Syn- and anti-conformations of 5′-deoxy- and 5′-O-methyl-uridine 2′,3′-cyclic monophosphate

Two uridine 2′,3′-cyclic monophosphate (cUMP) derivatives, 5′-deoxy (DcUMP) and 5′-O-methyl (McUMP), were studied by means of quantum chemical methods. Aqueous solvent effects were estimated based on the isodensity-surface polarized-continuum model (IPCM). Gas phase calculations revealed only slight energy differences between the syn- and anti-conformers of both compounds: the relative energies of the syn-structure are −0.9 and 0.2 kcal mol^-1 for DcUMP and McUMP, respectively. According to the results from the IPCM calculations, however, both syn-conformers become about 14 kcal mol^-1 more stable in aqueous solution than their corresponding anti-structures. Additionally, the effects of a countercation and protonation on DcUMP were studied, revealing that the syn-structure is also favored over the anti-one for these systems.     

Simultaneous inhibition of guinea pig brain 2′, 3′-cyclic nucleotide 3′-phosphohydrolase and myelin protein synthesis by 2′-adenosine monophosphate

Although the function of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNPase) in myelin is unknown, the enzyme has been implicated in the metabolism of myelin proteins. Using 2′-AMP to inhibit CNPase, we examined the effect of reduced enzyme activity on the $\text{in vitro}$ incorporation of ¹⁴C-leucine into brain proteins. The results of this study revealed that (1) guinea pig brain homogenates incorporate leucine into protein from a sucrose medium in a linear fashion, (2) all brain fractions (cytosol, myelin, and microsomes) are labelled within 1 hr, (3) 2′-AMP inhibition of CNPase by 50% results in a similar inhibition of brain protein synthesis, and (4) the reduced protein synthesis is accompanied by a shift in label from myelin proteins to those found in the microsomes. These results are consistent with a role for CNPase in myelin protein synthesis.     

Chemical synthesis and biological activity of novel brominated 7-deazaadenosine-3′,5′-cyclic monophosphate derivatives

Synthetic derivatives of cyclic adenosine monophosphate, such as halogenated or other more hydrophobic analogs, are widely used compounds, to investigate diverse signal transduction pathways of eukaryotic cells. This inspired us to develop cyclic nucleotides, which exhibit chemical structures composed of brominated 7-deazaadenines and the phosphorylated ribosugar. The synthesized 8-bromo- and 7-bromo-7-deazaadenosine-3′,5′-cyclic monophosphates rank among the most potent activators of cyclic nucleotide-regulated ion channels as well as cAMP-dependent protein kinase. Moreover, these substances bind tightly to exchange proteins directly activated by cAMP.     

Calcitonin gene-related peptide receptor independently stimulates 3′,5′-cyclic adenosine monophosphate and Ca2+ signaling pathways

The inhibition of ion transporting ATPases (Na⁺,K⁺-ATPase, Ca²⁺,Mg²⁺- and Ca²⁺-ATPase) by two amphiphilic drugs e.g. chlorpromazine (antipsychotic) and chloroquine (antimalarial) are found to be competitive in nature in vitro with respect to the substrate. Two binding sites - high and low affinity are found to exist on all the three ATPases toward these drugs as evident from the plot of F/F₀ vs. different drug concentrations of tryptophan fluorescence of the enzymes. Circular dichroism analysis suggest that binding of drugs to the high affinity site does not involve any change in conformation of ATPase molecules which occur only when drug binds to the low affinity sites. The drug binding sites and possible effect on conformational change of ATPase molecules of these two drugs have been described in this report.     

Geometry of the dry-state oligomerization of 2′,3′-cyclic phosphates

Summary Evaporation of a solution of thymidine plus either theexo or theendo diastereomer of uridine cyclic 2,3-O, O-phosphorothioate (U > p(S) in 1,2-diaminoethane hydrochloride buffer gave the 2,5 and 3,5 isomers of (P-thio) uridylylthymidine (Up(S)dT) in a ratio of 1:2 with a combined yield of about 20%. These isomers were re-converted to U > p(S) and dT by a reaction that is known to proceed by an in-line mechanism. Both the 2,5 and 3,5 isomers gave as product the same diastereomer of U > p(S) that had been used originally in their formation. These dry-state prebiotic reactions (Verlander, Lohrmann, and Orgel 1973) are thus shown to be stereospecific, and both the 2,5 and 3,5 internucleotide bonds are formed by an in-line mechanism.Abbreviations DAE 1,2-diaminoethane - HPLC high pressure liquid chromatography - RNase bovine pancreatic ribonuclease A, EC 3.1.4.22 - TEAB triethylammonium bicarbonate - tris tris(hydroxymethyl)aminomethane - UMP(S) uridine monophosphorothioate - U > p uridine cyclic 2,3-phosphate - U > p(S) uridine cyclic 2,3-O, O-phosphorothioate - Up(S)dT (P-thio)uridylylthymidine - U2p(R_p-S)5dT (P-thio)uridylylthymidine with theR configuration at phosphorous, and a 2,5 internucleotide linkage     

