The effects ofS-adenosylhomocysteine andS-adenosylmethionine on some purine- and pyrimidine-metabolizing systems

The effects of S-adenosylhomocysteine and S-adenosyl-methionine on some purine- and pyrimidine-metabolizing systems have been examined. Both compounds were capable of acting as relatively good inhibitors of adenosine deaminase, nucleoside phosphorylase, and adenylate deaminase activities but as relatively poor inhibitors of myokinase and nucleoside monophosphate kinase. The inhibitory effects were freely reversible. 5'-Nucleotidase, orotidine 5'- phosphate, and phospho-diesterase were unaffected. Nucleoside phosphorylase was competitively inhibited by both compounds, whereas mixed inhibitory effects occurred with adenosine deaminase.     

Activation of steroidogenesis and adenylate cyclase by adenosine in adrenal and Leydig tumor cells.

Steroidogenesis by Y-1 adrenal tumor cells in culture is stimulated by ATP, adenyl-5'-yl imidodiphosphate (App(NH)), adenosine 5'(beta, alpha-methylene)triphosphate (App(CH2)p), ADP, AMP, NAD, FAD, and adenosine but not by adenine or other nucleoside triphosphates. ATP, App(NH)p, App(CH2)p, and adenosine are active in the micromolar range. Like adrenocorticotropic hormone (ACTH), the onset of stimulation is immediate and occurs to the same extent. Also active are 2'- and 5'-deoxyadenosine and 2-chloroadenosine whereas adenine xyloside, L-riboside, or arabinoside have very low activity. Stimulation is accompanied by rounding of the cells. Dipyridamole, an inhibitor of adenosine transport, increased the response to low concentrations of adenosine, suggesting that adenosine acts externally. Stimulation of steroidogenesis by adenosine or phosphorylated adenosine compounds fails to occur in the presence of crystalline adenosine deaminase, and the effect of the enzyme on adenosine, ATP, or NAD stimulation is reversed by the competitive inhibitor erythro-9-[3-(nonane-2-ol)]adenine. This suggests that the enzyme acts specifically on adenosine and a requirement for the conversion of the above compounds to adenosine seems probable. The inhibition of cAMP effects by adenosine deaminase suggests that some of its effects are also mediated by conversion to adenosine. Similar stimulation is seen in I-10 Leydig tumor cells, but an ACTH-resistant mutant of Y-1 cells, called OS-3, is relatively resistant to adenosine. Adenosine and 2-chloroadenosine stimulate adenylate cyclase in membranes from Y-1 and I-10 cells at concentrations slightly greater than are effective for steroidogenesis. Other nucleosides are ineffective. Like the NH2-terminal 24 residues of adrenocorticotropic hormone (1-24 ACTH), the adenosine effect in Y-1 membranes is rapid and is on the Vmax intercept (versus ATP) and not on the Km. In contrast to steroidogenesis, adenosine is only a partial agonist for adenylate cyclase. It effect occurs in the presence of ITP, GTP, or guanyl-5'-yl imidodiphosphate (Gpp(NH)p). Theophylline inhibits adenosine-stimulated steroidogenesis. Inhibition of adenylate cyclase occurs in the same concentration range but is of the mixed type.     

Catabolism of adenine nucleotides in adenosine deaminase deficient erythrocytes

$\text{In vitro}$ incubation studies using fluoride and iodoacetate as glycolytic inhibitors have been carried out on red cells of the two subjects with adenosine deaminase deficiency. For comparison, similar studies have also been carried out on red cells from a normal subject and from a child with severe combined immunodeficiency with normal adenosine deaminase activity. The adenosine formed in the adenosine deaminase deficient red cells is a measure of adenosine 5′-phosphate breakdown initiated by 5′-nucleotidase, whereas inosine 5′-phosphate, inosine and hypoxanthine formation is a measure of adenosine 5′-phosphate breakdown initiated by adenylate deaminase. With fluoride as inhibitor, nearly all of the adenosine 5′-phosphate breakdown proceeded by way of adenylate deaminase, while with iodoacetate as inhibitor, 20–30% of the adenosine 5′-phosphate breakdown was initiated by 5′-nucleotidase acting on adenosine 5′-phosphate. In addition, significant amounts of adenine were produced in adenosine deaminase deficient red cells in the presence of the glycolytic inhibitors. Possible explanations for the findings noted in this study are discussed and related to recent studies on the properties of the pertinent purine nucleotide catabolic enzymes.     

