The brain in vivo expresses the 2',3'-cAMP-adenosine pathway

Although multiple biochemical pathways produce adenosine, studies suggest that the 2',3'-cAMP-adenosine pathway (2',3'-cAMP→2'-AMP/3'-AMP→adenosine) contributes to adenosine production in some cells/tissues/organs. To determine whether the 2',3'-cAMP-adenosine pathway exists in vivo in the brain, we delivered to the brain (gray matter and white matter separately) via the inflow perfusate of a microdialysis probe either 2',3'-cAMP, 3',5'-cAMP, 2'-AMP, 3'-AMP, or 5'-AMP and measured the recovered metabolites in the microdialysis outflow perfusate with mass spectrometry. In both gray and white matter, 2',3'-cAMP increased 2'-AMP, 3'-AMP and adenosine, and 3',5'-cAMP increased 5'-AMP and adenosine. In both brain regions, 2'-AMP, 3-AMP and 5'-AMP were converted to adenosine. Microdialysis experiments in 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) wild-type mice demonstrated that traumatic brain injury (controlled cortical impact model) activated the brain 2',3'-cAMP-adenosine pathway; similar experiments in CNPase knockout mice indicated that CNPase was involved in the metabolism of endogenous 2',3'-cAMP to 2'-AMP and to adenosine. In CSF from traumatic brain injury patients, 2',3'-cAMP was significantly increased in the initial 12 h after injury and strongly correlated with CSF levels of 2'-AMP, 3'-AMP, adenosine and inosine. We conclude that in vivo, 2',3'-cAMP is converted to 2'-AMP/3'-AMP, and these AMPs are metabolized to adenosine. This pathway exists endogenously in both mice and humans.     

Expression of the 2',3'-cAMP-adenosine pathway in astrocytes and microglia

Many organs express the extracellular 3',5'-cAMP-adenosine pathway (conversion of extracellular 3',5'-cAMP to 5'-AMP and 5'-AMP to adenosine). Some organs release 2',3'-cAMP (isomer of 3',5'-cAMP) and convert extracellular 2',3'-cAMP to 2'- and 3'-AMP and convert these AMPs to adenosine (extracellular 2',3'-cAMP-adenosine pathway). As astrocytes and microglia are important participants in the response to brain injury and adenosine is an endogenous neuroprotectant, we investigated whether these extracellular cAMP-adenosine pathways exist in these cell types. 2',3'-, 3',5'-cAMP, 5'-, 3'-, and 2'-AMP were incubated with mouse primary astrocytes or primary microglia for 1 h and purine metabolites were measured in the medium by mass spectrometry. There was little evidence of a 3',5'-cAMP-adenosine pathway in either astrocytes or microglia. In contrast, both cell types converted 2',3'-cAMP to 2'- and 3'-AMP (with 2'-AMP being the predominant product). Although both cell types converted 2'- and 3'-AMP to adenosine, microglia were five- and sevenfold, respectively, more efficient than astrocytes in this regard. Inhibitor studies indicated that the conversion of 2',3'-cAMP to 2'-AMP was mediated by a different ecto-enzyme than that involved in the metabolism of 2',3'-cAMP to 3'-AMP and that although CD73 mediates the conversion of 5'-AMP to adenosine, an alternative ecto-enzyme metabolizes 2'- or 3'-AMP to adenosine.     