Adenosine 3′:5′-cyclic monophosphate and catabolite repression in Escherichia coli

1. Both permanent and transient catabolite repression of beta-galactosidase synthesis in Escherichia coli are abolished by 5mm-3':5'-cyclic-AMP when elicited by glucose, but not when caused by a mixture of glucose, glucose 6-phosphate, gluconate and casein hydrolysate (casamino acids). 2. Glucose uptake is slightly increased by 3':5'-cyclic-AMP. 3. No significant effects of the nucleotide were found on the synthesis of protein and RNA, either in exponential growth on one substrate, or during a growth shift from glycerol to glycerol plus glucose. 4. Marked changes in the soluble-protein profiles of cells growing in glycerol and glucose were caused by the presence of 3':5'-cyclic-AMP. 5. Measurements of (14)CO(2) release from specifically-labelled glucose showed that 3':5'-cyclic-AMP greatly stimulated glycolytic activity while having a minor depressing effect on the metabolic flow through the pentose phosphate cycle. 6. The concentrations of several metabolic intermediates, particularly fructose 1,6-diphosphate, were greatly affected by the presence of 3':5'-cyclic-AMP. 7. Several metabolites partially relieved glucose repression of beta-galactosidase synthesis in EDTA-treated cells; three out of five of these metabolites reversed the effect more effectively than did 3':5'-cyclic-AMP. 8. The evidence for and against a direct role for 3':5'-cyclic-AMP is discussed. It is concluded that the evidence for indirect action is at least as strong as that for direct action.     

Immunohistochemical localization of 2′,3′-cyclic nucleotide 3′-phosphodiesterase in adult bovine cerebrum and cerebellum

We have previously shown that 2,3-cyclic nucleotide 3-phosphodiesterase (CNP; EC 3.1.4.37) in rat central nervous tissues can be immunohistochemically stained with anti-bovine CNP serum. However, the anti-bovine CNP serum prepared in our laboratory has only weak cross-reactivity with rat CNP. Sections of bovine nervous tissues were found to be stained effectively with the serum, and the localization of CNP has been revealed in greater detail. We describe here the immunohistochemical localization of CNP in adult bovine cerebrum and cerebellum. CNP stained was localized in myelin sheaths, oligodendrocytes, and the processes of oligodendrocytes; astrocytes and neurons were negative. All myelinated nerve fibers appeared to be stained with the anti-CNP serum. Perineuronal and perivascular oligodendrocytes, and oligodendrocytes extending their processes to isolated myelin fibers were stained. Interfascicular oligodendrocytes, however, did not react or reacted faintly to the anti-CNP serum; only their processes were reactive. Comparison with the stain for S-100 protein was helpful to distinguish oligodendrocytes from astrocytes particularly when both glial cells were situated together at the perineuronal and perivascular positions.Dedicated to Professor Yasuzo Tsukada.     

Implication of guanosine 3′,5′-cyclic monophosphate,adenosine 3′,5′-cyclic monophosphate,adenosine 5′-mono-, di- and triphosphate and fructose-2,6-bisphosphate in the regulation of the glycolytic pathway in hypoxic/anoxic mussel,Mytilus galloprovincialis

The change in the content of cyclic GMP, cyclic AMP, ATP, ADP, AMP and fructose-2,6-bisphosphate that occurred in the mantle of the mussel Mytilus galloprovincialis Lmk when specimens of this mollusk were subjected to a hypoxia/anoxia situation were assessed. After the early 24 h in anaerobiosis, a clear decrease was observed in the ATP content, which remained close to that value for the rest of the time. AMP content doubled during the early 24 h in anaerobiosis and, from that time on, it remained close to that value. Fructose-2,6-bisphoshate and cyclic GMP showed a similar behavior. The levels of these compounds rose significantly during the early hours in anaerobiosis, and then fell to values similar to those of aerobiosis, remaining constant for the rest of the time. Neither ADP nor cAMP showed significant variations.     

Influence of theophylline on concentrations of cyclic 3′,5′-adenosine monophosphate and cyclic 3′,5′-guanosine monophosphate of rat brain

The influence of theophylline (2.5–100 mg/kg p.o.) on cyclic 3,5-adenosine monophosphate (cAMP) and cyclic 3,5-guanosine monophosphate (cGMP) in brain of Sprague-Dawley rats (0.5–3.0 hr after administration of theophylline) was investigated. It was found that theophylline increases cAMP and cGMP levels when administered in a dose of 25 mg/kg or higher. A significant decrease of cGMP level was observed after administration of 10 mg/kg. The results of this study suggest that the influence of theophylline on cyclic nucleotide levels of rat brain is the result of two factors: (a) inhibitory properties of theophylline on cAMP and cGMP phosphodiesterases and (b) competition of theophylline with adenosine.     

