5''-Nucleotidase activity and adenosine formation in stimulated, hypoxic and underperfused rat heart.

Changes in 5'-nucleotidase activity were calculated on the basis of alterations in ATP, ADP, phosphocreatine, Pi, Mg2+, IMP and AMP, determined by using 31P n.m.r. spectroscopy and h.p.l.c., during isoprenaline infusion, graded hypoxia and graded underperfusion in isolated rat heart. Calculated activity changes were compared with the total efflux of purines (adenosine + inosine + hypoxanthine) in order to assess the involvement of various 5'-nucleotidases in formation of adenosine. Purine efflux exhibited an exponential relation with cytosolic [AMP] during isoprenaline infusion and hypoxia (r = 0.92 and 0.95 respectively), supporting allosteric activation of 5'-nucleotidase under these conditions. Purine efflux displayed a linear relation with cytosolic [AMP] during graded ischaemia (r = 0.96), supporting substrate regulation in the ischaemic heart. The calculated activities of membrane-bound ecto-5'-nucleotidase were similar to the observed relations between purine efflux and cytosolic [AMP] in all hearts. The calculated activities of the ATP-activated cytosolic and lysosomal enzymes and of the ATP-inhibited cytosolic 5'-nucleotidase could not explain the observed release of purines under the conditions examined. These results indicate that the kinetic characteristics of the membrane-bound ecto-enzyme are consistent with an important role in the formation of extracellular adenosine, whereas the characteristics of the other 5'-nucleotidases are inconsistent with roles in adenosine formation under the conditions of the present study.     

Mechanisms of hydrolysis of adenosine 5'-triphosphate, adenosine 5'-diphosphate, and inorganic pyrophosphate in aqueous perchloric acid

The acid-catalyzed hydrolysis of adenosine 5'-triphosphate (ATP) has been found to give rise both to adenosine 5'-diphosphate (ADP) and inorganic phosphate and to adenosine 5'-phosphate (AMP) and inorganic pyrophosphate. Kinetic and isotope studies on the mechanism of hydrolysis of ATP therefore depend on a knowledge of the mechanism of hydrolysis of the polyphosphate products, ADP and inorganic pyrophosphate. The latter reactions have been studied over the acidity range 1--5 M perchloric acid at 25 degrees C while the more complex problem of the hydrolysis of ATP has been followed at a single acidity (3 M perchloric acid). The positions of bond fission have been determined for both ATP and ADP.     

Phosphorylation of adenosine 5'-monophosphate by a dialyzed cell-free extract from Escherichia coli.

A cell-free extract of Escherichia coli, even after exhaustive dialysis, was found capable of phosphorylating adenosine 5'-monophosphate (AMP) to adenosine 5'-diphosphate (ADP) and adenosine 5'-triphosphate (ATP). Centrifugation at 100 000 g for 3h sedimented most of the capacity to phosphorylate AMP to ATP, while the supernatant retained a significant capacity to phosphorylate AMP to ADP. The pellet contained a greater amount of phosphate polymers (which were neither DNA, RNA, nor proteins) than did the supernatant. The addition of authentic inorganic polyphosphates to the supernatant restored the phosphorylating capacity of the original extracts. It is concluded that the observed phosphorylation is partly due to inorganic polyphosphate.     

A specific inhibitor of heart cytosolic 5'-nucleotidase I attenuates hydrolysis of adenosine 5'-monophosphate in primary rat myocytes

ATP breakdown was triggered in primary rat myocytes in the presence of coformycin to force the catabolism of AMP through hydrolysis to adenosine. Selective inhibitors of the cytosolic 5'-nucleotidase I (c-N-I) from myocardium were used to measure the intracellular contribution of this enzyme to AMP hydrolysis under these conditions. The selective inhibitor 5-ethynyl-2',3'-dideoxyuridine inhibited the hydrolysis of AMP to adenosine in a concentration-dependent manner with an IC50 value of 20 microM. Maximal inhibition prevented 76% of the conversion of AMP to adenosine, indicating that under these conditions the majority of AMP hydrolysis in rat myocytes occurs through this enzyme. When ATP breakdown was triggered in the presence of thymidine 5'-phosphonate, a more potent inhibitor of the purified cytosolic 5'-nucleotidase, less inhibition of AMP hydrolysis occurred and only after prolonged preincubation of the myocytes with the inhibitor. These data demonstrate that the selective nucleoside inhibitors of c-N-I can effectively block the hydrolysis of AMP inside myocytes. Thus, these inhibitors may be useful tools in identifying the role of c-N-I during ATP catabolism in whole tissue and animal experiments.     