Extracellular cAMP-adenosine pathways in the mouse kidney

The renal extracellular 2',3'-cAMP-adenosine and 3',5'-cAMP-adenosine pathways (extracellular cAMPs→AMPs→adenosine) may contribute to renal adenosine production. Because mouse kidneys provide opportunities to investigate renal adenosine production in genetically modified kidneys, it is important to determine whether mouse kidneys express these cAMP-adenosine pathways. We administered (renal artery) 2',3'-cAMP and 3',5'-cAMP to isolated, perfused mouse kidneys and measured renal venous secretion rates of 2',3'-cAMP, 3',5'-cAMP, 2'-AMP, 3'-AMP, 5'-AMP, adenosine, and inosine. Arterial infusions of 2',3'-cAMP increased (P < 0.0001) the mean venous secretion of 2'-AMP (390-fold), 3'-AMP (497-fold), adenosine (18-fold), and inosine (adenosine metabolite; 7-fold), but they did not alter 5'-AMP secretion. Infusions of 3',5'-cAMP did not affect venous secretion of 2'-AMP or 3'-AMP, but they increased (P < 0.0001) secretion of 5'-AMP (5-fold), adenosine (17-fold), and inosine (6-fold). Energy depletion (metabolic inhibitors) increased the secretion of 2',3'-cAMP (8-fold, P = 0.0081), 2'-AMP (4-fold, P = 0.0028), 3'-AMP (4-fold, P = 0.0270), 5'-AMP (3-fold, P = 0.0662), adenosine (2-fold, P = 0.0317), and inosine (7-fold, P = 0.0071), but it did not increase 3',5'-cAMP secretion. The 2',3'-cAMP-adenosine pathway was quantitatively similar in CD73 -/- vs. +/+ kidneys. However, 3',5'-cAMP induced a 6.7-fold greater increase in 5'-AMP, an attenuated increase (61% reduction) in inosine and a similar increase in adenosine in CD73 -/- vs. CD73 +/+ kidneys. In mouse kidneys, 1) 2',3'-cAMP and 3',5'-cAMP are metabolized to their corresponding AMPs, which are subsequently metabolized to adenosine; 2) energy depletion activates the 2',3'-cAMP-adenosine, but not the 3',5'-cAMP-adenosine, pathway; and 3) although CD73 is involved in the 3',5'-AMP-adenosine pathway, alternative pathways of 5'-AMP metabolism and reduced metabolism of adenosine to inosine compensate for life-long deficiency of CD73.     

The 2',3'-cAMP-adenosine pathway

Our recent studies employing HPLC-tandem mass spectrometry to analyze venous perfusate from isolated, perfused kidneys demonstrate that intact kidneys produce and release into the extracellular compartment 2',3'-cAMP, a positional isomer of the second messenger 3',5'-cAMP. To our knowledge, this represents the first detection of 2',3'-cAMP in any cell/tissue/organ/organism. Nuclear magnetic resonance experiments with isolated RNases and experiments in isolated, perfused kidneys suggest that 2',3'-cAMP likely arises from RNase-mediated transphosphorylation of mRNA. Both in vitro and in vivo kidney experiments demonstrate that extracellular 2',3'-cAMP is efficiently metabolized to 2'-AMP and 3'-AMP, both of which can be further metabolized to adenosine. This sequence of reactions is called the 2',3'-cAMP-adenosine pathway (2',3'-cAMP → 2'-AMP/3'-AMP → adenosine). Experiments in rat and mouse kidneys show that metabolic poisons increase extracellular levels of 2',3'-cAMP, 2'-AMP, 3'-AMP, and adenosine; however, little is known regarding the pharmacology of 2',3'-cAMP, 2'-AMP, and 3'-AMP. What is known is that 2',3'-cAMP facilitates activation of mitochondrial permeability transition pores, a process that can lead to apoptosis and necrosis, and inhibits proliferation of vascular smooth muscle cells and glomerular mesangial cells. In summary, there is mounting evidence that at least some types of cellular injury, by triggering mRNA degradation, engage the 2',3'-cAMP-adenosine pathway, and therefore this pathway should be added to the list of biochemical pathways that produce adenosine. Although speculative, it is possible that the 2',3'-cAMP-adenosine pathway may protect against some forms of acute organ injury, for example acute kidney injury, by both removing an intracellular toxin (2',3'-cAMP) and increasing an extracellular renoprotectant (adenosine).     

A phosphate-binding histidine of binuclear metallophosphodiesterase enzymes is a determinant of 2',3'-cyclic nucleotide phosphodiesterase activity