Mechanism of the adenylate cyclase reaction. Stereochemistry of the reaction catalyzed by the enzyme from Brevibacterium liquefaciens

Adenylate cyclase from Brevibacterium liquefaciens (ATCC 14929) catalyzes the formation of the RP-diastereomer of adenosine 3':5'-cyclic monophosphorothioate from the SP-diastereomer of adenosine-5'-(1-thiotriphosphate). The reaction catalyzed by this adenylate cyclase proceeds with inversion of configuration at phosphorus, indicating that the cyclization reaction is direct and does not involve formation of an adenylated enzyme intermediate.     

Transition-state stabilization by adenosine deaminase: 1,6-addition of water to purine ribonucleoside, the enzyme's affinity for 6-hydroxy-1,6-dihydropurine ribonucleoside, and the effective concentration of substrate water at the active site

Positions of equilibria of highly unfavorable addition reactions, whose products are present at concentrations below the limits of detection, can be determined from equilibria of combination of anionic nucleophiles with quaternized enamines. Applied to the newly prepared 1-methylpurinium ribonucleoside cation, this method yields approximate equilibrium constants of 2 X 10(-9) M-1 for addition of water and 4 X 10(-5) M-1 for addition of N-acetylcysteine to neutral purine ribonucleoside, in dilute aqueous solution. Positions of 13C magnetic resonances and UV absorption maxima of the above complexes and comparison with those of adenosine deaminase complexes strongly suggest that purine ribonucleoside is bound by adenosine deaminase as the 1,6 covalent hydrate, not as a covalently bonded complex formed by addition of a thiol group at the active site. The favorable position of equilibrium of the hydration reaction on the enzyme, together with its extremely unfavorable position in free solution, indicates that the effective activity of substrate water at the active site is in the neighborhood of 10(10) M. The Ki value of the active diastereomer of 6-hydroxy-1,6-dihydropurine ribonucleoside is estimated as 1.6 X 10(-13) M, more than 8 orders of magnitude lower than the apparent dissociation constants of enzyme complexes with the substrate adenosine or the product inosine. The enzyme's remarkable affinity for this hydrated species, which is vanishingly rare in free solution, seems understandable in terms of the hydrate's close resemblance to a hydrated intermediate approaching the transition state in direct water attack on adenosine.     

Substrate specificity of adenosine deaminase. The role of methyl groups at 2', 3', and 5'-carbon atoms of adenosine

The substrate specifity of adenosine deaminase has been studied using C'-methyl derivatives of adenosine. On the basis of the correlation revealed between conformations of 2'- and 3'-C-methyladenosine and their substrate properties, a modified stereochemical model is suggested: the enzyme accepts the substrate within a N-type conformational range (4E----4T3----3E) of the furanose ring. The model was analysed in details using a number of C3'-modified adenosines and 5'-C-methyladenosine analogues with D-allo- and L-talo-configuration.     

Aspects of purine metabolism in the gill epithelium of rainbow trout, Salmo gairdneri Richardson.

1. Enzymes interconnecting the adenylate pool were present in high concentration. 2. AMP and adenosine were easily deaminated by the corresponding enzymes whose high levels were detected. 3. Adenylate was hydrolyzed either by deamination to yield IMP which was further dephosphorylated to inosine or by dephosphorylation to adenosine followed by deamination to inosine. 4. Incubation of gill extract with [-14C]-AMP in the presence and absence of ATP but with adenosine deaminase inhibitors allowed demonstration that ATP controlled the balance between these pathways. 5. Some biochemical properties of 5'-nucleotidase. AMP deaminase and adenosine deaminase were defined. 6. Purine salvage enzymes were also estimated.     