Structure and mechanism of E. coli RNA 2′,3′-cyclic phosphodiesterase

2H (two-histidine) phosphoesterase enzymes are distributed widely in all domains of life and are implicated in diverse RNA and nucleotide transactions, including the transesterification and hydrolysis of cyclic phosphates. Here we report a biochemical and structural characterization of the Escherichia coli 2H protein YapD, which was identified originally as a reversible transesterifying “nuclease/ligase” at RNA 2′,5′-phosphodiesters. We find that YapD is an “end healing” cyclic phosphodiesterase (CPDase) enzyme that hydrolyzes an _HORNA>p substrate with a 2′,3′-cyclic phosphodiester to a _HORNAp product with a 2′-phosphomonoester terminus, without concomitant end joining. Thus we rename this enzyme ThpR (two-histidine 2′,3′-cyclic phosphodiesterase acting on RNA). The 2.0 Å crystal structure of ThpR in a product complex with 2′-AMP highlights the roles of extended histidine-containing motifs ⁴³HxTxxF⁴⁸ and ¹²⁵HxTxxR¹³⁰ in the CPDase reaction. His43-Nε makes a hydrogen bond with the ribose O3′ leaving group, thereby implicating His43 as a general acid catalyst. His125-Nε coordinates the O1P oxygen of the AMP 2′-phosphate (inferred from geometry to derive from the attacking water nucleophile), pointing to His125 as a general base catalyst. Arg130 makes bidentate contact with the AMP 2′-phosphate, suggesting a role in transition-state stabilization. Consistent with these inferences, changing His43, His125, or Arg130 to alanine effaced the CPDase activity of ThpR. Phe48 makes a π–π stack on the adenine nucleobase. Mutating Phe28 to alanine slowed the CPDase by an order of magnitude. The tertiary structure and extended active site motifs of ThpR are conserved in a subfamily of bacterial and archaeal 2H enzymes.     

Some properties of adenosine 3′,5′-cyclic monophosphate phosphodiesterase in the superior cervical ganglion of the guinea pig

Adenosine 3,5-cyclic monophosphate (cAMP) phosphodiesterase activity in crude guinea-pig superior cervical ganglion homogenates was assayed under a variety of experimental conditions. Two forms of cAMP phosphodiesterase were found, one with high and the other with low affinity for the substrate. TheK _m values were about 1 and 110 M respectively. Imidazole slightly but constantly stimulated the former enzyme form over a wide range of concentrations and l-methyl-3-isobutylxanthine was a weak competitive inhibitor with aK _i value of 90 M. Low affinity cAMP phosphodiesterase activity was increased by calmodulin and Ca²⁺. This stimulation was not observed in the presence of trifluoperazine, a specific inhibitor of calmodulin. On the other hand, neither [d-Ala²]met-enkephalinamide nor prostaglandin E₂, alone or in combination, influenced high affinity cAMP phosphodiesterase.     

Binding proteins for adenosine 3′:5′-cyclic monophosphate in bovine adrenal cortex

Five peaks of cyclic AMP-binding activity could be resolved by DEAE-cellulose chromatography of bovine adrenal-cortex cytosol. Two of the binding peaks co-chromatographed with the catalytic activities of cyclic AMP-dependent protein kinases (ATP-protein phosphotransferase, EC 2.7.1.37) of type I or type II respectively. A third binding protein was eluted between the two kinases, and appeared to be the free regulatory moiety of protein kinase I. Two of the binding proteins for cyclic AMP, sedimenting at 9S in sucrose gradients, could also bind adenosine. They bound cyclic AMP with an apparent equilibrium dissociation constant (K(d)) of about 0.1mum, and showed an increased binding capacity for cyclic AMP after preincubation in the presence of K(+), Mg(2+) and ATP. The two binding proteins differed in their apparent affinities for adenosine. The isolated regulatory moiety of protein kinase I had a very high affinity for cyclic AMP (K(d)<0.1nm). At low ionic strength or in the presence of MgATP, the high-affinity binding of cyclic AMP to the regulatory subunit of protein kinase I was decreased by the catalytic subunit. At high ionic strength and in the absence of MgATP the high-affinity binding to the regulatory subunit was not affected by the presence of catalytic subunit. Under all experimental conditions tested, dissociation of protein kinase I was accompanied by an increased affinity for cyclic AMP. To gain some insight into the mechanism by which cyclic AMP activates protein kinase, the interaction between basic proteins, salt and the cyclic nucleotide in activating the kinase was studied.     