Binuclear metallophosphoesterases are an enzyme superfamily defined by a shared fold and a conserved active site. Although many family members have been characterized biochemically or structurally, the physiological substrates are rarely known, and the features that determine monoesterase versus diesterase activity are obscure. In the case of the dual phosphomonoesterase/diesterase enzyme CthPnkp, a phosphate-binding histidine was implicated as a determinant of 2',3'-cyclic nucleotide phosphodiesterase activity. Here we tested this model by comparing the catalytic repertoires of Mycobacterium tuberculosis Rv0805, which has this histidine in its active site (His(98)), and Escherichia coli YfcE, which has a cysteine at the equivalent position (Cys(74)). We find that Rv0805 has a previously unappreciated 2',3'-cyclic nucleotide phosphodiesterase function. Indeed, Rv0805 was 150-fold more active in hydrolyzing 2',3'-cAMP than 3',5'-cAMP. Changing His(98) to alanine or asparagine suppressed the 2',3'-cAMP phosphodiesterase activity of Rv0805 without adversely affecting hydrolysis of bis-p-nitrophenyl phosphate. Further evidence for a defining role of the histidine derives from our ability to convert the inactive YfcE protein to a vigorous and specific 2',3'-cNMP phosphodiesterase by introducing histidine in lieu of Cys(74). YfcE-C74H cleaved the P-O2' bond of 2',3'-cAMP to yield 3'-AMP as the sole product. Rv0805, on the other hand, hydrolyzed either P-O2' or P-O3' to yield a mixture of 3'-AMP and 2'-AMP products, with a bias toward 3'-AMP. These reaction outcomes contrast with that of CthPnkp, which cleaves the P-O3' bond of 2',3'-cAMP to generate 2'-AMP exclusively. It appears that enzymic features other than the phosphate-binding histidine can influence the orientation of the cyclic nucleotide and thereby dictate the choice of the leaving group.     

Self-association of adenosine 5'-monophosphate (5'-AMP) as a function of pH and in comparison with adenosine, 2'-AMP and 3'-AMP

The concentration dependence of the chemical shifts for protons H-2, H-8, and H-1' of adenosine (Ado), 2'-AMP, 3'-AMP and 5'-AMP was measured in D2O at 27 degrees C under several degrees of protonation. All results are consistent with the isodesmic model of indefinite noncooperative stacking. The association constants for Ado decrease with increasing protonation: Ado (K = 15 M-1) greater than D(Ado)+/Ado (6.0 M-1) greater than D(Ado)+ (0.9 M-1). In contrast, a maximum is observed with 5'-AMP: 5'-AMP2- (K = 2.1 M-1) less than D(5'-AMP)- (3.4 M-1) less than D2(5'-AMP) +/- /D(5'-AMP)- (5.6 M-1) greater than D2(5'-AMP) +/- (approximately 2 M-1) greater than D3(5'-AMP)+ (less than or equal to 1 M-1). Self-stacking is most pronounced here if 50% of the adenine residues are protonated at N-1; complete base protonation reduces the stacking tendency drastically. Comparing the self-association of 2'-, 3'- and 5'-AMP shows that there is no influence of the phosphate-group position in the 2-fold negatively charged species, i.e., K congruent to 2 M-1 for all three AMP2- species. More importantly, there is also no significant influence observed if the stacking tendency of the three D2(AMP) +/- /D(AMP)-1:1 mixtures is compared (K congruent to 6-7 M-1); moreover, the measured association constants are within experimental error identical with the constant determined for D(Ado)+/Ado (K = 6.0 M-1). This indicates that any coulombic contribution between the -PO3(H)- group and the H+ (N-1) unit of the adenine residue to the stability of the mentioned stacks in D2O is small. However, experiments in 50% (v/v) dioxane-D8/D2O with the D2(5'-AMP) +/- /D(5'-AMP)- 1:1 system reveal, despite its low solubility, that coulombic interactions contribute to the self-association in an environment with a reduced polarity (compared to that of water). The implications of these observations for biological systems are briefly indicated.     

Effect of Vacuum Ultraviolet Irradiation on Abiogenous Synthesis of Adenine Nucleotides in the Presence of Lunar Ground

Vacuum ultraviolet (VUV) radiation, a powerful energy source in free Space, have been established to promote synthesis of natural nucleotides. It is shown that lunar ground protects from destruction (up to 1.5–2.0% ) the nucleotides formed after VUV-irradiation of dry films (adenosine and inorganic phosphate). Identification and quantitative determination of the products of synthesis and destruction is performed by the method of high performance liquid chromatography. The following products of synthesis are found: 5'-AMP > 2'3'-cAMP > 2'-AMP > 3'5'-cAMP > 3'-AMP. The obtained results are discussed from the viewpoint of hypothesis about the Space (extraterrestrial) origin of biologically important compounds that were initial for evolution on the Earth.     