Regulation of platelet adenylate cyclase by adenosine

The stimulatory and inhibitory effects of adenosien of the adenylate cyclases of human and pig platelets were studied. Stimulation occurred at lower concentrations than did inhibition, and stimulatory effect was prevented by methylxanthines. Stimulation by adenosine was immediate in onset and was reversible, under conditions when cyclic AMP formation was linear with respect to time and protein concentration.The stimulatory and inhibitory effects could be distinguished further by the use of various analogues of adenosine and could be prevented by adenosine deaminase. The data suggest that both stimulation and inhibition were due to adenosine itself and not one of its degradation products and that in the platelet preparation, neither formation nor degradation of adenosine during the adenylate cyclase incubation appreciably influenced measured activity.Stimulation by adenosine was additive with the effects of GMP-P(NH)P, and α- or β-adrenergic stimulation, but was abolished by prostaglandin E₁ or by NaF. Prostaglandin E₁ and NaF increased the sensitivity of adenylate cyclase to inhibition by adenosine. The data suggests that guanly-5′-yl(β-γ imino)diphosphate and/or adrenergic stimulation and adenosine exert their effects on adenylate cyclase by distinct mechanisms, but that prostaglandin E₁ or F^? and adenosine increase enzyme activity by mechanisms which may involve common intermediates in the coupling to adenylate cyclase.     

A Kinetic Study of the Rat Liver Adenosine Kinase Reverse Reaction

Adenosine kinase is an enzyme catalyzing the reaction: adenosine + ATP → AMP + ADP. We studied some biochemical properties not hitherto investigated and demonstrated that the reaction can be easily reversed when coupled with adenosine deaminase, which transforms adenosine into inosine and ammonia. The overall reaction is: AMP + ADP → ATP + inosine + NH₃. The exoergonic ADA reaction shifts the equilibrium and fills the energy gap necessary for synthesis of ATP. This reaction could be used by cells under particular conditions of energy deficiency and, together with myokinase activity, may help to restore physiological ATP levels.     

A kinetic study of the rat liver adenosine kinase reverse reaction

Adenosine kinase is an enzyme catalyzing the reaction: adenosine + ATP --> AMP + ADP. We studied some biochemical properties not hitherto investigated and demonstrated that the reaction can be easily reversed when coupled with adenosine deaminase, which transforms adenosine into inosine and ammonia. The overall reaction is: AMP + ADP --> ATP + inosine + NH(3). The exoergonic ADA reaction shifts the equilibrium and fills the energy gap necessary for synthesis of ATP. This reaction could be used by cells under particular conditions of energy deficiency and, together with myokinase activity, may help to restore physiological ATP levels.     

Stimulation of guinea-pig brain adenylate cyclase by adenosine analogues with potent pharmacological activity in vivo

1-Methylisoguanosine, a marine natural product with potent muscle-relaxant and cardiovascular actions $\text{in vivo}$, interacts directly with adenosine receptors in guinea-pig brain slices to stimulate adenylate cyclase. These effects are blocked by theophylline. Comparison of the $\text{in vivo}$ pharmacological activity of a number of synthetic analogues of 1-methylisoguanosine with $\text{in vitro}$ adenylate cyclase-stimulating ability indicates that compounds lacking the latter biochemical activity have little muscle-relaxant activity. Adenosine is a potent stimulator of adenylate cyclase but is inactive $\text{in vivo}$ because of rapid removal from the extracellular environment by uptake and deamination. Unlike adenosine, 1-methylisoguanosine is resistant to deamination and is only poorly accumulated by brain tissue slices or homogenates containing synaptosomes. Since it is an extremely weak competitive inhibitor of adenosine deaminase and only a weak inhibitor of adenosine uptake, it is unlikely to act by potentiating the effects of adenosine itself at extracellular receptors. Thus, the pharmacological effects of 1-methylisoguanosine are apparently due to its actions as a long-lasting adenosine analogue.     

Synthesis and properties of diastereoisomers of adenosine 5'-(O-1-thiotriphosphate) and adenosine 5'-(O-2-thiotriphosphate).

The chemical synthesis of adenosine 5'-(O-1-thiotriphosphate) (ATPalphaS) and adenosine 5'-(O-2-thiotriphosphate) (ATPbetaS) is described. Both exist as a pair of diastereomers, A and B. The isomers of ATPalphaS can be distinguished on the basis of their different reaction rates with myokinase as well as nucleoside diphosphate kinase. With both enzymes, isomer A reacts fast whereas isomer B reacts considerably more slowly. Phosphorylation of a mixture of isomers of ADPalphaS with pyruvate or acetate kinase yields ATPalphaS, isomer A, whereas the phosphoryl transfer with creatine or arginine kinase yields isomer B. The isomers of ATPbetaS differ in their reactivity with myosin. Isomer A is readily hydrolyzed, whereas isomer B is not. However, isomer B reacts faster with nucleoside diphosphate kinase and ADP than isomer A. Phosphoryl transfer with pyruvate kinase onto ADPbetaS yields ATPbetaS, isomer A, with acetate kinase, isomer B.     

