibition of both bacterial adenylyl cyclases by 2'd3'AMP was competitive with respect to substrate MgATP. The data indicate basic differences in susceptibilities of these bacterial adenylyl cyclases to inhibition by poly(A), by adenosine analogs, and the effects of divalent cations. Although the potency of 2'd3'AMP and the metal-dependent nature of inhibition of Bacillus anthrasis adenylyl cyclase shared characteristics of "P"-site-mediated inhibition, the fact that inhibition of both bacterial adenylyl cyclases was competitive with respect to substrate strongly suggests that this inhibition was at the catalytic site and that these bacterial enzymes do not contain a distinct "P"-site.

Adenosine↗
PubMed1990

Kinetics of "P"-site-mediated inhibition of adenylyl cyclase and the requirements for substrate.

The kinetics of "P"-site-mediated inhibition of adenylyl cyclase was studied with the detergent-solubilized enzyme from rat brain. Mn2(+)-activated adenylyl cyclase exhibited typical noncompetitive inhibition by 2'-d3'-AMP or 2',5'-dideoxyadenosine (2',5'-ddAdo). However, enzyme that was preactivated with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or proteolytically with ninhibin (+ GTP gamma S) exhibited apparently uncompetitive inhibition with either 2'-d3'-AMP or 2',5'-ddAdo and with either MgATP or MgApp(NH)p (adenosine 5'-(beta gamma-imino)triphosphate) as substrate. Inhibition increased with increasing substrate concentration, consistent with distinct domains for catalysis and the P-site and the formation of a 2'-d3'-AMP.C.MgATP complex. This conclusion was supported by the kinetics of product inhibition. For both cAMP and inorganic pyrophosphate (MgPPi) inhibition was mixed, suggesting that product release is likely random sequential. Although MgPPi enhanced inhibition in the presence of P-site agonist, it did not affect the dissociation constant for P-site agonist. The uncompetitive character of P-site-mediated inhibition and the independence of inhibition by MgPPi and P-site agonist imply that the P-site binding domain is distinct from the substrate binding domain. Given the structural requirements for catalysis and for P-site-mediated inhibition, these domains would be expected to be homologous. Sensitivity to P-site-mediated inhibition was also dependent on the structure of ATP, with the following IC50 values for 2'-d3'-AMP: ATP approximately 2'-dATP (approximately 1 microM); adenosine 5'-O-(3-thiotriphosphate) (approximately 5 microM); App(NH)p (approximately 30 microM); adenosine 5'-(beta gamma-methylene)triphosphate (approximately 300 microM). The differing effectiveness of the ATP analogs to support P-site inhibition was not due to their binding at the P-site. This effect of substrate was also observed with the platelet enzyme and was independent of the means by which the enzyme was activated, whether by Mn2+ or proteolytically by ninhibin/GTP gamma S, suggesting it is a general characteristic of P-site-mediated inhibition. The data suggest a structure for activated adenylyl cyclase such that one nucleotide binding domain, selective for ATP vis-à-vis other ATP analogs, allosterically modulates a proximate P-site domain.

Adenosine Triphosphate↗
PubMed1990

Inhibition of adenylate cyclase by polyadenylate.

The effects of ribo- and deoxyribonucleic acids on the activity of detergent-dispersed adenylate cyclases from rat and bovine brain were examined. Mn2+ (10 mM)-activated adenylate cyclase was inhibited by micromolar concentrations of poly(A) (IC50 congruent to 0.45 microM). This inhibition was directly due to poly(A) and was not mediated by: (a) protein contamination of the poly(A) preparation, (b) metal chelation, (c) formation of an acid-soluble inhibitor of adenylate cyclase, (d) effects on the specific activity of [alpha-32P]ATP, (e) competition with MnATP for binding to adenylate cyclase, or (f) diversion of substrate to an alternate polymerase reaction. Inhibition of adenylate cyclase by poly(A) was on the enzyme's catalytic unit, as purified preparations of the enzyme from bovine brain were inhibited by poly(A). This inhibition by poly(A) was not likely mediated via the enzyme's "P"-site, through which activated forms of the enzyme are selectively inhibited by specific adenosine phosphates. In contrast with inhibition by the "P"-site agonist 3' AMP, inhibition of adenylate cyclase by poly(A) was slow in onset and was not reversible by dilution and showed a different metal-dependence. Inhibition of adenylate cyclase was relatively specific for poly(A) as poly(U) caused less than 50% inhibition and deoxyribonucleic acids had no effect. The potency and specificity of the inhibition of adenylate cyclase by poly(A) imply a biochemically interesting interaction that is possibly also of physiological significance.

Adenylyl Cyclase Inhibitors↗
PubMed1990

Tissue levels, source, and regulation of 3'-AMP: an intracellular inhibitor of adenylyl cyclases.