An effect of insulin on adipose-tissue adenosine 3′:5′-cyclic monophosphate phosphodiesterase

1. 3':5'-Cyclic nucleotide phosphodiesterase activity was measured in homogenates prepared from epididymal fat-pads and isolated fat-cells incubated in the absence and presence of insulin. 2. Homogenates of insulin-treated tissues showed an increase in phosphodiesterase activity compared with controls. No effect of insulin was observed when the hormone was added directly to homogenates. 3. There was kinetic evidence for the presence of two 3':5'-cyclic nucleotide phosphodiesterases in adipose tissue. Insulin raised the maximal velocity of the low-K(m) enzyme and lowered the K(m) of the higher-K(m) enzyme. 4. It is suggested that the effect of insulin on adipose tissue phosphodiesterase accounts for the ability of this hormone to lower cyclic-AMP concentration in the tissue.     

Synthesis of 2′,3′-Didehydro-2′,3′-Dideoxy-3′-C-Methyl Substituted Nucleosides

Abstract

1-(2,3-Dideoxy-3-C-hydroxmethyl-β-D-threo-pentofuranosyl) -,1- (2,3-didehydro-2,3-dideoxy-3-C-hydroxymethyl-β-D-glycero- pentofuranosyl) -and 1-(3-C-azidomethyl-2,3-dideoxy-3-C-hydroxymethyl-β-D-glycero- pentofuranosyl)uracil, thymine and cytosine were synthesized and evaluated for anti-HIV activity. The synthetic strategy was based on an allylic alcohol transposition of the corresponding 3′-C-methylene-nucleoside analogues.     

Phosphonates Derivatives of 2′,3′-Dideoxy-2′,3′-didehydro-pentopyranosyl Nucleosides

Abstract

Adenine and thymine derivatives of 2′,3′-dideoxy-2′,3′-didehydropento-pyranosyl nucleosides carrying a phosphonomethyl moiety at their 4′-O-position and in a cis relationship with the heterocyclic base have been synthesized.     

Anti-anabolic effects of adenosine 3′:5′-cyclic monophosphate. Inhibition of protein synthesis

1. Evidence is presented that cyclic AMP inhibits the incorporation of l-[4,5-(3)H]leucine into protein in a cell-free system from rat liver. This inhibition occurs after aminoacyl-tRNA formation. 2. Microsomal fractions, isolated after the incubation of postmitochondrial supernatant with cyclic AMP and ATP, show a diminished ability to synthesize protein. Both cyclic AMP and ATP are required for this effect. 3. A possible physiological role for the anti-anabolic action of cyclic AMP is discussed in terms of the control of gluconeogenesis.     

Interaction of myelin basic protein and 2′,3′-cyclic nucleotide phosphodiesterase with mitochondria

The content and distribution of myelin basic protein (MBP) isoforms (17 and 21.5 kDa) as well as 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) were determined in mitochondrial fractions (myelin fraction, synaptic and non-synaptic mitochondria) obtained after separation of brain mitochondria by Percoll density gradient. All the fractions could accumulate calcium, maintain membrane potential, and initiate the opening of the permeability transition pore (mPTP) in response to calcium overloading. Native mitochondria and structural contacts between membranes of myelin and mitochondria were found in the myelin fraction associated with brain mitochondria. Using Western blot, it was shown that addition of myelin fraction associated with brain mitochondria to the suspension of liver mitochondria can lead to binding of CNPase and MBP, present in the fraction with liver mitochondria under the conditions of both closed and opened mPTP. However, induction of mPTP opening in liver mitochondria was prevented in the presence of myelin fraction associated with brain mitochondria (Ca²⁺ release rate was decreased 1.5-fold, calcium retention time was doubled, and swelling amplitude was 2.8-fold reduced). These results indicate possible protective properties of MBP and CNPase.     

Synthesis and release of adenosine 3′: 5′-cyclic monophosphate by Chlamydomonas reinhardtii

One of the labeled compounds synthesized by Chlamydomonas reinhardtii when ³²Pi was supplied was isolated from both the cells and the medium in which the cells had grown. This compound copurified with authentic [8-³H]cAMP by TLC to a constant ratio of ³²P/³H. The compound was degraded by beef heart cyclic nucleotide phosphodiesterase to a product which cochromatographed with authentic 5′AMP, at the same rate as the hydrolysis of authentic cAMP-[³H] to 5′AMP-[³H]. In both cases, 1-Me-3-isoBu-xanthine, a specific inhibitor of the phosphodiesterase, totally blocked the reaction. It is concluded that the compound synthesized by C. reinhardtii was cAMP, 85% of which was released into the medium.