Selective adenosine release from human B but not T lymphoid cell line

Intracellular adenosine formation and release to extracellular space was studied in WI-L2-B and SupT1-T lymphoblasts under conditions which induce or do not induce ATP catabolism. Under induced conditions, B lymphoblasts but not T lymphoblasts, release significant amounts of adenosine, which are markedly elevated by adenosine deaminase inhibitors. In T lymphoblasts, under induced conditions, only simultaneous inhibition of both adenosine deaminase activity and adenosine kinase activities resulted in small amounts of adenosine release. Under noninduced conditions, neither B nor T lymphoblasts release adenosine, even in the presence of both adenosine deaminase or adenosine kinase inhibitors. Comparison of B and T cell's enzyme activities involved in adenosine metabolism showed similar activity of AMP deaminase, but the activities of AMP-5'-nucleotidase, adenosine kinase and adenosine deaminase differ significantly. B lymphoblasts release adenosine because of their combination of enzyme activities which produce or utilize adenosine (high AMP-5'-nucleotidase and relatively low adenosine kinase and adenosine deaminase activities). Accelerated ATP degradation in B lymphoblasts proceeds not only via AMP deamination, but also via AMP dephosphorylation into adenosine but its less efficient intracellular utilization results in the release of adenosine from these cells. In contrast, T lymphoblasts release far less adenosine, because they contain relatively low AMP-5'-nucleotidase and high adenosine kinase and adenosine deaminase activities. In T lymphoblasts, AMP formed during ATP degradation is not readily dephosphorylated to adenosine but mainly deaminated to IMP by AMP deaminase. Any adenosine formed intracellularly in T lymphoblasts is likely to be efficiently salvaged back to AMP by an active adenosine kinase. In general, these results may suggest that adenosine can be produced only by selective cells (adenosine producers) whereas other cells with enzyme combination similar to SupT1-T lymphoblasts can not produce significant amounts of adenosine even in stress conditions.     

Stereochemistry of hydrolysis of adenosine 3':5'-cyclic phosphorothioate by the cyclic phosphodiesterase from beef heart.

Adenosine 3':5'-cyclic phosphorothioate, Sp-diastereomer was hydrolyzed by cyclic phosphodiesterase from beef heart in the presence of [18O]water to [18O]adenosine 5'-phosphorothioate. This was phosphorylated by myokinase and pyruvate kinase to [18O]adenosine 5'-(1-thiotriphosphate),Sp-diastereomer. The position of 18O was determined to be in a nonbridging position. This result indicates that the hydrolysis proceeded with inversion of configuration at phosphorus.     

The binding of [3H]adenosine to synaptosomal and other preparations from the mammalian brain.

1. A high-affinity adenosine-binding site with Kd(adenosine) 0.5-1.3 microM was demonstrated in particulate and synaptosomal fractions isolated from the cerebral cortex of guinea pig, rat and ox. 2. Binding of [3H]adenosine to this site was inhibited by theophylline and by 2-chloroadenosine, but not by four other adenosine analogues. 3. Endogenous adenosine, found to be present in some preparations at approx. 1 pmol/mg of protein, diminished the binding capacity of the preparations for [3H]adenosine. 4. Addition of the adenosine deaminase inhibitor erythro-9-[1-(1-hydroxyethyl)heptyl]-adenine revealed the presence of a second lower affinity binding site with Kd (adenosine) 5-9 microM and a higher maximal adenosine-binding capacity. The inhibitor partially blocked binding to the high-affinity site in preparations from which adenosine deaminase had been removed by washing. 5. To preparations of particulate fractions maintained under iso-osmotic conditions, adenosine attachment was non-saturable and temperature-dependent, indicating the existence of an active uptake process. 6. The location and binding constant of the high-affinity adenosine-binding site suggest that it corresponds to the receptor site for adenosine-activated adenylate cyclase.     