Activation of the alternative oxidase of Yarrowia lipolytica by adenosine 5'-monophosphate

The study of the effect of nucleoside phosphates on the activity of cyanide-resistant oxidase in the mitochondria and the submitochondrial particles of Yarrowia lipolytica showed that adenosine monophosphate (5'-AMP, AMP) did not stimulate the respiration of the intact mitochondria. The incubation of the mitochondria at room temperature (25 degrees C) for 3-5 h or their treatment with ultrasound, phospholipase A, and detergent Triton X-100 at a low temperature inactivated the cyanide-resistant alternative oxidase. The inactivated alternative oxidase could be reactivated by AMP. The reactivating effect of AMP was enhanced by azolectin. Some other nucleoside phosphates also showed reactivating ability in the following descending order. AMP = GMP > GDP > GTP > XMP > IMP. The apparent reaction rate constant Km for AMP upon the reactivation of the alternative oxidase of mitochondria treated with Triton X-100 or incubated at 25 degrees C was 12.5 and 20 microM, respectively. The Km for AMP upon the reactivation of the alternative oxidase of submitochondrial particles was 15 microM. During the incubation of yeast cells under conditions promoting the development of alternative oxidase, the content of adenine nucleotides (AMP, ADP, and ATP) in the cells and their respiration tended to decrease. The subsequent addition of cyanide to the cells activated their respiration, diminished the intracellular content of ATP three times, and augmented the content of AMP five times. These data suggest that the stimulation of cell respiration by cyanide may be due to the activation of alternative oxidase by AMP.     

Affinity labeling of AMP-ADP sites in heart phosphofructokinase by 5-p-fluorosulfonylbenzoyl adenosine.

The purine nucleotide derivative, 5′-p-fluorosulfonylbenzoyl adenosine (5′-FSO₂B_ZAdo) functions as an affinity label for the allosteric sites of phosphofructokinase. The modified enzyme at pH 6.9 is insensitive to allosteric inhibition by ATP, activation by AMP, c-AMP, ADP and shows no sigmoidal kinetics for fructose-6-P. The reaction does not appear to occur at the catalytic site since modification of the enzyme does not significantly affect its specific activity nor its Michaelis constant at pH 8.2. ADP, and to a much lesser degree AMP and ATP, protects the enzyme from modification by the adenosine reagent. The modified enzyme essentially does not bind significant amounts of AMP, c-AMP, ADP, but still binds an analog of ATP, AppNHp. The adenosine affinity label will be of value in studies on the nature of the AMP-ADP allosteric sites.     

A continuous spectrophotometric assay for monitoring adenosine 5′-monophosphate production

A number of biologically important enzymes release adenosine 5′-monophosphate (AMP) as a product, including aminoacyl–tRNA synthetases, cyclic AMP (cAMP) phosphodiesterases, ubiquitin and ubiquitin-like ligases, DNA ligases, coenzyme A (CoA) ligases, polyA deadenylases, and ribonucleases. In contrast to the abundance of assays available for monitoring the conversion of adenosine 5′-triphosphate (ATP) to ADP, there are relatively few assays for monitoring the conversion of ATP (or cAMP) to AMP. In this article, we describe a homogeneous assay that continuously monitors the production of AMP. Specifically, we have coupled the conversion of AMP to inosine 5′-monophosphate (IMP) (by AMP deaminase) to the oxidation of IMP (by IMP dehydrogenase). This results in the reduction of oxidized nicotine adenine dinucleotide (NAD⁺) to reduced nicotine adenine dinucleotide (NADH), allowing AMP formation to be monitored by the change in the absorbance at 340 nm. Changes in AMP concentrations of 5 μM or more can be reliably detected. The ease of use and relatively low expense make the AMP assay suitable for both high-throughput screening and kinetic analyses.     

An adenosine 3':5'-monophosphate-adenosine binding protein from mouse liver.