Evidence against the presence of 3',5'-cyclic adenosine monophosphate and relevant enzymes in Lactobacillus plantarum

Analysis of cells of Lactobacillus plantarum, starved or undergoing induction, showed no 3', 5'-cyclic adenosine monophosphate (cAMP). Neither adenyl cyclase nor 3', 5'-cAMP phosphodiesterase was detected in extracts. Extracts of L. plantarum did not inhibit these two enzymes of Escherichia coli K-12, strain W1435. Incubation of adenosine triphosphate (ATP)-U-(14)C with cells or various cell-free fractions of L. plantarum did not produce labeled 3', 5'-cAMP. Of various 3', 5'-cyclic and acyclic nucleotides tested, only 3', 5'-cAMP, ATP, and yeast adenylic acid stimulated l-arabinose isomerase. Yeast adenylic acid was two to four times as effective as 3', 5'-cAMP or ATP. 2', 3'-cAMP was not effective.     

Identification of 2',3'-cyclic nucleotide 3'-phosphodiesterase in bovine adrenal medulla

The adrenal medulla contains an enzyme which catalyzes the hydrolysis of 2',3'-cAMP to 2'-AMP. For the parameters which have been examined, the adrenal medulla 2',3'-cAMP phosphodiesterase appears to be similar to brain 2',3'-cyclic nucleotide 3'-phosphodiesterase (also commonly referred to as CNPase). The apparent Km of the adrenal medulla CNPase for 2',3'-cAMP is 0.88 mM. The enzyme activity is unaltered by either EDTA, MgCl2 or CaCl2 in the presence or absence of calmodulin. The apparent molecular weight is 102,500 daltons. The function of the enzyme in either the brain or the adrenal medulla is, at the present time, unknown.     

Irreversible inactivation of adenylyl cyclase by the "P"-site agonist 2',5'-dideoxy-,3'-p-fluorosulfonylbenzoyl adenosine

2',5'-Dideoxy,3'-p-fluorosulfonylbenzoyl Adenosine (2',5'-dd3'-FSBA) was synthesized and found to be an agonist and affinity label for the "P"-site of adenylyl cyclase. This compound irreversibly inactivated both a crude detergent-dispersed adenylyl cyclase from rat brain and the partially purified enzyme from bovine brain. The irreversible inactivation by 100 to 200 microM 2',5'-dd3'-FSBA was blocked in a concentration-dependent manner by several established P-site inhibitors of adenylyl cyclase, 2',5'-dideoxyadenosine, 2'-d3'-AMP, adenosine, and 2'-deoxyadenosine, but not by inosine, N6-(phenylisopropyl)adenosine, adenine, 2'-d3':5'-cAMP, or 5'-AMP, agents known not to act at the P-site. Moreover, irreversible inactivation by 2',5'-dd3'-FSBA occurred in the presence of ATP at concentrations up to 3 mM, making it unlikely that inactivation was due to an effect on the enzyme's catalytic site. Adenylyl cyclase was also irreversibly inactivated by 5'-FSBA, although modestly (less than 20%) and apparently nonspecifically. Dithiothreitol protected the enzyme from irreversible inactivation by 2',5'-dd3'-FSBA, but reversible inhibition of the enzyme was still observed, although with reduced potency. When 2 mM dithiothreitol was added after a 30-min preincubation with 2',5'-dd3'-FSBA, the rat brain enzyme was partially (approximately 80%) reactivated. The data suggest that 2',5'-dd3'-FSBA may irreversibly inactivate adenylyl cyclase by reacting with a cysteinyl moiety in proximity to the P-site domain of the enzyme. These data together with results of studies of P-site inhibition kinetics published elsewhere (Johnson, R. A., and Shoshani, I. (1990) J. Biol. Chem. 265, 11595-11600) strongly suggest that the P-site and catalytic site are distinct domains on the enzyme. 2',5'-dd3'-FSBA, and especially its radiolabeled analog, should prove to be a useful probe for structural studies of adenylyl cyclase, particularly with regard to the P-site.     

