Biomedical subjects

I Shoshani

Publications and source records attributed to I Shoshani.

6 recordsLinked to original sources

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↗