A cyclic AMP-adenosine binding protein from mouse liver has been purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis in the absence and presence of sodium dodecyl sulfate and by analytical ultracentrifugation. The binding protein had a Stokes radium of 48 A based on gel chromatography. Both the purified binding protein and the binding activity in fresh cytosol sedimented as 9 S on sucrose gradient centrifugation. The homogeneous protein had a sedimentation coefficient (S20, w) of 8.8 x 10-13 s, as calculated from sedimentation velocity experiments. By use of the Stokes radius and S20, w', the molecular weight was calculated to be 180,000. The protein was composed of polypeptides having the same molecular weight of 45,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and thus appeared to consist of four subunits of equal size. The isoelectric point, pI = 5.7. The binding capacity for cyclic AMP increased by preincubating the receptor protein in the presence of Mg2+ ATP. This process, tentatively termed activation, was studied in some detail and was shown not be be be accompanied by dissociation, aggregation, or phosphorylation of the binding protein. Cyclic AMP was bound to the protein with an apparent dissociation constant (Kd) of 1.5 x 10-7 M. The binding of cyclic AMP was competitively inhibited by adenosine, AMP, ADP, and ATP whose inhibition constants were 8 x 10-7 M, 1.2X 10-6 M, 1.5 X 10-6 M, and higher than 5 x 10-6 M respectively. A hyperbolic Scatchard plot was obtained for the binding of adenosine to the activated binding protein, indicating more than one site for adenosine. The binding of adenosine to the site with the highest affinity (Kd=2 x 10-7 M) for this nucleoside was not suppressed by excess cyclic AMP and was thus different from the aforementioned cyclic AMP binding site. Cyclic GMP, GMP, guanosine, cyclic IMP, IMP, and inosine did not inhibit the binding of either cyclic AMP or adenosine. The binding protein had no cyclic AMP phosphodiesterase, adenosine deaminase, phosphofructokinase, or protein kinase activities, nor does it inhibit the catalytic subunit of the cyclic AMP-dependent protein kinase.     

Identification and partial characterization of an adenosine(5'')tetraphospho(5'')adenosine hydrolase on intact bovine aortic endothelial cells.

The biologically active dinucleotides adenosine(5')tetraphospho(5')adenosine (Ap4A) and adenosine(5')-triphospho(5')adenosine (Ap3A), which are both releasable into the circulation from storage pools in thrombocytes, are catabolized by intact bovine aortic endothelial cells. 1. Compared with extracellular ATP and ADP, which are very rapidly hydrolysed, the degradation of Ap4A and Ap3A by endothelial ectohydrolases is relatively slow, resulting in a much longer half-life on the endothelial surface of the blood vessel. The products of hydrolysis are further degraded and finally taken up as adenosine. 2. Ap4A hydrolase has high affinity for its substrate (Km 10 microM). 3. ATP as well as AMP transiently accumulates in the extracellular fluid, suggesting an asymmetric split of Ap4A by the ectoenzyme. 4. Mg2+ or Mn2+ at millimolar concentration are needed for maximal activity; Zn2+ and Ca2+ are inhibitory. 5. The hydrolysis of Ap4A is retarded by other nucleotides, such as ATP and Ap3A, which are released from platelets simultaneously with Ap4A.     

Formation of adenosine 3',5'-monophosphate from adenosine in mouse thymocytes.

The effect of adenosine on the mouse thymocyte adenylate cyclase-adenosine 3':5'-monophosphate (cyclic AMP) system was examined. Adenosine, like prostaglandin E1, can cause 5-fold or greater increases in thymocyte cyclic AMP content in the presence but not in the absence of certain cyclic phosphodiesterase inhibitors. Two non-methylxanthine inhibitors potentiated the prostaglandin E1 and adenosine responses, while methylxanthines selectively inhibited the adenosine response. Adenosine increased cyclic AMP content significantly within 1 min and was maximal by 10 to 20 min with approx. 2 and 10 muM adenosine being minimal and half-maximal effective doses, respectively. Combinations of prostaglandin E1, isoproterenol and adenosine were near additive and not synergistic. Of the adenosine analogues tested, only 2-chloro- and 2-fluoroadenosine significantly increased cyclic AMP. Thymocytes prelabeled with [14C]adenine exhibited dramatic increases in cyclic [14C]AMP 10 min after addition of adenosine or prostaglandin E1 which corresponded to simultaneously determined increases in total cyclic AMP. Using [14C]adenosine, the percent of total cyclic AMP increase due to adenosine was only 16%. Adenosine was also shown to elicit a 40% increase in particulate thymocyte adenylate cyclase activity. Therefore, the increased content of cyclic AMP seen in mouse thymocytes after incubation with adenosine was due primarily to stimulation of adenylate cyclase and only partially to conversion of adenosine to cyclic AMP. The increased cellular content of cyclic AMP may be, in part, responsible for various immunosuppressive effects of adenosine.     