Interaction of 8-substituted derivatives and adenosine-3',5'-cyclophosphate esters with protein kinase from pig brain]

A synthesis of previously unknown 8-substituted derivatives and alkyl esters of cyclic adenosine-3',5'-monophosphate, containing reactive groups, was carried out. The interaction of the compounds obtained with a homogeneous preparation of protein kinase from pig brain was studied. It was found that all compounds, with the exception of neutral esters of 3',5'-AMP, activate the enzyme and competitively inhibit 3H-labelled 3',5'-cAMP binding by the regulatory subunit of protein kinase. The activating effect and affinity of 8-(beta-aminoethylamino)-3',5'-cAMP for protein kinase was 10 times lower than that for 3',5'-cAMP and other 8-substituted derivatives of the cyclic nucleotide. It was found that 8-(N-chloroacetylaminoethylamino)-3',5'-cAMP interaction with the enzyme is of irreversible type, which suggest covalent blocking of the nucleophilic group of the 3',5'-cAMP binding site of protein kinase. The data obtained indicate that the 3',5'-cAMP molecule is bound to the regulatory site of protein kinase in the syn-conformation. The previously made assumption on the crucial importance of the negative charge in the 3',5'-cyclophosphate system for the interaction of cyclic AMP with the regulatory subunit of protein kinase has been thus confirmed.     

An investigation of 2':3'-cyclic nucleotide 3'-phosphodiesterase (EC 3.1.4.37, CNP) in peripheral blood elements and CNS myelin

Activity of 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNP) of human erythrocyte membranes was determined in the presence of various brain CNP inhibitory compounds. Also, the hydrolysis of 2':3'-cAMP and 2':3'-cCMP by CNP of human platelets and lymphocytes was confirmed by thin layer chromatography and CNP activity was measured in lymphocytes, platelets, erythrocytes and CNS myelin. Human erythrocyte CNP activity was reduced 75 percent by the organomercurial p-chloromercuriphenyl sulfonate (1 X 10(-4) M), 46 percent by thimerosal (1 X 10(-4) M) and 35 percent by cupric chloride (1 X 10(-3) M). The 2'-AMP or 2'-CMP isomer was produced, exclusively, by the hydrolysis of 2':3'-cAMP or 2':3'-cCMP, respectively, by CNP of human lymphocytes and platelets and indicates a CNP-like activity is not only present in erythrocytes and the central and peripheral nervous systems, but also platelets and lymphocytes. CNP activities of human erythrocytes, human human and rat lymphocytes and human platelets were less than 4 percent of the activity of human and bovine CNS myelin.     

Repressible extracellular phosphodiesterases showing cyclic 2'',3''- and cyclic 3'',5''-nucleotide phosphodiesterase activities in Neurospora crassa.

Two molecular species of repressible extracellular phosphodiesterases showing cyclic 2',3'- and cyclic 3',5'-nucleotide phosphodiesterase activities were detected in mycelial culture media of wild-type Neurospora crassa and purified. The two molecular species were found to be monomeric and polymeric forms of an enzyme constituted of identical subunits having molecular weights of 50,000. This enzyme had the same electrophoretic mobility as repressible acid phosphatase. The enzyme designated repressible cyclic phosphodiesterase showed pH optima of 3.2 to 4.0 with a cyclic 3',5'-AMP substrate and 5.0 to 5.6 with a cyclic 2',3'-AMP substrate. Repressible cyclic phosphodiesterase was activated by MnCl2 and CoCl2 with cyclic 2',3'-AMP as substrate and was slightly activated by MnCl2 with cyclic 3',5'-AMP. The enzyme hydrolyzed cyclic 3',5'- and cyclic 2',3'-nucleotides, in addition to bis-rho-nitrophenyl phosphate, but not certain 5' -and 3'-nucleotides. 3'-GMP and 3'-CMP were hydrolyzed less efficiently. Mutant strains A1 (nuc-1) and B1 (nuc-2), which cannot utilize RNA or DNA as a sole source of phosphorus, were unable to produce repressible cyclic phosphodiesterase. The wild type (74A) and a heterocaryon between strains A1 and B1 produced the enzyme and showed growth on orthophosphate-free media containing cyclic 2',3'-AMP or cyclic 3',5'-AMP, whereas both mutants showed little or no growth on these media.     