Ubiquitin adenylate: structure and role in ubiquitin activation

The acid precipitate of the ubiquitin activating enzyme after reaction with ATP and ubiquitin contains one enzyme equivalent of ubiquitin adenylate in which the carboxyl-terminal glycine of ubiquitin and AMP are in an acyl-phosphate linkage. The recovered ubiquitin adenylate has the catalytic properties proposed for it as a reaction intermediate. Thus, upon reaction with fresh enzyme in the absence of Mg2+ or ATP, the product complex, E-ubiquitin . AMP-ubiquitin, is formed. This complex is capable of generating ubiquitin-protein isopeptide derivatives when added to a reticulocyte fraction that catalyzes protein conjugation. This reproduces the effect previously shown to require ubiquitin, ATP, and Mg2+. In the presence of activating enzyme, ubiquitin adenylate is converted to ATP and free ubiquitin in a step requiring PPi and Mg2+. On the basis of studies of [32P]PPi/nucleoside triphosphate exchange, the activating enzyme could be used to generate 2'-deoxy-AMP-, 2'-deoxy-IMP-, and 2'-deoxy-GMP-ubiquitin but not pyrimidine nucleotide-ubiquitin derivatives. The enzyme shows a modest preference for the pro-S diastereomers of adenosine 5'-O-(1-thiotriphosphate) and adenosine 5'-O-(2-thiotriphosphate). Inorganic phosphate, arsenate, methyl phosphate, and tripolyphosphate, but not nucleoside triphosphates, can serve as alternate substrates in place of PPi in the reverse of ubiquitin adenylate formation. Therefore, the enzyme catalyzes the unusual reaction ATP + Pi in equilibrium ADP + PPi in the presence of ubiquitin.     

Synthesis of diadenosine 5',5'-P1,P4-tetraphosphate (AppppA) from adenosine 5'-phosphosulfate and adenosine 5'-triphosphate catalyzed by yeast AppppA phosphorylase

A novel way of enzymatic synthesis of diadenosine 5',5"'-P1,P4-tetraphosphate (AppppA), which does not involve aminoacyl-tRNA synthetases, has been discovered. Yeast AppppA alpha, beta-phosphorylase catalyzes irreversible conversion of adenosine 5'-phosphosulfate (APS) and ATP into AppppA according to the equation APS + ATP----AppppA + sulfate. In this reaction, the enzyme exhibits a broad pH optimum (between 6 and 8) and requires Mn2+, Mg2+, or Ca2+ ions for activity, with Mn2+ being twice as effective as Mg2+ or Ca2+ at optimal concentration (0.5 mM). The Km values computed for APS and ATP are 80 microM and 700 microM, respectively. The rate constant for the AppppA synthesis is 3 s-1 (pH 8.0, 30 degrees C, 0.5 mM MgCl2). Some ATP analogues like ppppA, GTP, adenosine 5'-(alpha, beta-methylenetriphosphate), and adenosine 5'-(beta, gamma-methylenetriphosphate), but not dATP, UTP, or CTP, are also substrates for AppppA phosphorylase and accept adenylate from APS with the formation of AppppA, AppppG, Appp(CH2)pA, and App(CH2)ppA, respectively. Functional versatility of yeast AppppA phosphorylase may provide a link between metabolism of AppppA on one hand and metabolism of APS and phosphate on the other and raises the possibility of participation of AppppA in regulation of metabolism of APS and/or inorganic phosphate in yeast.     

Comparative enzymology of AMP deaminase,adenylate kinase,and creatine kinase in vertebrate heart and skeletal muscle: the characteristic AMP deaminase levels of skeletal versus cardiac muscle are reversed in the North American toad