Kinetic characterization of ATP diphosphohydrolase and 5'-nucleotidase activities in cells cultured from submandibular salivary glands of rats

The participation of ecto-ATP diphosphohydrolase (CD39; ecto-NTPDase) and ecto-5'-nucleotidase (CD73) activities in the nucleotide hydrolysis by salivary gland cells from rats was evaluated. We investigated the biochemical characteristics of these ectoenzymes in cells cultured from submandibular salivary glands of rats. The V(max) for the hydrolysis of ATP, ADP and AMP were 2275+/-153 (mean+/-SEM, n = 4), 941+/-96 (mean+/-SEM, n = 5) and 175+/-5 (mean+/-SEM, n = 5) nmol Pi liberated per min per mg of protein, respectively. The K(m) values for ATP, ADP and AMP were 224+/-8 microM (mean+/-SEM, n = 4), 163+/-15 microM (mean+/-SEM, n = 5) and 117+/-5 microM (mean+/-SEM, n = 5), respectively. The competition plot showed that ATP and ADP were hydrolyzed at the same active site on the enzyme. It may be postulated that the physiological role for this ecto-enzyme cascade is to terminate the action of the co-transmitter ATP, generating adenosine.     

Regulation of adenosine 3' :5'-monophosphate efflux from rat glioma cells in culture*.

Rat glioma cells grown in culture secrete cyclic adenosine 3':5'-monophosphate (cyclic AMP) into the culture medium following stimulation by beta-agonistic catecholamines. Agents which reduced cellular ATP levels such as valinomycin, oligomycin, and uncouplers of oxidative phosphorylation, inhibited cyclic AMP efflux. Secretion of cyclic AMP was also prevented by prostaglandin A-1 and pharmacological agents including probenecid and papaverine. Of the latter agents, only papaverine reduced ATP levels. These results suggest that the transport of cyclic AMP across animal cell membranes is energy-dependent and subject to regulation.     

Synthesis and biological activity of 5'-capped derivatives of 5'-triphosphoadenylyl(2'----5')adenylyl(2'----5')adenosine

The oligonucleotides A5'pp5'A2'p5'A2'p5'A and A5'ppp5'A2'p5'A2'p5'A were prepared by reaction of AMP or ADP, respectively, with the 5'-(phosphoimidazolidate) of A2'p5'A2'p5'A. A5'pppp5'A2'(p5'A)n (n = 1-3) were synthesized by reaction of p5'A2'(p5'A)n (n = 1-3) with adenosine 5'-trimetaphosphate. All structures were confirmed by enzyme digestion and 1H and 31P nuclear magnetic resonance (NMR). The products A5'pppp5'A2'p5'A and A5'pppp5'A2'p5'A2'p5'A were found to be identical with two of the products of the 2-5A synthetase catalyzed reaction of Ap4A with ATP, thus confirming the structural assignments made by earlier investigators. In extracts of mouse L cells programmed with encephalomyocarditis virus RNA, A5'pppp5'A2'p5'A2'p5'A2'p5'A and A5'pppp5'A2'p5'A2'p5'A were equipotent with 2-5A itself as inhibitors of translation. The oligomers A5'ppp5'A2'p5'A2'p5'A and A2'pppp5'A2'p5'A were about 100 times less active than 2-5A, and A5'pp5'A2'p5'A2'p5'A was without translational inhibitory activity. When affinity for the 2-5A-dependent endonuclease was determined (by displacement of 2-5A[32P]pCp from endonuclease), all of the analogues, as well as 2-5A itself, had similar affinities for the endonuclease except for A5'pppp5'A2'p5'A, which was bound approximately 100 times less effectively. Under conditions of the radiobinding assay, A5'pppp5'A2'p5'A2'p5'A was degraded (t1/2 = 2 h) to ATP, ADP, AMP, ppp5'A2'p5'A2'p5'A, and p5'A2'p5'A2'p5'A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vectorial production of adenosine by 5'-nucleotidase in the perfused rat heart

Rat hearts were perfused simultaneously with [8-3H] AMP and [8-14C]adenosine. [8-3H] AMP was hydrolzyed by 5'-nucleotidase to produce intra- and extracellular [8-3H] adenosine. Comparison of the specific activities of [3H]- and [14C]adenosine in the heart cells with the specific activities of [3H]- and [14C]adenosine in the effluent perfusate showed that much more [3H]adenosine accumulated in the tissue than would be expected if extracellular adenosine were the immediate precursor of intracellular adenosine. Conversely, perfusion of rat hearts with [8-14C]AMP and [8-3H]adenosine led to a much greater accumulation of intracellular [14C]adenosine than would be expected from an uptake of adenosine from the perfusate. These results are interpreted to be due to hydrolysis of extracellular AMP by 5'-nucleotidase, located in the plasma membrane, and release of the resulting adenosine inside the cell. Measurements of the specific activities of 3H and 14C in ATP, ADP, AMP, and inosine support this interpretation.     