Cyclic 3',5'-AMP phosphodiesterase in Physarum polycephalum. I. Chemotaxis toward inhibitors and cyclic nucleotides.

The effect of several inhibitors of the enzyme cyclic 3',5'-AMP phosphodiesterase as chemoattractants in Physarum polycephalum was examined. Of the compounds tested, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Roche 20-1724/001) and 1-ethyl-4-(isopropylidinehydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid ethyl ester, hydrochloride (Squibb 20009) were the most potent attractants. 3-Isobutyl-1-methyl xanthine, theophylline, and morin (a flavanoid) were moderate attractants and sometimes gave negative chemotaxis at high concentrations. Cyclic 3',5'-AMP was an effective, but not potent attractant. A repellent effect following the positive chemotactic action was sometimes observed with cyclic 3',5'-AMP at concentrations as high as 1 . 10(-2) M. Dibutyryl cyclic AMP appeared to be a somewhat more potent attractant than cyclic 3',5'-AMP. The 8-thiomethyl and 8-bromoderivatives of cyclic AMP, which are poorly hydrolyzed by the phosphodiesterase, were not attractants in Physarum. Possible participation of cyclic 3',5'-AMP in the directional movement in P. polycephalum is discussed.     

Effect of cyclic 3':5'-AMP derivatives prostaglandins and related agents on human chorionic gonadotropin secretion in human malignant trophoblast in culture.

The secretion of human chorionic gonadotropin (hCG) is stimulated by addition of N6, O2'-dibutyryl cyclic 3':5'-AMP (dbcAMP) or theophylline to normal term placenta and human malignant trophoblast cells in vitro. To understand better the specificity of this process, malignant trophoblast cultures were incubated with 3':5'-cyclic AMP (cAMP) derivatives, prostaglandins and other agents for 1 to 3 days, and the secretion of radioimmunoassayable hCG was measured. Whereas dbcAMP was the most potent agent in stimulating secretion of hCG, the N6--and O2'-monobutyryl derivatives of cAMP and phosphodiesterase inhibitors (theophylline, papaverine, 3-isobutyl-1-methylxanthine) also increased the secretion of the hormone. A slight increase in hCG secretion was observed following addition of adenine. By contrast, butyrate, cAMP, cyclic 3':5'-GMP (cGMP), dbcGMP, 5'-AMP, adenosine, L-epinephrine and prostaglandins E1, E2, F1a and F2a were ineffective. Particulate fractions from sonicates of malignant trophoblast cultures contained adenylate cyclase activity which was stimulated more than 10-fold by NaF, but not by either catecholamines or prostaglandins. The relatively specific stimulation of hCG secretion suggested that a regulatory process involving cAMP may have physiological significance in the trophoblast.     

Identification of 2':3'-cyclic nucleotide 3'-phosphodiesterase in the vertebrate retina

The enzyme, 2':3'-cyclic nucleotide 3'-phosphodiesterase (2':3'-cNMP-3'-ase) has been used as a marker in the nervous system for the presence of myelin membrane or myelin-producing glial cells. In this study, goldfish and bovine neural retinas are found to have high levels of such a diesterase activity. Analysis of retinal tissue incubated with 2':3'-cAMP shows only 2'-AMP as the reaction product, indicating the selective hydrolysis of the cyclic nucleotide. Microdissection of the goldfish retina demonstrates the highest 2':3'-cNMP-3'-ase activity in the region of the photoreceptors. A fraction enriched in bovine rod outer segments has about a 5-fold increase in specific enzyme activity when compared to whole retina preparations. These data suggest that 2':3'-cNMP-3'-ase is either closely associated with or is an intrinsic feature of vertebrate photoreceptor elements. The retina, which contains this enzyme, may serve as a model to investigate the influence of 2':3'-cyclic nucleotides on a function of the nervous system.     