Tissue levels of 3'-AMP were measured in several rat tissues and the sensitivities of the respective adenylyl cyclases were compared with respect to "P" site-mediated inhibition by 3'-AMP2'-deoxy-3'AMP (2'd3'-AMP), and 2',5'-dideoxyadenosine. IC50 values for these P site inhibitors of adenylyl cyclases varied widely among tissues, e.g., with skeletal muscle being least sensitive to 3'-AMP (IC50 greater than 170 microM) and brain being most sensitive (IC50 approximately 10 microM). These differences were noted when activation was with Mn2+ but diminished with Mn2+ plus forskolin and conceivably may reflect the distribution of different isozymes of adenylyl cyclase. 3'-AMP levels also varied significantly among rat tissues, with spleen having the highest levels (approximately 280 nmol/g), kidney, liver, heart, and brain having decreasing 3'-AMP content, and skeletal muscle levels being immeasureably low (less than 0.1 nmol/g). When rats were made diabetic with streptozotocin, the 3'-AMP content of livers increased from approximately 47 nmol/g in control animals to approximately 84 nmol/g, a change largely reversed by maintenance of diabetic animals with insulin. The data suggest that tissue 3'-AMP levels may be regulated and in certain tissues may be sufficient to inhibit adenylyl cyclase in vivo. Three potential sources of 3'-AMP and 2'd3'-AMP, the most potent naturally occurring P site inhibitors of adenylyl cyclase, were examined. No evidence was found for the formation of either nucleotide from the respective cyclic nucleotide by a unique cyclic nucleotide phosphodiesterase or from the respective nucleoside by a hypothetical adenosine 3'-kinase and ATP. Substantial 3'-AMP and 2'd3-AMP were formed by spleen and liver homogenates from the respective oligonucleotides (RNA, mRNA, and DNA) in a time- and protein-dependent manner. The data imply the existence of enzymes in these tissues to catalyze the formation of 3'-AMP and 2'd3'-AMP from nucleic acids and suggest that these activities may account for the formation of P site agonists under in vivo conditions. The data suggest that these P site inhibitors are a potential link between fluctuations in nucleic acid metabolism and altered sensitivity of membrane-bound adenylyl cyclase to stimulatory signals.

Adenosine Monophosphate↗
PubMed1989

Ammonium ions enhance proteolytic activation of adenylate cyclase and decrease its sensitivity to inhibition by "P"-site agonists.

A detergent-dispersed adenylate cyclase from rat brain was used to study the effects of ammonium salts and polyamines on the proteolytic activation of the enzyme by a sperm protease and on the sensitivity of adenylate cyclase to inhibition via its "P"-site. A purified preparation of a trypsin-like, serine protease from bovine sperm was used to activate solubilized adenylate cyclase in the presence of guanosine 5'-O-(3-thiotriphosphate (GTP gamma S). The proteolytically activated form of adenylate cyclase was found to be particularly sensitive to further activation by ammonium bicarbonate. The activation by NH4HCO3 was found to be due to the NH+4 cation and was characterized by an increased Vmax and by a decreased sensitivity of adenylate cyclase to inactivation by elevated concentrations of the sperm protease or by trypsin. NH4Cl and (NH4)2SO4 also caused biphasic effects on adenylate cyclase, which mimicked but were less effective than those caused by NH4HCO3. Consistent with observations of others, adenylate cyclase activity was enhanced by ammonium ions whether in the presence of reversible (Mn2+) or irreversible (GTP gamma S) activators. Mn2+- and GTP gamma S-stimulated activities were similarly optimally enhanced by 30 mM (NH4)2SO4 and by 30 to 150 mM NH4Cl or NH4HCO3. Ammonium ions did not increase the activity of the purified catalytic unit. Moreover, the effect of ammonium ions was not accompanied by an increased rate of activation by GTP gamma S, suggesting that the activation of Gs (guanine nucleotide-dependent stimulatory component) may not be the primary cause of stimulation by ammonium salts. Several polyamines at millimolar concentrations blocked the stimulatory effect of NH+4. This was observed when adenylate cyclase was activated by Mn2+, but not when it was activated by GTP gamma S or by the sperm protease + GTP gamma S. The inhibitory effect of polyamines was not due to the formation of a complex with ATP. Both the increase in Vmax of the Mn2+-stimulated enzyme by NH+4 and the decrease in Vmax caused by spermine were accompanied by an increase in the enzyme's Km MnATP app. Spermine increased the IC50 for inhibition of Mn2+-activated adenylate cyclase by 2',5'-dideoxyadenosine (2',5'-ddAdo) from 0.75 to 4.6 microM, consistent with the idea that increased sensitivity of P-site-mediated inhibition is associated with increased enzyme activity. In contrast, activation of Mn2+-stimulated adenylate cyclase by 30 mM (NH4)2SO4 also reduced sensitivity to inhibition by 2',5'-ddAdo(IC50 1.1 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

Acrosin↗
PubMed1989

Cation and structural requirements for P site-mediated inhibition of adenylate cyclase.

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)

Adenosine↗
PubMedDate not supplied

Enzymatic preparation of 32P-labeled beta-L-2',3',-dd-5'ATP and its use as a high-affinity, conformation-specific ligand for labeling adenylyl cyclases.

An enzymatic method was developed for the preparation of unlabeled and [beta-32P]-labeled beta-L-2',3'-dd-5'ATP from the monophosphate with near quantitative yields. beta-L-2',3'-dd-5'ATP was a competitive and potent inhibitor of adenylyl cyclases (IC5 approximately 30 nM). Upon uv-irradiation beta-L-2',3'-dd-[beta-32P]-5'ATP directly crosslinked to a chimeric construct of this enzyme. Data suggest that this is a pre-transition state inhibitor and contrasts with the equipotent 2',5'-dd-3'ATP, a post-transition state, noncompetitive inhibitor.

Adenosine Triphosphate↗