Growth inhibition of transformed mouse fibroblasts by adenine nucleotides occurs via generation of extracellular adenosine

The growth of transformed mouse fibroblasts (3T6 cells) in medium containing 5% fetal bovine serum was inhibited after treatment with concentrations greater than 50 microM ATP, ADP, or AMP. Adenosine, the common catabolite of the nucleotides, had no effect on cell growth at concentrations below 1 mM. However, the following results indicate that the toxicity of ATP, ADP, and AMP is mediated by serum- and cell-associated hydrolysis of the nucleotides to adenosine. 1) ADP and AMP, but not ATP, were toxic to 3T6 cells grown in serum-free medium or medium in which phosphohydrolase activity of serum was inactivated. Under these conditions, the cells exhibited cell-associated ADPase and 5'-nucleotidase activity, but little ecto-ATPase activity. 2) Inhibition of adenosine transport in 3T6 cells by dipyridamole or S-(p-nitrobenzyl)-6-thioinosine prevented the toxicity of ATP in serum-containing medium and of ADP and AMP in serum-free medium. 3) A 16-24-h exposure to 125 microM AMP or ATP was needed to inhibit cell growth under conditions where serum- and cell-associated hydrolysis of the nucleotides generated adenosine in the medium continuously over the same time period. In contrast, 125 microM adenosine was completely degraded to inosine and hypoxanthine within 8-10 h. Furthermore, multiple doses of adenosine added to the cells at regular intervals over a 16-h period were significantly more toxic than an equivalent amount of adenosine added in one dose. Treatment of 3T6 cells with AMP elevated intracellular ATP and ADP levels and reduced intracellular UTP levels, effects which were inhibited by extracellular uridine. Uridine also prevented growth inhibition by ATP, ADP, and AMP. These and other results indicate that serum- and cell-associated hydrolysis of adenine nucleotides to adenosine suppresses growth by adenosine-dependent pyrimidine starvation.     

Influence of the protonation degree on the self-association properties of adenosine 5'-triphosphate (ATP)

The concentration dependence of the chemical shifts for the protons H-2, H-8 and H-1' of ATP has been measured in D2O at 27 degrees C under several degrees of protonation in the pD range from 1.5 to 8.4. The results at pD greater than 4.5 are consistent with the isodesmic model of indefinite noncooperative stacking, while those at pD less than 4.5 indicate a preference for the formation of dimeric stacks. The stacking tendency follows the series, ATP4- (K = 1.3 M-1) less than D(ATP)3- (2.1 M-1) less than 1:1 ratio of D(ATP)3-/D2(ATP)-2- (6.0 M-1) much less than D2(ATP)2- (approximately 200 M-1) much greater than D3(ATP)- (K approximately less than 17 M-1) (for reasons of comparison all constants are expressed in the isodesmic model). These results are compared with previous data for adenosine [Ado (K = 15 M-1) greater than 1:1 ratio of Ado/D(Ado)+ (6.0 M-1) greater than D(Ado)+ (0.9 M-1)] and AMP [AMP2- (K = 2.1 M-1) less than D(AMP)- (3.4 M-1) less than 1:1 ratio of D(AMP)-/D2(AMP) +/- (5.6 M-1) greater than D2(AMP) +/- (approximately equal to 2 M-1) greater than D3(AMP)+ (K less than or equal to 1 M-1)] to facilitate the interpretation of the results for the ATP systems. Stack formation of H2(ATP)2- is clearly favored by additional ionic interactions; this is confirmed by measuring via potentiometric pH titrations the acidity constants of H2(ATP)2- in solutions containing different concentrations of ATP. It is suggested that in the [H2(ATP)]4-(2) dimer intermolecular ion pairs (and hydrogen bonds) are formed between the H+(N-1) site of one H2(ATP)2- and the gamma-P(OH)(O)-2 group of the other; in this way (a) the stack is further stabilized, and (b) the positive charges at the adenine residues are compensated (otherwise repulsion would occur as is evident from the adenosine systems). A detailed structure for the [H2(ATP)4-(2) dimer is proposed and some implications of the described stacking properties of ATP for biological systems are indicated.     