The specific activity of three characteristic enzymes, adenylate deaminase, adenylate kinase, and creatine kinase, in the skeletal muscles and heart of a variety of vertebrate land animals, including the human, are surveyed. Data from this study and available studies in the literature suggest that adenosine monophosphate deaminase in land vertebrates is quite high in white skeletal muscle, usually somewhat lower in red muscle, and 15-to 500-fold lower in cardiac muscle. Adenosine monophosphate deaminase is active primarily under ischemic or hypoxic conditions which occur frequently in white muscle, only occasionally in red muscle, and ought never occur in heart muscle, and this may therefore account for observed enzyme levels. The common North American toad, Bufo americanus, provides a striking exception to the rule with cardiac adenosine monophosphate deaminase as high as in mammalian skeletal muscle, whereas its skeletal muscle level of adenosine monophosphate deaminase is several times lower. The exceptional levels in the toad are not due to a change in substrate binding and are not accompanied by comparable change in the level of adenylate or creatine kinase. Nor do they signal any major change in isozyme composition, since a human muscle adenosine monophosphate deaminase-specific antiserum reacts with toad muscle adenosine monophosphate deaminase, but not with toad heart adenosine monophosphate deaminase. They do not represent any general anuran evolutionary strategy, since the bullfrog (Rana catesbeiana) and the giant tropic toad (Bufo marinus) have the usual vertebrate pattern of adenosine monophosphate deaminase distribution. Lower skeletal muscle activities in anurans may simply represent the contribution of tonic muscle fiber bundles containing low levels of adenosine monophosphate deaminase, but the explanation for the extremely high adenosine monophosphate deaminase levels in heart ventricular muscle is not apparent.Abbreviations AK adenylate kinase - AMP adenosine monophosphate - AMPD, AMP deaminase - CPK creatine (phospho)kinase - EHNA erythro-9-(2-hydroxy-3-nonyl)-adenine-HCl     

Neuroblastoma cell adenylate cyclase: direct activation by adenosine and prostaglandins.

—Adenylate cyclase activity of permeabilized neuroblastoma cells was measured by the conversion of [α³²P]ATP into labelled cyclic AMP. Adenosine (10^?6 - 10^?4m ) induced a dose-dependent increase in cyclic AMP formation. This effect could not be accounted for either by an adenosine-induced inhibition of the phosphodiesterase activity present in the enzyme preparation, or by a direct conversion of adenosine into cyclic AMP. This indicates that the observed increase in cyclic AMP accumulation reflected an activation of adenylate cyclase. Adenosine is partially metabolized during the course of incubation with the enzyme preparation. However, none of the identified non-phosphorylated adenosine metabolites were able to induce an adenylate cyclase activation. This suggests that adenosine itself is the stimulatory agent. The apparent K_m of the adenylate cyclase for adenosine was 5 ± 10^?6-10^?5m . Maximal activation represented 3-4 times the basal value (10-100 pmol cyclic AMP formed/10 min/mg protein). The adenosine effect was stereospecific, since structural analogues of adenosine were inactive. Adenosine increased the maximal velocity of the adenylate cyclase reaction. The stimulatory effect of adenosine was inhibited by theophylline. Prostaglandin PGE₁ had a stimulatory effect much more pronounced than that of adenosine (6-10-fold the basal value at 10^?6m ). Dopamine and norepinephrine induced a slight adenylate cyclase activation which was not potentiated by adenosine. It is concluded that adenosine is able to activate directly neuroblastoma cell adenylate cyclase. It seems very likely that such a direct activation is also present in intact nervous tissue and account, at least partly, for the observed cyclic AMP accumulation in response to adenosine.     

Mechanism of activation of phenylalanine and synthesis of P1, P4-bis(5'-adenosyl) tetraphosphate by yeast phenylalanyl-tRNA synthetase

The activation of L-phenylalanine by yeast phenylalanyl-tRNA synthetase using adenosine 5'-[(S)-alpha-17O,alpha,alpha-18O2]triphosphate is shown to proceed with inversion of configuration at P alpha of ATP. This observation taken together with the lack of positional isotope exchange when adenosine 5'-[beta,beta-18O2]triphosphate is incubated with the enzyme in the absence of phenylalanine and in the presence of the competitive inhibitor phenylalaninol indicates that activation of phenylalanine occurs by a direct "in-line" adenylyl-transfer reaction. In the presence of Zn2+, yeast phenylalanyl-tRNA synthetase also catalyzes the phenylalanine-dependent hydrolysis of ATP to AMP and the synthesis of P1,P4-bis(5'-adenosyl) tetraphosphate (Ap4A). With adenosine 5'-[(S)-alpha-17O,alpha,alpha-18O2]triphosphate, the formation of AMP and Ap4A is shown to occur with inversion and retention of configuration, respectively. It is concluded that phenylalanyl adenylate is an intermediate in both processes, Zn2+ promoting AMP formation by hydrolytic cleavage of the C-O bond and Ap4A formation by displacement at phosphorus of phenylalanine by ATP.