Regulation of urocaninase activity in the liver: role of 3',5'-AMP]

A dependence of rat liver urocaninase activity on the agents affecting the adenylate cyclase system was studied in vitro and in vivo. Urocaninase is considerably activated after the injection of glucagone, NaF, theophylline and 3',5'-AMP. Under conditions optimal for the protein kinase activity of phosphorylase the urocaninase of liver extracts was activated 7-fold on the average. The nezyme retains its activity after gel-filtration through Sephadex G-25 and is capable of inactivation in the presence of Mg2+ and of reactivation after addition of ATP and 3',5'-AMP. These data suggest a possibility of regulation of mammalian liver urocaninase activity by 3',5'-AMP-dependent phosphorylation of the enzyme. Derivatives of hypoxanthine (theophylline and caffeine) in concentration 10(-4) M activate urocaninase in liver extracts 2--3 and 1.5-fold respectively. The activation is probably not due to the 3',5'-AMP phosphodiesterase inhibition, since another phosphodiesterase inhibitor--papaverine--has no activating effect on urocaninase.     

Montmorillonite: A multifunctional mineral catalyst for the prebiological formation of phosphate esters

Reaction of diiminosuccinonitrile (DISN) with 3'-AMP in the presence of alkali- and alkaline earth-montmorillonites results in the formation of 2',3'-cAMP in aqueous solution. Little or no 2', 3'-cAMP is produced when metal ion concentrations equivalent to that of the metal ion associated with the homoionic clays are used instead of mobntmorillionite. Yields comparable to those obtained with DISN are obtained when diaminomaleonitrile (DAMN) is used in place of DISN as the condensing agent. DAMN, a compound which is more stable than DISN in aqueous solution, is oxidized to DISN on the surface of the clay by Fe+3 in the clay lattice. DISN, the true condensing agent, is thus generated in the presence of the bound 3'-AMP on the montmorillonite surface. The montmorillonite catalyzes the DISN-mediated formation of 2', 3'-cAMP and this product, which binds much less strongly than does the 3'-AMP, is desorbed from the clay surface. This research established that the montmorillonite performs four different functions in its role as catalyst: (1) Binding one of the substrate molecules (3'-AMP) (2) Activating the second substrate (DAMN) (3) Catalyzing the formation of 2', 3'-cAMP (4) Releasing the reaction product so another substrate molecules can bind to the montmorillonite.     

Specific binding of 3',5'-AMP by rat liver mitochondria

It was found that 3':5'-AMP is bound by rat liver mitochondria with an affinity which corresponds to a physiological concentration of the nucleotide and a low capacity. The bound 3':5'-AMP rapidly dissociates upon dilution of mitochondrial suspensions. This finding points to the existence in mitochondria of a 3':5'-AMP receptor protein. The putative biological role of this protein is discussed.     

Transport of cyclic adenosine 3'',5''-monophosphate across Escherichia coli vesicle membranes.

The uptake and efflux of cyclic adenosine 3',5'-monophosphate (3',5'-cAMP) by Escherichia coli membrane vesicles were studied. Metabolic energy was not required for the uptake process and was found to actually decrease the amount of 3',5'-cAMP found in the vesicles. 3',5'-cAMP uptake exhibits saturation kinetics (Km = 10 mM, Vmax = 2.8 nmol/mg of protein per min) and was competitively inhibited by a number of 3',5'-cAMP analogs. The uptake of 3',5'-cAMP was found to be sharply affected by a membrane phase transition. The excretion of 3',5'-cAMP was studied by using everted membrane vesicles. Efflux in this system was dependent upon metabolic energy and was reduced or abolished by uncouplers. Different energy sources powered efflux at different rates, showing a relationship between the degree of membrane energization and rate of excretion of 3',5'-cAMP. The efflux process also displayed saturation kinetics (Km = 10.0 mM, Vmax = 0.98 nmol/mg of protein per min) and was competitively inhibited by the same 3',5'-cAMP analogs and to the same degree as was the uptake process. 3',5'-cAMP was found to be chemically unaltered by both the uptake and excretion processes. These data are interpreted as showing that the uptake and excretion of 3',5'-cAMP in E. coli membrane vesicles are carrier-mediated phenomena, possibly employing the same carrier system. Uptake is by facilitated diffusion whereas efflux is via an energy-dependent, active transport process. Evidence is presented showing that cells can regulate the number of 3',5'-cAMP transport carriers. The rate of 3',5'-cAMP excretion is possibly regulated by both the degree of membrane energization and the number of carriers present per cells.