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.     

Thermodynamics of the disproportionation of adenosine 5'-diphosphate to adenosine 5'-triphosphate and adenosine 5'-monophosphate, II. Experimental data

High-pressure liquid-chromatography and microcalorimetry have been used to determine equilibrium constants and enthalpies of reaction for the disproportionation reaction of adenosine 5′-diphosphate (ADP) to adenosine 5′-triphosphate (ATP) andadenosine 5′-monophosphate (AMP). Adenylate kinase was used to catalyze this reaction. The measurements were carried out over the temperature range 286 to 311 K, at ionic strengths varying from 0.06 to 0.33 mol kg⁻¹, over the pH range 6.04 to 8.87, and over the pMg range 2.22 to 7.16, where pMg = -log a(Mg²⁺). The equilibrium model developed by Goldberg and Tewari (see the previous paper in this issue) was used for the analysis of the measurements. Thus, for the reference reaction: 2 ADp³⁻ (ao) AMp²⁻ (ao)+ ATp⁻ (ao), K° = 0.225 ± 0.010, ΔG° = 3.70 +- 0.11 kJ mol ⁻¹, ΔH° = −1.5 ± 1. 5 kJ mol ⁻¹, °S ° = −17 ± 5 J mol⁻¹ K⁻¹, and ACP_p°≈ = −46 J mo1l⁻¹ K⁻¹ at 298.15 K and 0.1 MPa. These results and the thermodynamic parameters for the auxiliary equilibria in solution have been used to model the thermodynamics of the disproportionation reaction over a wide range of temperature, pH, ionic strength, and magnesium ion morality. Under approximately physiological conditions (311.15 K, pH 6.94, [Mg²⁺] = 1.35 × 10⁻³ mol kg⁻¹, and I = 0.23 mol kg⁻¹) the apparent equilibrium constant (K_A′ = m(ΣAMP)m(ΣATP)/[ m(ΣADP)]²) for the overall disproportionation reaction is equal to 0.93 ± 0.02. Thermodynamic data on the disproportionation reaction and literature values for this apparent equilibrium constant in human red blood cells are used to calculate a morality of 1.94 × 10⁻⁴ mol kg⁻¹ for free magnesium ion in human red blood cells. The results are also discussed in relation to thermochemical cycles and compared with data on the hydrolysis of the guanosine phosphates.     

Role of adenosine deaminase, ecto-(5''-nucleotidase) and ecto-(non-specific phosphatase) in cyanide-induced adenosine monophosphate catabolism in rat polymorphonuclear leucocytes.

1. The role of adenosine deaminase (EC 3.5.4.4), ecto-(5'-nucleotidase) (EC 3.1.3.5) and ecto-(non-specific phosphatase) in the CN-induced catabolism of adenine nucleotides in intact rat polymorphonuclear leucocytes was investigated by inhibiting the enzymes in situ. 2. KCN (10mM for 90 min) induced a 20-30% fall in ATP concentration accompanied by an approximately equimolar increase in hypoxanthine, ADP, AMP and adenosine concentrations were unchanged, and IMP and inosine remained undetectable ( less than 0.05 nmol/10(7) cells). 3. Cells remained 98% intact, as judged by loss of the cytoplasmic enzyme lactate dehydrogenase (EC 1.1.1.27). 4. Pentostatin (30 microM), a specific inhibitor of adenosine deaminase, completely inhibited hypoxanthine production from exogenous adenosine (55 microM), but did not black CN-induced hypoxanthine production or cause adenosine accumulation in intact cells. This implied that IMP rather than adenosine was an intermediate in AMP breakdown in response to cyanide. 5. Antibodies raised against purified plasma-membrane 5'-nucleotidase inhibited the ecto-(5'-nucleotidase) by 95-98%. Non-specific phosphatases were blocked by 10 mM-sodium beta-glycerophosphate. 6. These two agents together blocked hypoxanthine production from exogenous AMP and IMP (200 microM) by more than 90%, but had no effect on production from endogenous substrates. 7. These data suggest that ectophosphatases do not participate in CN-induced catabolism of intracellular AMP in rat polymorphonuclear leucocytes. 8. A minor IMPase, not inhibited by antiserum, was detected in the soluble fraction of disrupted cells.